nephrin and Proteinuria

Early detection of pre-eclampsia remains one of the major focuses of antenatal obstetric care. There is often a delay in the diagnosis, mainly due to the non-specific nature of the condition. Podocytes which play a pivotal role in glomerular function become injured in pre-eclampsia leading to subsequent proteinuria. Our aim was to review available studies to determine the clinical utility of biomarkers of podocyte injury in pre-eclampsia.. We used QUADAS (Quality Assessment of Diagnostic Accuracy Studies) criteria to perform a systematic review of the literature to determine the clinical utility of podocyte injury biomarkers in predicting pre-eclampsia.. This study identified five potential renal biomarkers including podocytes, nephrin, synaptopodin, podocin and podocalyxin. The pooled sensitivity of all biomarkers was 0.78 (95% CI 0.74-0.82) with a specificity of 0.82 (95% CI 0.79-0.85). The area under the Summary of Receiver Operating Characteristics Curve (SROC) was 0.926 (SE 0.30). Urinary nephrin achieved the highest diagnostic values with a sensitivity of 0.81 (95% CI 0.72-0.88) and specificity of 0.84 (95% CI 0.79-0.84).. Biomarkers of glomerular injury show promise as diagnostic aids in pre-eclampsia. A large-scale prospective cohort study is warranted before these biomarkers can be recommended for routine clinical care.

Topics: Biomarkers; Case-Control Studies; Female; Glomerular Filtration Barrier; Humans; Kidney Glomerulus; Membrane Proteins; Podocytes; Pre-Eclampsia; Pregnancy; Proteinuria; ROC Curve; Sensitivity and Specificity; Sialoglycoproteins

Podocyte biology is a developing science that promises to help improve understanding of the mechanistic nature of multiple diseases associated with proteinuria. Proteinuria in nephrotic syndrome has been linked to mechanistic dysfunctions in the renal glomerulus involving the function of podocyte epithelial cells, including podocyte foot process effacement.. Developments in imaging technology are improving knowledge of the detailed structure of the human renal glomerulus and cortex. Podocyte foot processes attach themselves to the glomerular capillaries at the glomerular basement membrane (GBM) forming intercellular junctions that form slit diaphragm filtration barriers that help maintain normal renal function. Damage in this area has been implicated in glomerular disease. Injured podocytes undergo effacement whereby they lose their structure and spread out, leading to a reduction in filtration barrier function. Effacement is typically associated with the presence of proteinuria in focal segmental glomerulosclerosis, minimal change disease, and diabetes. It is thought to be due to a breakdown in the actin cytoskeleton of the foot processes, complex contractile apparatuses that allow podocytes to dynamically reorganize according to changes in filtration requirements. The process of podocyte depletion correlates with the development of glomerular sclerosis and chronic kidney disease. Focal adhesion complexes that interact with the underlying GBM bind the podocytes within the glomerular structure and prevent their detachment. Key Messages: Knowledge of glomerular podocyte biology is helping to advance our understanding of the science and mechanics of the glomerular filtering process, opening the way to a variety of new potential applications for clinical targeting.

Topics: Actins; Humans; Membrane Proteins; Nephrotic Syndrome; Podocytes; Proteinuria

Urinary podocalyxin and nephrin are urine markers of podocyte dysfunction that may reflect the integrity of kidney's filtration barrier. Studies on their respective roles in glomerular diseases are still underway. However, the isolated and unsystematic manner in which they are being studied does not permit proper identification of their roles in each glomerular disease. As such, there is little or no appreciation of what research has already achieved and what remains to be achieved as the research direction is not clearly defined. We explored the recent studies and outlined the major findings regarding the value of both biomarkers in each of the three glomerular disease entities. Our review covered diabetic nephropathy, membranous nephropathy and IgA nephropathy.

Topics: Biomarkers; Diabetic Nephropathies; Glomerulonephritis, IGA; Humans; Membrane Proteins; Podocytes; Proteinuria; Sialoglycoproteins

Lupus is no longer an unknown chameleon of medicine. Significant progress has been made on unraveling the pathogenesis of lupus and lupus nephritis, and how to treat the disease. Here we provide an update on the pathophysiology of lupus and its related kidney disease, consider areas of controversy in disease management, and discuss the unmet needs of lupus nephritis and how to address these needs. We focus on rethinking how innovative therapies for lupus nephritis should be evaluated and evolving strategies to more efficiently mitigate irreversible nephron loss in patients with lupus nephritis.

Topics: Antibodies, Antinuclear; Autoimmunity; Biomarkers; Biopsy; Clinical Trials as Topic; Genetic Predisposition to Disease; Glucocorticoids; Humans; Immunosuppressive Agents; Kidney; Lupus Nephritis; Membrane Proteins; Mutation; Proteinuria

Topics: Actins; Adaptor Proteins, Signal Transducing; Animals; Cell Differentiation; Cytoskeletal Proteins; Glomerular Filtration Rate; Humans; Induced Pluripotent Stem Cells; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Mechanoreceptors; Membrane Proteins; Multiprotein Complexes; Podocytes; Proteinuria; rac1 GTP-Binding Protein; Renal Insufficiency, Chronic; Signal Transduction; TRPC Cation Channels; TRPC6 Cation Channel

Renal transplantation (RTx) is the only curative treatment for most cases of congenital and infantile nephrotic syndrome (NS) caused by genetic defects in glomerular podocyte proteins. The outcome of RTx in these children is usually excellent, with no recurrence of nephrotic syndrome. A subgroup of patients with the Finnish type of congenital nephrosis (CNF), shows, however, a clear risk for post-RTx proteinuria. Most of these patients have a homozygous truncating mutation (Fin-major mutation) in the nephrin gene (NPHS1), leading to total absence of the major podocyte protein, nephrin. After RTx, these patients develop anti-nephrin antibodies resulting in nephrotic range proteinuria. Plasma exchange combined with cyclophosphamide and anti-CD20 antibodies has proved to be successful therapy for these episodes. NS recurrence has also occurred in a few patients with mutations in the podocin gene (NPHS2). No anti-podocin antibodies have been detectable, and the pathophysiology of the recurrence remains open. While most of these episodes have resolved, the optimal therapy remains to be determined.

Topics: Autoantibodies; Autoantigens; Humans; Kidney Transplantation; Membrane Proteins; Mutation; Nephrotic Syndrome; Proteinuria; Recurrence

Podocytes are postmitotic renal glomerular cells with multiple ramifications that extend from the cell body. Processes departing from a podocyte interdigitate with corresponding projections from neighboring cells and form an intricate web that enwraps the glomerular capillary completely. Podocyte processes are interconnected by the slit diaphragm, an adhesion junction mostly formed by Ig-like molecules, cadherins/protocadherins, ephrin/eph, and neurexin molecules organized in an assembly that resembles synaptic junctions. Podocyte failure is primarily or secondarily implicated in all forms of proteinuric glomerular diseases, as confirmed by the morphological changes of their elaborate cell architecture detectable by electron microscopy. Importantly, mutations of podocyte proteins are responsible for the most severe forms of congenital nephrotic syndrome. In the last 15 years, progressive technological advances have aided the study of podocyte biology and pathology, confirming the relevance of podocyte molecules and signaling pathways for the function of the glomerular filter. This review will examine the most important and newest discoveries in the field, which is rapidly evolving, hopefully leading to a detailed knowledge of this fascinating cell and to the development of specific therapeutic options for proteinuric diseases.

Topics: Animals; Humans; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Mice; Models, Biological; Podocytes; Proteinuria; Signal Transduction

Heritable causes of proteinuria are rare and account for a relatively small proportion of all cases of proteinuria affecting children and adults. Yet, significant contributions to understanding the mechanistic basis for proteinuria have been made through genetic and molecular analyses of a small group of syndromic and nonsyndromic proteinuric disorders which are caused by mutations encoding structural components of the glomerular filtration barrier. Technological advances in genomic analyses and improved accessibility to mutational screening at clinically approved laboratories have facilitated diagnosis of proteinuria in the clinical setting. From a clinical standpoint, it may be argued that a genetic diagnosis mitigates exposure to potentially ineffective and harmful treatments in instances where a clear genotype-phenotype correlation exists between a specific gene mutation and treatment nonresponsiveness. However, cautious interpretation of risk may be necessitated in cases with phenotypic heterogeneity (eg, variability in clinical or histological presentation). This review summarizes gene mutations which are known to be associated with proteinuric glomerulopathies in children and adults.

Topics: Adult; Animals; Apolipoprotein L1; Apolipoproteins; Autoantibodies; Child; Complement System Proteins; Female; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Lipoproteins, HDL; Male; Membrane Proteins; Mice; Molecular Motor Proteins; Mutation; Myosin Heavy Chains; Nephrotic Syndrome; Proteinuria; Rats

Several recent studies have demonstrated that the slit diaphragm of the glomerular epithelial cell (podocyte) is the structure likely to be the principal barrier in the glomerular capillary wall. Nephrin identified as a gene product mutated in congenital nephrotic syndrome located at the outer leaflet of plasma membranes of the slit diaphragm. The anti-nephrin antibody is capable of inducing massive proteinuria, which indicates that nephrin is a key functional molecule in the slit diaphragm. Expression of nephrin was reduced in glomeruli of minimal change nephrotic syndrome. Some recent studies demonstrated that podocin, CD2-associated protein and NEPH1 are also functional molecules in the slit diaphragm, and their expressions are altered in membranous nephropathy and also in focal glomerulosclerosis. These observations suggested that the alteration of the molecular arrangement in the slit diaphragm is involved in the development of proteinuria in several kinds of glomerular diseases. Recent studies of our group have demonstrated that type 1 receptor-mediated angiotensin II action reduced the expression of the slit diaphragm-associated molecules and that type 1 receptor blockade ameliorated proteinuria by preventing the function of angiotensin II on the slit diaphragm. By the subtraction hybridization techniques using glomerular cDNA of normal and proteinuric rats, we detected that synaptic vesicle protein 2B and ephrin B1 are involved in the maintenance of the barrier function of the slit diaphragm.

Topics: Adaptor Proteins, Signal Transducing; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Cytoskeletal Proteins; Ephrin-B1; Glomerular Filtration Rate; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Membrane Proteins; Nephrotic Syndrome; Nerve Tissue Proteins; Podocytes; Proteinuria; Receptor, Angiotensin, Type 1

This review deals with podocyte proteins that play a significant role in the structure and function of the glomerular filter. Genetic linkage studies has identified several genes involved in the development of nephrotic syndrome and contributed to the understanding of the pathophysiology of glomerular proteinuria and/or focal segmental glomerulosclerosis. Here, we describe already well-characterized genetic diseases due to mutations in nephrin, podocin, CD2AP, alpha-actinin-4, WT1, and laminin beta2 chain, as well as more recently identified genetic abnormalities in TRPC6, phospholipase C epsilon, and the proteins encoded by the mitochondrial genome. In addition, the role of the proteins which have shown to be important for the structure and functions by gene knockout studies in mice, are also discussed. Furthermore, some rare syndromes with glomerular involvement, in which molecular defects have been recently identified, are briefly described. In summary, this review updates the current knowledge of genetic causes of congenital and childhood nephrotic syndrome and provides new insights into mechanisms of glomerular dysfunction.

Topics: Actinin; Adaptor Proteins, Signal Transducing; Cytoskeletal Proteins; Genetic Markers; Genome; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Laminin; Membrane Proteins; Mutation; Phosphoinositide Phospholipase C; Podocytes; Proteinuria; TRPC Cation Channels; TRPC6 Cation Channel; WT1 Proteins

Remarkable advances have been made in the past decade in understanding the pathophysiology of idiopathic nephrotic syndrome. Although the initiating events leading to the onset of proteinuria still are not well defined, it has become increasingly clear that many glomerular diseases can be classified as podocytopathies, with injury to the podocyte playing a major role in the development and progression of disease. A complex interaction of immune system mediators, slit diaphragm signal transduction, podocyte injury and conformational change, and mediators of apoptosis and fibrosis determine the extent and nature of proteinuria and progression of glomerulosclerosis. New insights into the pathogenesis of idiopathic nephrotic syndrome likely will lead to innovative therapies and new approaches to management and prevention.

Topics: Child; Child, Preschool; Disease Progression; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nephrosis, Lipoid; Nephrotic Syndrome; Podocytes; Proteinuria; Signal Transduction

Idiopathic nephrotic syndrome is the most frequent glomerular disease in children. While genetic analyses have provided new insights into disease pathogenesis through the discovery of several podocyte genes mutated in distinct forms of inherited nephrotic syndrome, the molecular bases of minimal change nephrotic syndrome (MCNS) and focal and segmental glomerulosclerosis (FSGS) with relapse remain unclear. Although immune cell disorders, which may involve both innate and adaptive immunity, appear to play a role in the pathogenesis of steroid sensitive MCNS, the mechanisms by which they induce podocyte dysfunction remain unresolved. It was postulated that podocyte injury results from a circulating factor secreted by abnormal T cells, but the possibility that bipolarity of the disease results from a functional disorder shared by both cell systems is not excluded. MCNS relapses are associated with an activation of the immune system, including an expansion of T and B cell compartments and production of growth factors as well as many cytokines. Dysfunction of T cells is supported by three main findings: (1) inhibition of a type III hypersensitivity reaction ; (2) defects in immunoglobulin switch ; (3) unclassical T helper polarization resulting from transcriptional interference between Th1 and Th2 transcriptional factors.

Topics: Adrenal Cortex Hormones; Cell Membrane Permeability; Cytokines; Glomerulosclerosis, Focal Segmental; Humans; Hypoalbuminemia; Immunosuppressive Agents; Kidney Glomerulus; Lymphocyte Subsets; Membrane Proteins; Nephrosis, Lipoid; Nephrotic Syndrome; Podocytes; Proteinuria; Proto-Oncogene Proteins c-fyn; Recurrence

Microalbuminuria is the earliest detectable clinical abnormality in diabetic glomerulopathy. On a molecular level, metabolic pathways activated by hyperglycemia, glycated proteins, hemodynamic factors, and oxidative stress are key players in the genesis of diabetic kidney disease. A variety of growth factors and cytokines are then induced through complex signal transduction pathways. Transforming growth factor-beta 1 (TGF-beta1) has emerged as an important downstream mediator for the development of renal hypertrophy and the accumulation of mesangial extracellular matrix components, but there is limited evidence to support its role in the development of albuminuria. The loss of proteoglycans in the glomerular basement membrane (GBM) has been recently questioned as causative of the albuminuria, and current research has focused on the podocyte as a central target for the effects of the metabolic milieu in the development and progression of diabetic albuminuria. Podocyte-derived vascular endothelial growth factor (VEGF), a permeability and angiogenic factor whose expression is increased in diabetic kidney disease, is perhaps a major mediator of the increased protein filtration. Decreased podocyte number and/or density as a result of apoptosis or detachment, GBM thickening with altered matrix composition, and a reduction in nephrin protein in the slit diaphragm with podocyte foot process effacement, all comprise the principal features of diabetic podocytopathy that clinically manifests as albuminuria and proteinuria. Many of these events are mediated by angiotensin II whose local concentration is stimulated by high glucose, mechanical stretch, and proteinuria itself. Angiotensin II in turn stimulates podocyte-derived VEGF, suppresses nephrin expression, and induces TGF-beta1 leading to podocyte apoptosis and fostering the development of glomerulosclerosis. Proteinuria can then induce in tubular cells a genetic program leading to tubulointerstitial inflammation, fibrosis and tubular atrophy. Besides direct effects of albuminuria on tubular cells, pathophysiological changes in the ultrafiltration barrier lead to an increased tubular filtration of various growth factors (TGF-beta1, insulin-like growth factor I) that may further alter the function of tubular cells. Moreover, angiotensin II also stimulates uptake of ultrafiltered proteins into tubular cells and enhances the production of proinflammatory and profibrotic cytokines within the cells. Migration of macro

Topics: Angiotensin II; Diabetic Nephropathies; Endothelial Cells; Endothelium, Vascular; Humans; Kidney Glomerulus; Membrane Proteins; Models, Biological; Podocytes; Proteinuria; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Inherited forms of proteinuria constitute a rare and heterogeneous group of diseases, the most prominent of which is glomerular dysfunction, which leads to proteinuria. Investigation of the genetic background underlying these diseases has provided significant data on the normal operation of the glomerular filter. Among the different components of the glomerulus, the podocyte slit diaphragm is considered the main source for genetically derived protein alteration, which leads in turn to proteinuria. Investigation of the different proteins revealed that the lack of nephrin and podocin is the leading cause of several inherited forms of proteinuria. It was also proposed that the lack of podocin is linked to cardiac anomalies. This review suggests that the absence of slit diaphragm proteins and the open zipper phenomenon are associated with cardiac anomalies.

Topics: Heart Failure; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nephrotic Syndrome; Podocytes; Proteinuria

Topics: Animals; Cadherins; Cell Adhesion; Humans; Kidney; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Mice; Models, Biological; Mutation; Podocytes; Proteinuria; Signal Transduction

Topics: Actinin; Adaptor Proteins, Signal Transducing; Animals; Collagen Type IV; Cyclin-Dependent Kinase Inhibitor Proteins; Cytoskeletal Proteins; Drug Design; Growth Substances; Heparan Sulfate Proteoglycans; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Laminin; Membrane Proteins; Microfilament Proteins; Podocytes; Proteins; Proteinuria; Receptor, Angiotensin, Type 1

Topics: Albumins; Animals; Diet, Sodium-Restricted; Glomerular Filtration Rate; Hemopexin; Humans; Immunosuppressive Agents; Membrane Proteins; Nephrosis, Lipoid; Proteinuria; Sodium Potassium Chloride Symporter Inhibitors; Th1 Cells; Th2 Cells

Topics: Animals; Antigen-Antibody Complex; Complement Activation; Complement Membrane Attack Complex; Glomerulonephritis; Glomerulonephritis, Membranous; Humans; Immunoglobulin G; Intracellular Signaling Peptides and Proteins; Low Density Lipoprotein Receptor-Related Protein-2; Membrane Proteins; Neprilysin; Proteinuria

Topics: Actinin; Actins; Adaptor Proteins, Signal Transducing; Angiotensin II Type 1 Receptor Blockers; Animals; Apoptosis; Cytoskeletal Proteins; Humans; Intracellular Signaling Peptides and Proteins; Kidney Failure, Chronic; Membrane Proteins; Podocytes; Proteins; Proteinuria; Receptor, Angiotensin, Type 1; Tetrazoles; Valine; Valsartan

The aim of the review is to discuss recent investigations on the glomerular filtration barrier. The barrier consists of three layers: the vascular endothelium, the glomerular basement membrane and the slit diaphragm located between podocyte foot processes. The main components of the slit diaphragm are nephrin, the product of NPHS1 gene and podocin, the product of NPHS2 gene. Mutations in NPHS1 lead to congenital nephrotic syndrome of the Finnish type (CNF), whereas NPHS2 mutations result in focal segmental glomerulosclerosis (FSGS). In both cases massive proteinuria is accompanied by the effacement of podocyte foot processess. Reduced expression and redistribution of nephrin and podocin are also seen in podocytes of patients with acquired glomerulopathies. The results suggest that those proteins play a pivotal role in the processes responsible for glomerular filtration. Together with podocin and CD2AP (CD2-associated protein), nephrin forms a complex determining the integrity of the slit diaphragm. Its function has not yet been fully understood and the pathways of signal transduction need to be elucidated.

Topics: Adaptor Proteins, Signal Transducing; Cytoskeletal Proteins; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Nephrosis, Lipoid; Proteins; Proteinuria

Topics: Adult; Age Factors; Animals; Basement Membrane; Blotting, Western; Child; Diagnosis, Differential; Fluorescent Antibody Technique; Glomerular Filtration Rate; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Mutation; Nephrotic Syndrome; Proteinuria; Rats; Reverse Transcriptase Polymerase Chain Reaction

Topics: Angiotensin II; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Benzimidazoles; Biphenyl Compounds; Cognition Disorders; Heart Failure; Humans; Hypertension; Kidney Diseases; Losartan; Membrane Proteins; Mineralocorticoid Receptor Antagonists; Proteins; Proteinuria; Randomized Controlled Trials as Topic; Stroke; Tetrazoles

Proteinuria has been demonstrated to be not only a representative sign of renal lesion but also a risk factor for the progression to renal failure through its injurious effects on tubulointerstitium. The responsible gene for Finnish type congenital nephrotic syndrome was identified and its product was named 'nephrin' which is located on slit membrane between foot processes of glomerular epithelial cells and is considered to be concerned also in the induction of acquired renal lesions with proteinuria. The monoclonal antibody against rat nephrin can induce proteinuria. These facts suggest that the important role for final barrier against macromolecules is played by the slit membrane. Understanding the proteinuria mechanism at molecular level is expected to lead to the establishment of appropriate treatments. Nephrin is regarded as the most promising and attractive molecule for the development of new therapeutic strategy. Many nephrologists are now much interested in the intimate relationship between nephrin and angiotensin II.

Topics: Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Antibodies, Monoclonal; Disease Models, Animal; Humans; Membrane Proteins; Nephrotic Syndrome; Proteins; Proteinuria; Rats

In minimal change nephrotic syndrome (MCNS), selective proteinuria is associated with structural alterations of the glomerular epithelial cells, such as effacement of the foot process. Although the pathogenesis of MCNS has not been completely clarified, clinical and experimental observations suggest that it results from T cell dysfunction in the pathogenesis. Recently, it has been proposed that the occurrence of MCNS has been associated with type 2 Th (Th2) lymphocyte-dependent conditions and some vascular permeability factors, which are induced by T cell disorder. In general, MCNS has the good long-term outcome with sustained remission and preserved renal function, because almost cases are responsible for the treatment. However, some patients show frequent relapses or resistance to this treatment and need large doses of immunosuppressive agents for a long time. Therefore, we should be care for the complications associated with prolonged these therapies.

Topics: Drug Hypersensitivity; Humans; Immunosuppressive Agents; Kidney; Lymphoma; Membrane Proteins; Nephrosis, Lipoid; Prednisolone; Prognosis; Proteins; Proteinuria; Th1 Cells; Th2 Cells; Vascular Endothelial Growth Factor A

Although congenital nephrotic syndromes (CNS) form a heterogenous group of disease characterized by proteinuria shortly after birth, the most common type of CNS is the congenital NS of the Finnish type (CNF). CNF is an autosomal recessive disease, and caused by mutations in the gene (NPHS1) for nephrin which is a key component of the podocyte slit diaphragm. In this review, some special issues concerning clinical and molecular diagnosis for CNS and optimal management of CNF patients were briefly summarized.

Topics: Albumins; Angiotensin-Converting Enzyme Inhibitors; Drug Therapy, Combination; Genes, Recessive; Humans; Indomethacin; Infant; Kidney Transplantation; Membrane Proteins; Mutation; Nephrectomy; Nephrotic Syndrome; Proteins; Proteinuria; Thyroid Hormones

Mutations of NPHS1, NPHS2, or WT1 may be responsible for severe forms of nephrotic syndrome in children, progressing to end-stage renal failure. Recent studies have shown that congenital nephrotic syndrome may be secondary to mutations of one of these three genes and that some patients have a digenic inheritance of NPHS1 and NPHS2 mutations. The clinical spectrum of NPHS2 mutations has broadened, with the demonstration that mutations in the respective gene podocin may be responsible for nephrotic syndrome occurring at birth, in childhood, or in adulthood. It is now well recognized that podocin mutations are found in 10%-30% of sporadic cases of steroid-resistant nephrotic syndrome with focal segmental glomerulosclerosis. Data from large cohorts indicate that the risk of recurrence of nephrotic syndrome after renal transplantation in patients with podocin mutations is very low.

Topics: Child; Humans; Intracellular Signaling Peptides and Proteins; Kidney Transplantation; Membrane Proteins; Mutation; Nephrotic Syndrome; Proteins; Proteinuria; Recurrence; WT1 Proteins

Great progress has been made of late in understanding the mechanisms of proteinuria, the structure and function of the slit diaphragm, and the genetic background of congenital nephrotic syndromes in new borns and infants. This paper presents recent achievements of molecular genetics in unraveling the causes of inherited disorders, e.g. Finnish-type nephrotic, Denys-Drash and Frasier's syndromes, as well as sporadic focal-segmental glomerulosclerosis. A change in the routine policy used in evaluating the causes of childhood nephrotic syndrome is discussed.

Topics: Actinin; Denys-Drash Syndrome; Frasier Syndrome; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mutation; Nephrotic Syndrome; Proteinuria; WT1 Proteins

Topics: Albuminuria; Angiotensin II; Animals; Diabetic Angiopathies; Humans; Hypertension; Membrane Proteins; Proteins; Proteinuria

Topics: Animals; Humans; Kidney Diseases; Membrane Proteins; Proteins; Proteinuria

Topics: Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Proteins; Proteinuria

Topics: Animals; Humans; Membrane Proteins; Nephrotic Syndrome; Proteins; Proteinuria

Proteinuria is a major cause of progression in renal disease. The glomerular ultrafiltration barrier, containing highly differentiated podocytes, normally restricts protein access to the urine. Patients with urinary protein in the nephrotic range (>3.5 g daily) often have effaced podocyte foot-processes. Slit diaphragms span the gaps between foot processes as a barrier to macromolecules. Nephrin and podocin are slit-diaphragm proteins identified in families with congenital nephrotic syndromes. CD2-associated protein (CD2AP) is an adapter protein originally identified as a novel ligand interacting with the T-cell-adhesion protein CD2. CD2AP knockout (-/-) mice develop a congenital nephrotic syndrome with podocyte foot-process effacements and die at 6 weeks of age from renal failure. CD2AP localises to the slit diaphragm and links nephrin and podocin to phosphoinositide 3-OH kinase; this complex has cell-signalling properties.. The CD2AP +/- heterozygous mice developed by Jeong Kim and colleagues (Science 2003; 300: 1298-300) are haploinsufficient and develop glomerular changes at 9 months of age with a histological pattern similar to that in human focal segmental glomerulosclerosis. These researchers found that 2 of 30 African-American patients with idiopathic focal segmental glomerulosclerosis had a CD2AP mutation that ablated expression of one allele. WHERE NEXT? Further studies should address the normal distribution of the CD2AP heterozygous mutation in different ethnic populations, because the association with human idiopathic focal segmental glomerulosclerosis could be accidental. Decreased expression of CD2AP in podocytes of individuals with the CD2AP heterozygous mutation would help to understand how the haploinsufficiency translates into increased susceptibility to renal disease. Transfection of podocytes with mutated CD2AP or study of cultured podocytes from CD2AP +/- mice would provide further insight into whether the nephrin-podocin-CD2AP signal-transduction pathway is altered and leads to increased apoptosis of podocytes. Assuming that a decrease in CD2AP attenuates clearance of glomerular immune complexes, patients with other types of idiopathic glomerulonephritis should also have a CD2AP mutation. However, first studies looking at the most common form of glomerulonephritis, IgA nephropathy, have failed to show decreased renal CD2AP expression.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Cytoskeletal Proteins; Glomerulosclerosis, Focal Segmental; Heterozygote; Humans; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Knockout; Mutation; Proteins; Proteinuria; Signal Transduction; src Homology Domains

Nephrin, a newly described protein, has been localized to the slit membrane between adjacent podocytes of the glomerulus. Its discovery followed the demonstration of the gene NPHS1 and its mutation, resulting in the absence of the protein product, nephrin, in the congenital nephrotic syndrome of the Finnish type. The link between permutations in nephrin expression and proteinuria has been shown in animal models by using neutralizing antibodies or studying mice with inactivation of the nephrin gene. Moreover, the expression of nephrin has been shown to be reduced in various animal models of proteinuric renal disease. The relationship between changes in nephrin expression and proteinuric renal disease in humans is not fully elucidated, with a reduction in expression of this protein reported in a range of renal diseases. Diabetic nephropathy, one of the major causes of end-stage renal disease, is associated with substantial proteinuria and in experimental models with a reduction in slit pore density. In experimental models of diabetes, nephrin expression has been described as being transiently increased in the first 8 weeks of diabetes, followed in longer-term studies with reduced nephrin expression in association with increasing proteinuria. An angiotensin II-receptor blocker has been shown to prevent depletion in glomerular nephrin expression in the diabetic kidney. Human studies in both type 1 and type 2 diabetes suggest down-regulation of nephrin expression in the diabetic kidney and it has been postulated that these changes may play a role in the pathogenesis of diabetic nephropathy, specifically the development of proteinuria in this condition. Although there are other proteins involved in the structure of the epithelial podocyte and specifically the slit pore, nephrin seems to play a pivotal role in preventing passage of protein through the glomerular barrier. Furthermore, it is suggested that the antiproteinuric effects of inhibition of the renin-angiotensin system may partly relate to the effects of these agents on nephrin expression.

Topics: Animals; Diabetic Nephropathies; Humans; Kidney Diseases; Membrane Proteins; Proteins; Proteinuria

It is well established that the glomerular capillary wall consists of three layers: endothelial cell, glomerular basement membrane, and the slit diaphragm bridging foot processes of glomerular epithelial cell. Which structure in the glomerular capillary wall represents the primary filter for retaining plasma proteins is not clearly elucidated. An anti-slit diaphragm monoclonal antibody (mAb) 5-1-6 causes massive proteinuria in rats by single intravenous injection, which clearly indicates that the slit diaphragm plays a critical role for maintaining the barrier function of the glomerular capillary wall. Recently, we concluded that mAb 5-1-6 recognized a rat homolog of nephrin, a gene product of NPHS1. The expression of nephrin decreased in puromycin aminonucleoside nephropathy and adriamycin nephropathy as well as mAb 5-1-6-induced nephropathy, which suggested that nephrin was involved in the development of proteinuria in these proteinuric states. In mAb 5-1-6 nephropathy, the slit diaphragm was maintained morphologically normal, although nephrin expression dramatically decreased. The finding suggested that nephrin was not a sole component of the slit diaphragm. To better understand the structure of the slit diaphragm, it is particularly important to identify other components that build up the structure of the slit diaphragm together with nephrin.

Topics: Animals; Antibodies, Monoclonal; Epithelial Cells; Humans; Kidney Glomerulus; Membrane Proteins; Proteins; Proteinuria

Recent discoveries in kidney research have given new insights into the molecular make-up of the glomerular filter and mechanisms of permselectivity. The identification of mutations in the genes for glomerular basement membrane type IV collagen has thus demonstrated the central role of the glomerular basement membrane as the structural skeleton of the glomerular capillary. Regional deterioration of this framework not only leads to proteinuria, but also to significant leakage of red blood cells into the urinary space. Tracer studies and the characterization of other glomerular basement membrane components, such as proteoglycans, have also emphasized the role of the glomerular basement membrane in the permselectivity process. However, more recent studies on nephrin, a key component of the slit diaphragm, as well as the podocyte and slit diaphragm-associated intracellular proteins, CD2-associated protein, podocin and alpha-actinin-4, have emphasized the role of the slit diaphragm as a central size-selective filtration barrier. These data have provided a completely new understanding of the mechanisms of proteinuria, both in inherited and acquired diseases. In this review, we present the recent progress made in the characterization of proteins that are important for glomerular permselectivity.

Topics: Adaptor Proteins, Signal Transducing; Animals; Basement Membrane; Capillaries; Cytoskeletal Proteins; Humans; Kidney Glomerulus; Membrane Proteins; Microscopy, Electron; Models, Biological; Mutation; Permeability; Proteins; Proteinuria

The sieving of plasma components occurs in the kidney through the glomerular capillary wall. This filter is composed of three layers: endothelium, glomerular basement membrane (GBM), and podocyte foot processes connected by slit diaphragms. Defects in this barrier lead to proteinuria and nephrotic syndrome. Previously, defective GBM was regarded to be responsible for proteinuria. However, recent work on genetic diseases has indicated that podocytes and the slit diaphragm are crucial in restricting protein leakage. Congenital nephrotic syndrome of the Finnish type (NPHS1) is caused by mutations in a novel NPHS1 gene, which encodes for a cell adhesion protein, nephrin. This protein is synthesized by podocytes, and seems to be a major component of the slit diaphragm. In severe NPHS1, lack of nephrin leads to missing slit diaphragm. The role of nephrin in acquired kidney diseases remains unknown. In addition to nephrin, other podocyte proteins (podocin, alpha-actinin-4, CD2AP, FAT) have recently been identified and associated with the development of proteinuria. It seems that the slit diaphragm and its interplay with the podocyte cytoskeleton is critical for the normal sieving process, and defects in one of these components easily lead to proteinuria.

Topics: Glomerulosclerosis, Focal Segmental; Humans; Kidney Glomerulus; Membrane Proteins; Mutation; Nephrotic Syndrome; Phosphoproteins; Proteins; Proteinuria

Proteinuria, reflecting increased glomerular permeability to macromolecules is a characteristic feature of diabetic nephropathy. Nephrin, a 1241-residue transmembrane protein is a key component of the podocyte slit pore membrane and a major contributor of the glomerular filtration barrier. We investigated the expression of nephrin in human kidney tissue from patients with diabetic nephropathy to elucidate its relationship with proteinuria and the effects of anti-proteinuric therapy with angiotensin converting enzyme inhibition.. Renal biopsies were examined from 14 patients with Type II (non-insulin-dependent) diabetes mellitus and proteinuria who had been randomised to receive treatment with the ACE inhibitor, perindopril (4 mg/day) or placebo for the preceding 2 years. These specimens were compared with control human tissue sections, obtained from areas of normal renal cortex following nephrectomy for malignancy. Proteinuria was measured, specimens were examined histologically for injury and the expression of nephrin messenger RNA was assessed by quantitative in situ hybridisation.. Glomeruli from placebo-treated patients with diabetic nephropathy, showed a 62% reduction in nephrin expression compared with control subjects (p=0.0003). In contrast, nephrin RNA in glomeruli from perindopril treated patients was similar to that in the non-diabetic control group. In both placebo and perindopril treated patients, a close inverse correlation was noted between the magnitude of nephrin gene expression and the degree of proteinuria (placebo: r=0.86, p=0.013, perindopril: r=0.91, p=0.004).. Modulation in nephrin expression is related to the extent of proteinuria in diabetic nephropathy. These changes define, at a molecular level alterations in the glomerulus that occur in relation to proteinuria in diabetes and the effects of anti-proteinuric treatment with ACE inhibition.

Topics: Angiotensin-Converting Enzyme Inhibitors; Biopsy; Blood Pressure; Creatinine; Diabetic Nephropathies; Gene Expression Regulation; Glycated Hemoglobin; Humans; In Situ Hybridization; Membrane Proteins; Perindopril; Placebos; Proteins; Proteinuria

Pre-eclampsia (PE) is characterized by new-onset hypertension and proteinuria. Damage of podocyte cells has been reported in pre-eclamptic women, thus podocyte specific proteins such as nephrin and podocalyxin could be useful biomarkers in PE.. To investigate the role of urinary nephrin (u-nephrin) and urinary podocalyxin (u-PDX) levels in predicting PE in women with a high-risk pregnancy.. We included 101 pregnant women in this study and allocated them into three groups: group 1 included pregnant women at high risk of developing PE (n=41), group 2 - pregnant women with PE (n=30), and group 3 was the controls including healthy pregnant women (n=30). The inclusion criteria for women with PE were de novo hypertension >140/90 mm Hg, proteinuria >300 mg/24 hours, and presence of edema after 20 weeks of gestation, while the exclusion criteria were a history of renal diseases and pregnant women younger than 18. Inclusion criteria for the group of women with a high-risk pregnancy was gestational week >15, a history of PE in a previous pregnancy, pre-existing diabetes type 1 or 2, pre-existing hypertension, multiple gestations, prior placental abruption, obesity women, nulliparity, maternal age >35 years, and a family history of PE. The study was conducted from March 2016 to May 2017 in the Medical Faculty at the Institute of Medical and Experimental Biochemistry in Skopje. Urine samples were used to measure the nephrin and podocalyxin levels using immunoenzyme assay, creatinine and microalbumin. Blood samples were collected for biochemical analyses.. U-nephrin levels were elevated in 96.7% of women with PE, and 73% of women with a high-risk pregnancy. U-PDX levels were elevated in 63% of the women with PE and 100% of the women with a high-risk pregnancy. U-nephrin and u-PDX levels were significantly increased in women with a high-risk pregnancy and women with PE compared with a control group (p.

Topics: Adult; Biomarkers; Female; Humans; Hypertension; Membrane Proteins; Placenta; Pre-Eclampsia; Pregnancy; Pregnant Women; Proteinuria; Sialoglycoproteins

Topics: Age of Onset; alpha-Fetoproteins; Child; Female; Glomerulosclerosis, Focal Segmental; Humans; Membrane Proteins; Mutation; Pregnancy; Proteinuria; Time Factors

Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified. To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these. Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.

Topics: Animals; Disease Models, Animal; Endoplasmic Reticulum; Female; Kidney Glomerulus; Male; Membrane Proteins; Mice; Nephrotic Syndrome; Podocytes; Proteinuria

Huangqi Guizhi Wuwu Decoction(HQGZWWD) is a Traditional Chinese Medicine formula from Synopsis of Golden Chamber used to treat blood arthralgia. According to the principle that the same treatment can be used for different diseases, HQGZWWD has proven effective for IgA nephropathy (IgAN) associated with spleen and kidney yang deficiency.. In this study, we investigated the mechanism by which HQGZWWD alleviates proteinuria and protects renal function in rats with IgAN by regulating the AT1R/Nephrin/c-Abl pathway.. Rats were randomly divided into six groups: control, IgAN model, IgAN model treated with low-dose HQGZWWD, IgAN model treated with medium-dose HQGZWWD, IgAN model treated with high-dose HQGZWWD, and IgAN model treated with valsartan. IgAN was induced using bovine γ-globulin. We evaluated the mediating effects of HQGZWWD on podocyte cytoskeletal proteins, the AT1R/Nephrin/c-Abl pathway, upstream tumor necrosis factor-α (TNF-α), and TNF-α receptor-1 (TNFR1).. The IgAN rats displayed proteinuria, IgA deposition in the mesangial region, and podocyte cytoskeletal protein damage. The expression of TNF-α, TNFR1, AT1R, and c-Abl was increased in the IgAN rat kidney, whereas the expression of nephrin, podocin, ACTN4, and phosphorylated nephrin (p-nephrin) was reduced. HQGZWWD treatment significantly alleviated podocyte cytoskeletal protein damage in the IgAN rats, upregulated the expression of nephrin, podocin, and ACTN4, and the colocalized expression of F-actin and nephrin. This study demonstrates that HQGZWWD attenuates podocyte cytoskeletal protein damage by regulating the AT1R-nephrin- c-Abl pathway, upregulating the expression of p-nephrin, and downregulating the expression of AT1R and c-Abl.. These results indicate that HQGZWWD attenuates podocyte cytoskeletal protein damage in IgAN rats by regulating the AT1R/Nephrin/c-Abl pathway, providing a potential therapeutic approach for IgAN.

Topics: Actinin; Actins; Animals; Cytoskeletal Proteins; Disease Models, Animal; Down-Regulation; Drugs, Chinese Herbal; Glomerulonephritis, IGA; Immunoglobulin A; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Podocytes; Protective Agents; Proteinuria; Proto-Oncogene Proteins c-abl; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; Tumor Necrosis Factor-alpha

Numerous disease-causing gene mutations have been identified in proteinuric diseases, such as nephrotic syndrome and glomerulosclerosis. This report describes the results of comprehensive genetic diagnosis of Japanese patients with severe proteinuria. In addition, the report describes the clinical characteristics of patients with monogenic disease-causing mutations. We conducted comprehensive gene screening of patients who had either congenital nephrotic syndrome, infantile nephrotic syndrome, steroid-resistant nephrotic syndrome, or focal segmental glomerular sclerosis. Using targeted next-generation sequencing, 60 podocyte-related genes were screened in 230 unrelated patients with proteinuria. A retrospective review of clinical data was conducted for these patients. We detected monogenic disease-causing mutations in 30% (69 of 230) of patients among 19 of the screened genes. Common genes with disease-causing mutations were WT1 (25%), NPHS1 (12%), INF2 (12%), TRPC6 (10%), and LAMB2 (9%). With various immunosuppressive or renoprotective therapies, remission of proteinuria in patients with unknown causative mutations was observed in 26% of patients, whereas only 5% of patients with monogenic disease-causing mutations exhibited complete remission. We assessed the genetic backgrounds of Japanese patients with severe proteinuria. The proportion of patients with gene defects was similar to that of other reports, but the disease-causing gene mutation frequency was considerably different.

Topics: Adolescent; Adult; Child; Child, Preschool; Female; Genetic Testing; Glomerular Filtration Rate; Glomerulosclerosis, Focal Segmental; Humans; Infant; Infant, Newborn; Japan; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Odds Ratio; Proteinuria; Renal Insufficiency, Chronic; Retrospective Studies; Severity of Illness Index; WT1 Proteins; Young Adult

Podocyte injury is the main cause of proteinuria in lupus nephritis (LN). Nestin, an important cytoskeleton protein, is expressed stably in podocytes and is associated with podocyte injury. However, the role of nestin in the pathogenesis of proteinuria in LN remains unclear. The correlations among nestin, nephrin and proteinuria were analyzed in LN patients and MRL/lpr lupus-prone mice. The expression of nestin in mouse podocyte lines (MPCs) and MRL/lpr mice was knocked down to determine the role of nestin in podocyte injury. Inhibitors and RNAi method were used to explore the role of mitophagy and oxidative stress in nestin protection of podocyte from damage. There was a significantly negative correlation between nestin and proteinuria both in LN patients and MRL/lpr mice, whereas the expression of nephrin was positively correlated with nestin. Knockdown of nestin resulted in not only the decrease of nephrin, p-nephrin (Y1217) and mitophagy-associated proteins in cultured podocytes and the podocytes of MRL/lpr mice, but also mitochondrial dysfunction in podocytes stimulated with LN plasma. The expression and phosphorylation of nephrin was significantly decreased by reducing the level of mitophagy or production of reactive oxygen species (ROS) in cultured podocytes. Our findings suggested that nestin regulated the expression of nephrin through mitophagy and oxidative stress to protect the podocytes from injury in LN.

Topics: Adult; Animals; Female; Humans; Kidney; Lupus Nephritis; Male; Membrane Proteins; Mesenchymal Stem Cells; Mice; Middle Aged; Nestin; Oxidative Stress; Podocytes; Protective Agents; Proteinuria

ShenYanXiaoBai granules is a traditional Chinese herbal medicine, It is used widely for the treatment of proteinuria caused by various kidney diseases.. This study investigated the mechanism of Shenyan Xiaobai Granule in the treatment of nephritis proteinuria.. 100 male wistar rats were divided into a blank group (n = 20) and a nephropathy group (n = 80) using random number table after 1 week adaptive feeded. Rats were injected with adriamycin (6.5 mg/kg) via the tail vein to induce nephropathy except for blank group. Every rat's urine protein was checked with urine protein dipstick test after three days that showed all rats in nephropathy group were successful modelled. Nephropathy group was divided into model group, benazepril group, ShenYanXiaoBai low dose group, ShenYanXiaoBai high dose group equally. Blank and model group were given distilled water 2 ml as control, then benazepril group received benazepril 0.90 mg/kg, ShenYanXiaoBai low dose group received ShenYanXiaoBai granules 1.80 g/kg as high dose group was given 3.60 g/kg, gavage for 6 days a week last for seven weeks. Urinary albumin/urinary creatinine were measured in seventh day every week. Three rats were randomly selected from each group to be executed in 3th and 5th weekend to detect the mRNA and protein expression level in kidney. The rest rats were as well.. The therapeutic effect of ShenYanXiaoBai high dose group was better than the two other treated groups from the 5th week to the 7th week, the comparison had a significant difference. The therapeutic effect of benazepril group was better than the ShenYanXiaoBai low dose group in the 7 weeks and the comparison had a significant difference.

Topics: Animals; Creatinine; Doxorubicin; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Kidney Function Tests; Male; Medicine, Chinese Traditional; Membrane Proteins; Podocytes; Protective Agents; Proteinuria; Random Allocation; Rats, Wistar

Podocytes are specialized cells with a limited capacity for cell division that do not regenerate in response to injury and loss. Insults that compromise the integrity of podocytes promote proteinuria and progressive renal disease. The aim of this study was to evaluate the potential renoprotective and regenerative effects of mesenchymal stromal cells (mSC) in a severe form of the podocyte injury model induced by intraperitoneal administration of puromycin, aggravated by unilateral nephrectomy. Bone derived mSC were isolated and characterized according to flow cytometry analyses and to their capacity to differentiate into mesenchymal lineages. Wistar rats were divided into three groups: Control, PAN, and PAN+ mSC, consisting of PAN rats treated with 2 × 10

Topics: Animals; Cell Differentiation; Cell Division; Down-Regulation; Glomerulosclerosis, Focal Segmental; Hypertension; Inflammation; Intracellular Signaling Peptides and Proteins; Kidney Diseases; Male; Membrane Proteins; Mesenchymal Stem Cells; Microfilament Proteins; Nephrectomy; Podocytes; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Wistar; Regeneration; Sialoglycoproteins; Vascular Endothelial Growth Factor A

Management of children with congenital nephrotic syndrome (CNS) is challenging. Bilateral nephrectomies followed by dialysis and transplantation are practiced in most centres, but conservative treatment may also be effective.. We conducted a 6-year review across members of the European Society for Paediatric Nephrology Dialysis Working Group to compare management strategies and their outcomes in children with CNS.. Eighty children (50% male) across 17 tertiary nephrology units in Europe were included (mutations in NPHS1, n = 55; NPHS2, n = 1; WT1, n = 9; others, n = 15). Excluding patients with mutations in WT1, antiproteinuric treatment was given in 42 (59%) with an increase in S-albumin in 70% by median 6 (interquartile range: 3-8) g/L (P < 0.001). Following unilateral nephrectomy, S-albumin increased by 4 (1-8) g/L (P = 0.03) with a reduction in albumin infusion dose by 5 (2-9) g/kg/week (P = 0.02). Median age at bilateral nephrectomies (n = 29) was 9 (7-16) months. Outcomes were compared between two groups of NPHS1 patients: those who underwent bilateral nephrectomies (n = 25) versus those on conservative management (n = 17). The number of septic or thrombotic episodes and growth were comparable between the groups. The response to antiproteinuric treatment, as well as renal and patient survival, was independent of NPHS1 mutation type. At final follow-up (median age 34 months) 20 (80%) children in the nephrectomy group were transplanted and 1 died. In the conservative group, 9 (53%) remained without dialysis, 4 (24%; P < 0.001) were transplanted and 2 died.. An individualized, stepwise approach with prolonged conservative management may be a reasonable alternative to early bilateral nephrectomies and dialysis in children with CNS and NPHS1 mutations. Further prospective studies are needed to define indications for unilateral nephrectomy.

Topics: Albumins; Child; Child, Preschool; Europe; Female; Humans; Infant; Male; Membrane Proteins; Mutation; Nephrectomy; Nephrology; Nephrotic Syndrome; Pediatrics; Prospective Studies; Proteinuria; Retrospective Studies; Sepsis; Thrombosis

Topics: Actins; Adult; Angiotensin I; Animals; Blood Pressure; Cell Line; Down-Regulation; Female; Humans; Hypertension, Pregnancy-Induced; Membrane Proteins; Mice; Peptide Fragments; Podocytes; Pre-Eclampsia; Pregnancy; Proteinuria; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled; Renin-Angiotensin System; Zonula Occludens-1 Protein

Glomerulonephritis (GN) is a common cause of end-stage kidney disease and is characterized by glomerular inflammation, hematuria, proteinuria, and progressive renal dysfunction. Transforming growth factor (TGF)-β is involved in glomerulosclerosis and interstitial fibrosis. TGF-β activates multiple signaling pathways, including the canonical SMAD pathway. We evaluated the role of SMAD signaling in renal injury and proteinuria in a murine model of GN. SMAD3

Topics: Animals; Anti-Glomerular Basement Membrane Disease; Autoantibodies; Cell Line; Disease Models, Animal; Endothelial Cells; Fibrosis; Kidney Glomerulus; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Paracrine Communication; Podocytes; Proteinuria; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Wenshen Jianpi recipe (WSJPR), a blended traditional Chinese medicine, is considered to have the possible beneficial effect on the progression of diabetic nephropathy (DN). This present study was designed to elucidate this protective activity in a rat model with streptozotocin (STZ)-induced DN and to explore the possible underlying mechanism.. Adult Sprague Dawley (SD) rats were induced to develop DN through intraperitoneal injection of STZ (60 mg/kg). Animals were orally administered saline, WSJPR at 7.5, 15, 30 g/kg, and valsartan (25 mg/kg) daily for 8 weeks. Blood and 24-h urine samples of each rat were collected for biochemical examination at 2-week intervals. Microcirculatory blood flow in the renal cortex and hemorheology index were also measured. At the end of 8 weeks, all rats were sacrificed to obtain the kidney tissues for histological examination and reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the transcriptional levels of nephrin and podocin genes.. WSJPR could improve serum total protein (TP) and albumin (ALB), reduce the excretion rates of urine-TP (U-TP), urine-ALB (U-ALB) and urine urea nitrogen (UUN) (P < 0.05), although it did not significantly alter the hyperglycemia. In addition, treatment with WSJPR could strongly reduce blood flow, erythrocyte aggregation index, and ameliorate microcirculation. In histological measurement, WSJPR-treated rats showed a significant amelioration in glomerular hypertrophy and mesangial expansion. By RT-PCR, we found WSJPR up-regulated the nephrin and podocin expression at mRNA levels.. This study suggested that WSJPR could effectively relieve renal damage and improve renal function of DN rats by ameliorating metabolism disorder and increasing the gene expression of nephrin and podocin, which might be a useful approach for the treatment of DN.

Topics: Animals; Diabetic Nephropathies; Disease Models, Animal; Drugs, Chinese Herbal; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Male; Medicine, Chinese Traditional; Membrane Proteins; Proteinuria; Rats; Rats, Sprague-Dawley

Focal segmental glomerulosclerosis (FSGS) is considered a subset of the podocytopathies. The molecular pathogenesis of podocytopathy is still unknown. There has not been an experimental animal model of isolated podocytopathy induced by antibody in C57BL/6 strain, which is widely used as the genetic background. Nephrin is closely associated with the slit diaphragm of the glomerular podocyte, and has recently received attention as a potential therapeutic target. The function of nephrin, especially its role in FSGS development via podocytopathy, is being elucidated. We report our experience with a C57BL/6 FSGS model induced by polyclonal rabbit anti-mouse nephrin antibody (α-mNep Ab).. α-mNep Ab, which was generated by genetic immunization, was administered into C57BL/6 mice at once, intravenously. Urinary protein excretion, the development of glomerulosclerosis and the number of podocyte in mouse kidney were evaluated.. The α-mNep Ab-induced FSGS was associated with massive proteinuria and nephrotic syndrome. In periodic acid-Schiff staining, FSGS was observed from day 7 after antibody injection. Podocyte numbers and podocyte marker (anti-Wilms tumor 1 and anti-synaptopodin)-positive areas were clearly decreased. These results suggest that this FSGS mouse model reliably reproduces the human nephrotic syndrome and FSGS.. We succeeded in making the nephrotic syndrome model mice induced by α-mNep Ab using C57BL/6. This model may be useful for studying the mechanisms of podocytopathy.

Topics: Animals; Antibodies; Body Weight; Female; Glomerulosclerosis, Focal Segmental; HEK293 Cells; Humans; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Nephrotic Syndrome; Podocytes; Proteinuria; Rabbits; Urodynamics

Nephrin is a key structural component of the podocyte slit diaphragm, and proper expression of nephrin on the cell surface is critical to ensure integrity of the blood filtration barrier. Maintenance of nephrin within this unique cell junction has been proposed to require dynamic phosphorylation events and endocytic recycling, although the molecular mechanisms that control this interplay are poorly understood. Here, we investigated the possibility that the phosphotyrosine adaptor protein ShcA regulates nephrin turnover. Western blotting and immunostaining analysis confirmed that ShcA is expressed in podocytes. In immunoprecipitation and pulldown assays, ShcA,

Topics: Animals; Biotinylation; Cell Membrane; Cytosol; Endocytosis; HEK293 Cells; Humans; Kidney Diseases; Male; Membrane Proteins; Nephrosis; Phosphorylation; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Tyrosine; Up-Regulation

Congenital nephrotic syndrome (CNS) is primarily a monogenetic disease, with the majority of cases due to changes in five different genes: the nephrin (NPHS1), podocin (NPHS2), Wilms tumor 1 (WT1), laminin ß2 (LAMB2), and phospholipase C epsilon 1 (PLCE1, NPHS3) gene. Usually CNS is not responsive to immunosuppressive therapy, but treatment with ACE inhibitors, AT1 receptor blockade and/or indomethacin can reduce proteinuria. If the disease progresses to end-stage renal disease, kidney transplantation is the therapy of choice.. Here, we present the case of a 4-month-old girl with congenital nephrotic syndrome. Upon admission, the patient presented with life-threatening anasarca, hypoalbuminemia, proteinuria, and impaired growth. There was no evidence of an infectious or immunological etiology. The genetic evaluation revealed a heterozygous variant in NPHS1 (p.Arg207Trp), in NPHS2 (p.Ser95Phe) as well as in PLCE1 (p.Ala1045Ser) and did not explain CNS. In addition to daily parenteral albumin infusions plus furosemide, a pharmacological antiproteinuric therapy was started to reduce protein excretion. Based on the genetic results, immunosuppressive therapy with prednisolone was initiated, but without response. However, following cyclosporine A treatment, the patient achieved complete remission and now has good renal function, growth, and development.. A profound search for the cause of CNS is necessary but has its limitations. The therapeutic strategy should be adapted when the etiology remains unclear.

Topics: Cyclosporine; Drug Therapy, Combination; Female; Genetic Testing; Heterozygote; Humans; Immunosuppressive Agents; Infant; Intracellular Signaling Peptides and Proteins; Kidney; Membrane Proteins; Nephrotic Syndrome; Phosphoinositide Phospholipase C; Polymorphism, Single Nucleotide; Proteinuria; Treatment Outcome; Ultrasonography

Nephrin (Nphs1) is an adhesion protein that is expressed at the podocyte intercellular junction in the glomerulus. Nphs1 mutations in humans or deletion in animal genetic models results in a developmental failure of foot process formation. A number of studies have shown decrease in expression of nephrin in various proteinuric kidney diseases as well as in animal models of glomerular disease. Decrease in nephrin expression has been suggested to precede podocyte loss and linked to the progression of kidney disease. Whether the decrease in expression of nephrin is related to loss of podocytes or lead to podocyte detachment is unclear. To answer this central question we generated an inducible model of nephrin deletion (Nphs1Tam-Cre) in order to lower nephrin expression in healthy adult mice. Following tamoxifen-induction there was a 75% decrease in nephrin expression by 14 days. The Nphs1Tam-Cre mice had normal foot process ultrastructure and intact filtration barriers up to 4-6 weeks post-induction. Despite the loss of nephrin expression, the podocyte number and density remained unchanged during the initial period. Unexpectedly, nephrin expression, albeit at low levels persisted at the slit diaphragm up to 16-20 weeks post-tamoxifen induction. The mice became progressively proteinuric with glomerular hypertrophy and scarring reminiscent of focal and segmental glomerulosclerosis at 20 weeks. Four week-old Nphs1 knockout mice subjected to protamine sulfate model of podocyte injury demonstrated failure to recover from foot process effacement following heparin sulfate. Similarly, Nphs1 knockout mice failed to recover following nephrotoxic serum (NTS) with persistence of proteinuria and foot process effacement. Our results suggest that as in development, nephrin is necessary for maintenance of a healthy glomerular filter. In contrast to the developmental phenotype, lowering nephrin expression in a mature glomerulus resulted in a slowly progressive disease that histologically resembles FSGS a disease linked closely with podocyte depletion. Podocytes with low levels of nephrin expression are both susceptible and unable to recover following perturbation. Our results suggest that decreased nephrin expression independent of podocyte loss occurring as an early event in proteinuric kidney diseases might play a role in disease progression.

Topics: Animals; Cell Membrane; Gene Deletion; Kidney Glomerulus; Membrane Proteins; Mice; Podocytes; Protein Stability; Proteinuria

Mutations in NPHS1 can lead to disruption of the filtration barrier and cause proteinuria in nephrotic syndrome (NS). The aim of the study was to evaluate NPHS1 mutations, its susceptibility to the disease, and their association in children with steroid-resistant NS; mutation frequency of 9% was observed in patients with steroid-resistant NS, of which, six mutations and two single-nucleotide polymorphisms observed in the study population were found to be novel.

Topics: Child; Child, Preschool; Female; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Infant; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype; Polymorphism, Single Nucleotide; Proteinuria

Previous studies have shown that podocyte autophagy is an important trigger for proteinuria and glomerulosclerosis. The mammalian rapamycin target protein (mTOR) occupies a pivotal position in the autophagy pathway. In this study, we planned to clarify the mechanism of mTOR regulation of podocyte autophagy and the effect of rapamycin (RAPA).. All rats were randomly divided into normal control group (n = 8), DN group (n = 8), and RAPA group (n = 8). Blood and urine samples were collected at the 4th, 8th, and 12th weeks of the experiment. The serum creatinine (Scr), urine volume levels, and the 24 h urine protein (UP) levels were examined. The nephrin, podocin, mTOR, ribosomal S6 kinase 1 (S6K1), and autophagy marker light chain 3 (LC3II) expression levels were evaluated by immunohistochemistry, quantitative PCR, and immunoblotting.. The urine volume, 24 h UP, and Scr of the DN and RAPA groups increased significantly compared with the NC group (p < .05). Nephrin and podocin expression was decreased in the kidney tissues of the DN and RAPA groups compared with the NC group (p < .05). The expression levels of mTOR and S6K1 increased and LC3II expression decreased in the renal tissues of the DN and RAPA groups compared with the NC group (p < .05). After RAPA treatment, all the above indexes were improved compared with the DN group (p < .05), but were significantly abnormal compared with the NC group (p < .05).. The proteinuria and kidney function had improved after RAPA treatment. These results confirmed that RAPA specifically binds to mTOR kinase, and inhibits mTOR activity, thereby regulating the pathological autophagic process.

Topics: Animals; Autophagy; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Streptozocin; TOR Serine-Threonine Kinases

BACKGROUND The aim of this study was to determine whether senescence in renal glomeruli is involved in lupus nephritis (LN); the expression of senescence-associated β-galactosidase (SA-β-Gal) and its association with glomerular lesions were investigated in a mouse model of LN. MATERIAL AND METHODS Eighteen MRL/lpr mice with severe proteinuria were randomly divided into 2 equal groups and intraperitoneally injected with dexamethasone (DEX) or saline; 4 age-matched mice with mild proteinuria served as controls. Serum creatinine and urinary protein levels were analyzed, and kidney histological changes were observed by periodic acid-Schiff and Sirius Red staining. SA-β-Gal was detected via histochemistry. Glomerular expression of collagen IV, α-SMA, and nephrin was analyzed by immunohistochemistry, and glomerular complement C3 deposition was tested by immunofluorescence. The relationships between SA-β-Gal expression and renal function or glomerular lesion markers were determined by Spearman's correlation analysis. RESULTS Mice with severe proteinuria exhibited glomerular segmental sclerosis and endothelial cell proliferation. DEX administration suppressed these lesions but had no significant effect on 24-hour urinary protein levels. The elevated glomerular expression of SA-β-Gal in proteinuric mice was attenuated by DEX treatment. In addition, DEX treatment markedly downregulated glomerular C3 deposition and collagen IV and α-SMA expression, while significantly increasing nephrin expression. Furthermore, SA-β-Gal expression was positively correlated with urinary protein levels and expression of α-SMA. CONCLUSIONS Accelerated senescence of glomerular cells may contribute to glomerular injury in LN.

Topics: Actins; Animals; beta-Galactosidase; Cellular Senescence; Collagen Type IV; Creatinine; Dexamethasone; Disease Models, Animal; Female; Immunohistochemistry; Kidney Glomerulus; Lupus Nephritis; Membrane Proteins; Mice; Mice, Inbred MRL lpr; Proteinuria

This study aimed to explore the correlation between microRNA-155 (miR-155), interleukin 17A (IL-17), and late preeclampsia (PE) using biochemical parameters in maternal serum and urine.. Sixty patients with PE were recruited to this study and were divided into 3 groups according to levels of urinary protein: mild urinary protein group (group A); moderate urinary protein group (group B); and severe urinary protein group (group C). All subjects presented with late onset PE (≥32 weeks). In addition, 20 healthy pregnant women were recruited as a normal group (NP). Maternal serum and urine samples were obtained from all participants and assayed using immunofluorescence, transmission electron and immune electron microscopy, and western blot. Furthermore, placentas were also collected and miR-155 and IL-17 expression was measured using an enzyme-linked immunosorbent assay.. Levels of miR-155 and IL-17 expression were found to be increased in PE placentas and serum, compared to the normal group (P <.05). IL-17 levels and podocytes had a positive association in the serum of patients with PE. In addition, over-expression of miR-155 resulted in increased IL-17 production by CD4+ T cells in vitro, and nephrin expression was decreased in podocytes. Furthermore, IL-17 reduced nephrin expression in podocytes and podocyte apoptosis in a dose and time-dependent manner.. The results of this study demonstrate a correlation between miR-155 and IL-17 in the formation of proteinuria during late onset PE.

Topics: Apoptosis; Biomarkers; Birth Weight; CD4 Antigens; Cells, Cultured; Coculture Techniques; Female; Humans; Interleukin-17; Lymphocytes; Membrane Proteins; MicroRNAs; Placenta; Podocytes; Pre-Eclampsia; Pregnancy; Pregnancy Trimester, Third; Proteinuria; Severity of Illness Index

Injury of the glomerular filter causes proteinuria by disrupting the sensitive interplay of the glomerular protein network. To date, studies of the expression and trafficking of glomerular proteins have been mostly limited to in vitro or histologic studies. Here, we report a novel in vivo biotinylation assay that allows the quantification of surface expression of glomerular proteins in mice. Kidneys were perfused in situ with biotin before harvest. Afterwards glomeruli were isolated and lyzed. The protein of interest was separated by immunoprecipitation and the amount of surface-expressed protein was quantified by Western blot analysis with streptavidin staining. As proof-of-concept, we examined the presence of nephrin in the slit diaphragm in two well-established murine models of proteinuric kidney disease: nephrotoxic nephritis and adriamycin nephropathy. In proteinuric animals, significantly less nephrin was detected in the slit diaphragm. When proteinuria decreased once again during the course of disease, the amount of surface nephrin returned to the baseline. Our present results suggest that our assay is a valuable tool to study the glomerular filter in proteinuric kidney diseases. Note that the assay is not limited to proteins expressed in the slit diaphragm, and all surface proteins that are accessible to biotin perfusion and immunoprecipitation qualify for this analysis.

Topics: Albuminuria; Animals; Disease Models, Animal; Gene Expression; Kidney Diseases; Kidney Glomerulus; Male; Membrane Proteins; Mice; Nephritis; Proteinuria; Time Factors

Podocyte injury and glomerular basement membrane thickening have been considered as essential pathophysiological events in diabetic nephropathy. The aim of this study was to investigate the possible beneficial effects of vinpocetine on diabetes-associated renal damage. Male Wistar rats were made diabetic by injection of streptozotocin (STZ). Diabetic rats were treated with vinpocetine in a dose of 20mg/kg/day for 6 weeks. Treatment with vinpocetine resulted in a marked decrease in the levels of blood glucose, glycosylated haemoglobin, creatinine, blood urea nitrogen, urinary albumin and albumin/creatinine ratio along with an elevation in creatinine clearance rate. The renal contents of advanced glycation end-products, interleukin-10, tissue growth factor-β, nuclear factor (NF)-κB and Ras-related C3 botulinum toxin substrate 1 (Rac 1) were decreased. Renal nephrin and podocin contents were increased and their mRNA expressions were replenished in vinpocetine-treated rats. Moreover, administration of vinpocetine showed improvements in oxidative status as well as renal glomerular and tubular structures. The current investigation revealed that vinpocetine ameliorated the STZ-induced renal damage. This beneficial effect could be attributed to its antioxidant and antihyperglycemic effects parallel to its ability to inhibit NF-κB which eventually modulated cytokines production as well as nephrin and podocin proteins expression.

Topics: Animals; Blood Glucose; Body Weight; Diabetic Nephropathies; Disease Models, Animal; Fasting; Gene Expression Regulation; Glycated Hemoglobin; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; NF-kappa B; Podocytes; Proteinuria; rac1 GTP-Binding Protein; Rats; Rats, Wistar; RNA, Messenger; Vinca Alkaloids

Icariin has previously been demonstrated to attenuate hyperglycemia‑induced renal injury, however the renoprotective effects of icariin in a rat model of pregnancy‑induced hypertension (PIH) remain to be elucidated. The present study aimed to investigate the effect of icariin on PIH‑induced acute kidney injury (AKI) and proteinuria. Following 18 days of icariin treatment between day 1 and day 18 of gestation, which was combined with NG‑nitro‑L‑arginine methyl ester (L‑NAME) treatment between day 12 and day 18 of gestation to induce PIH, the 24 h urine protein level, blood urea nitrogen and serum creatinine were measured by using the Coomassie Brilliant Blue method, a commercial enzymatic kit and the picric acid method, respectively. Renal tissues were collected at day 18 of gestation for hematoxylin and eosin staining and immunohistochemistry. The mRNA expression of AGT and protein expression of angiotensin II (Ang II) in the kidneys of control and PIH rats was investigated by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively, to determine the effect of icariin on components of the renin‑angiotensin system. The results demonstrated that L‑NAME treatment in pregnant rats resulted in significant increases in systolic blood pressure (SBP) and diastolic blood pressure, in addition to the induction of severe proteinuria. The significant increase in SBP and proteinuria in PIH rats was prevented by icariin. L‑NAME‑induced AKI resulted in profound renal histological alterations, including mesangial expansion and glomerular lesions. L‑NAME administration exerted a marked decrease in the mRNA and protein expression levels of nephrin in the kidneys from PIH rats compared with control group. Furthermore, upregulation of circulating and renal Ang II levels in PIH rats was observed. However, icariin treatment significantly reversed the L‑NAME‑induced downregulation of nephrin and upregulation of circulating and renal Ang II levels in PIH rats. These results demonstrated that icariin administration improved urinary protein excretion levels and renal tissue damage in PIH rats, and the underlying mechanism was mediated in part, via upregulation of nephrin expression and downregulation of Ang II.

Topics: Acute Kidney Injury; Angiotensin II; Animals; Blood Pressure; Blood Urea Nitrogen; Creatinine; Disease Models, Animal; Down-Regulation; Female; Flavonoids; Gestational Age; Hypertension, Pregnancy-Induced; Kidney; Membrane Proteins; NG-Nitroarginine Methyl Ester; Pregnancy; Proteinuria; Rats; Rats, Wistar; Renin-Angiotensin System; Reproduction; Up-Regulation

Hypertrophy is a prominent feature of damaged podocytes in diabetic kidney disease (DKD). mTORC1 hyperactivation leads to podocyte hypertrophy, but the detailed mechanism of how mTORC1 activation occurs under pathological conditions is not completely known. Moreover, reduced nephrin tyrosine phosphorylation has been observed in podocytes under pathological conditions, but the molecular mechanism linking nephrin phosphorylation and pathology is unclear so far. In this study, we observed a significant increase in C1-Ten level in diabetic kidney and in high glucose-induced damaged podocytes. C1-Ten acts as a protein tyrosine phosphatase (PTPase) at the nephrin-PI3K binding site and renders PI3K for IRS-1, thereby activating mTORC1. Furthermore, C1-Ten causes podocyte hypertrophy and proteinuria by increasing mTORC1 activity in vitro and in vivo. These findings demonstrate the relationship between nephrin dephosphorylation and the mTORC1 pathway, mediated by C1-Ten PTPase activity. We suggest that C1-Ten contributes to the pathogenesis of DKD by inducing podocyte hypertrophy under high glucose conditions.

Topics: Animals; Diabetic Nephropathies; Glucose; HEK293 Cells; Humans; Hypertrophy; Insulin Receptor Substrate Proteins; Male; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mice; Phosphatidylinositol 3-Kinases; Phosphorylation; Podocytes; Protein Tyrosine Phosphatases; Proteinuria; Signal Transduction; Tensins

To investigate the effects of Huaiqihuang Granules (, HQH), a mixture of Chinese herbs including Trametes robiniophila Murr, Fructus Lycii and Polygonatum sibiricum, on adriamycininduced nephropathy (ADRN) in rats and its underlying mechanisms.. Rats with ADRN were divided into four groups: the sham group, the model group (distilled water), the low-dose HQH-treated (2 g/kg) group, and the high-dose HQH-treated (4 g/kg) group. Body weight and 24-h urinary protein (Upro) were checked every week. After 5-week intervention, at the end of the study, the rats were sacrificed and blood samples were collected for examination of biochemical parameters, including glomerular morphological makers, podocyte shape, cellular apoptosis, expressions of nephrin, inflammatory and apoptosis markers.. HQH ameliorated the rat's general status, proteinuria, renal morphological appearance and glomerulosclerosis. The decreased expression of nephrin in ADRN rats was increased by HQH, as well as the impaired podocyte foot process fusion. Cytosolic levels of p65 and inhibitor of nuclear factor κBα (IκBα) were decreased in ADRN rats, and recovered by the treatment of HQH. Consistently, the induced expression of tumor necrosis factor α (TNF-α), phosphorylated nuclear factor κB p65 (p-NFκB p65) and IκBα in ADRN were markedly suppressed by HQH. In addition, induction of Bax, cleaved caspase-3 and cytochrome C in ADRN rats were suppressed by HQH, indicating the amelioration of apoptosis.. HQH could ameliorate renal impairments in ADRN rats by increasing nephrin expression, inhibiting NF-κB signaling pathway via the down-regulation of p-NF-κB p65 and p-IκBα, and suppression of glomerular and tubular apoptosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Body Weight; Caspase 3; Chromatography, High Pressure Liquid; Cytochromes c; Doxorubicin; Drugs, Chinese Herbal; Kidney; Kidney Diseases; Kidney Glomerulus; Kidney Tubules; Male; Membrane Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Organ Size; Proteinuria; Rats, Sprague-Dawley; Signal Transduction; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Podocyte depletion in the kidney is associated with end-stage kidney disease (ESKD). Pre-eclampsia (PE) increases the risk of ESKD in later life. This study was performed to determine whether nephrinuria (soluble nephrin in the urine) is correlated with proteinuria and/or podocyturia (podocytes in the urine) in PE women.. Eighty-three urine samples, consisting of 45 and 38 samples from 27 normotensive and nine PE women, respectively, underwent simultaneous determination of nephrin, protein, and creatinine concentrations in the urine supernatant and quantitative analysis of podocyte-specific protein mRNA expression. This included podocin (Pod-mRNA) and nephrin (Nep-mRNA), using real-time polymerase chain reaction in the pelleted urine. Nephrinuria and proteinuria were corrected by creatinine concentration. Pod- and Nep-mRNA expression levels were corrected by GAPDH.. Nephrinuria, proteinuria, Pod-mRNA expression, and Nep-mRNA expression all increased with advancing gestation in PE women, while not in normotensive women. The nephrinuria was strongly correlated with proteinuria (R = 0.901, P <  0.001), Pod-mRNA expression level (R = 0.824, P < 0.001), and Nep-mRNA expression level (R  =  0.724, P <  0.001) in urine samples from PE women, while the nephrinuria was significantly correlated with proteinuria alone (R  =  0.419, P <  0.005) in urine samples from normotensive women.. Nephrinuria reflected well the degrees of proteinuria and podocyturia in PE women. This suggested that increased nephrinuria/proteinuria was associated with podocyte loss in the kidneys of PE women.

Topics: Adult; Creatine; Female; Gestational Age; Humans; Kidney Failure, Chronic; Membrane Proteins; Podocytes; Pre-Eclampsia; Pregnancy; Proteinuria; Risk Factors; Young Adult

The aim of this study was to elucidate whether genetic screening test results of pediatric patients with steroid-resistant nephrotic syndrome (SRNS) vary with ethnicity.. Using high-throughput DNA sequencing, 28 nephrotic syndrome-related genes were analyzed in 110 chil-dren affected by SRNS and 10 children with isolated proteinuria enrolled by 5 centers in China (67 boys, 53 girls). Their age at disease onset ranged from 1 day to 208 months (median, 48.8 months). Patients were excluded if their age at onset of disease was over 18 years or if they were diagnosed as having Alport syndrome.. A genetic etiology was identified in 28.3% of our cohort and the likelihood of establishing a genetic diagnosis decreased as the age at onset of nephrotic syndrome increased. The most common mutated genes were ADCK4 (6.67%), NPHS1 (5.83%), WT1 (5.83%), and NPHS2 (3.33%), and the difference in the frequencies of ADCK4 and NPHS2 mutations between this study and a study on monogenic causes of SRNS in the largest international cohort of 1,783 different families was significant. A case of congenital nephrotic syndrome was attributed to a homozygous missense mutation in ADCK4, and a de novo missense mutation in TRPC6 was detected in a case of infantile nephrotic syndrome.. Our results showed that, in the first and the largest multicenter cohort of Chinese pediatric SRNS reported to date, ADCK4 is the most common causative gene, whereas there is a low prevalence of NPHS2 mutations. Our data indicated that the genetic testing results for pediatric SRNS patients vary with different ethnicities, and this information will help to improve management of the disease in clinical practice.

Topics: Adolescent; Age of Onset; Asian People; Child; Child, Preschool; China; Cohort Studies; DNA Mutational Analysis; Drug Resistance; Female; Genetic Predisposition to Disease; Genetic Testing; Glucocorticoids; High-Throughput Nucleotide Sequencing; Homozygote; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mutation, Missense; Nephrotic Syndrome; Protein Kinases; Proteinuria; Sequence Analysis, DNA; TRPC6 Cation Channel; WT1 Proteins

Podocytes injury is involved in the development of diabetic nephropathy. This study was designed to confirm the reno- and podocyte-protective effects of pitavastatin in diabetic rats and clarify its mechanisms.. Wistar rats were divided into 4 treatment groups: control, streptozotocin (STZ; 55 mg/kg)-induced diabetes, STZ with pitavastatin (10 mg/kg/day), and STZ with tempol (1 mmol/l).. STZ-induced diabetic rats exhibited increases in urinary protein excretion and plasma creatinine, and a decrease in creatinine clearance. Pitavastatin significantly improved these parameters without reducing cholesterol levels, whereas tempol did not. The treatment with STZ-enhanced renal fibrosis, mesangial proliferation, transforming growth factor (TGF)-β, MCP-1 and suppressed Rho in association with decrement of bone morphogenetic protein (BMP)-7 expression in renal cortex. Moreover, STZ decreased podocyte related factors, podocin and nephrin, and BMP-7 in podocytes. Pitavastatin significantly ameliorated all these indices. On the other hand, improvement by tempol was found only in TGF-β, MCP-1 and histological changes.. Pitavastatin exhibited reno- and podocyte-protective effects accompanied by BMP-7 preservation and Rho suppression.

Topics: Animals; Bone Morphogenetic Protein 7; Chemokine CCL2; Creatinine; Diabetes Mellitus, Experimental; Gene Expression; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intracellular Signaling Peptides and Proteins; Kidney; Male; Malondialdehyde; Membrane Proteins; Nitrates; Nitrites; Protective Agents; Proteinuria; Quinolines; Rats, Wistar; rho-Associated Kinases; Transforming Growth Factor beta; Up-Regulation

Although calcineurin (CN) is distributed in many cell types and functions in regulating cell functions, the precise roles ofCNremained in each type of the cells are not well understood yet. ACNinhibitor (CNI) has been used for steroid-resistant nephrotic syndrome. ACNIis assumed to ameliorate proteinuria by preventing the overproduction of T-cell cytokines. However, recent reports suggest thatCNIhas a direct effect on podocyte. It is accepted that a slit diaphragm (SD), a unique cell-cell junction of podocytes, is a critical barrier preventing a leak of plasma protein into urine. Therefore, we hypothesized thatCNIhas an effect on theSD In this study, we analyzed the expression ofCNin physiological and in the nephrotic model caused by the antibody against nephrin, a critical component of theSD We observed thatCNis expressed at theSDin normal rat and human kidney sections and has an interaction with nephrin. The staining ofCNat theSDwas reduced in the nephrotic model, whileCNactivity in glomeruli was increased. We also observed that the treatment with tacrolimus, aCNI, in this nephrotic model suppressed the redistribution ofCN, nephrin, and otherSDcomponents and ameliorated proteinuria. These observations suggested that the redistribution and the activation ofCNmay participate in the development of theSDinjury.

Topics: Animals; Antibodies, Monoclonal; Calcineurin; Calcineurin Inhibitors; Cell Line; Child; Disease Models, Animal; Female; Humans; Intercellular Junctions; Male; Membrane Proteins; Mice; Nephrotic Syndrome; Podocytes; Protein Transport; Proteinuria; Rats, Wistar; Tacrolimus; Time Factors

Podocyte injury has been suggested to induce phenotypic alteration of glomerular podocytes and accelerate the detachment of podocytes from the glomeruli resulting in podocyturia. However, it is not clear whether podocyte phenotypic alteration occurs in the urine of women with preeclampsia (PE). Seventy-seven and 116 pelleted urine samples from 38 and 18 women at various stages of normal and PE pregnancies, respectively underwent quantitative analysis of podocyte-specific or associated protein mRNA expression, including podocin, nephrin, and synaptopodin using RT-PCR. Significant proteinuria in pregnancy (SPIP) is defined as protein:creatinine ratio (P/Cr, mg/mg) ≥ 0.27 in the urine supernatant. All three urine-pellet mRNAs expression levels were significantly positively correlated with P/Cr levels, suggesting that podocyturia increased with proteinuria. The podocin:nephrin mRNA ratio (PNR) and synaptopodin:nephrin mRNA ratio (SNR) increased significantly with increasing P/Cr, while the podocin:synaptopodin mRNA ratio (PSR) did not change significantly according to P/Cr, resulting in significantly higher PNR and SNR, but not PSR levels, in urine from PE women with than without SPIP. The PNR, SNR, and PSR in urine from PE women before onset of SPIP were comparable to those from controls. Thus, nephrin mRNA expression was reduced in the podocytes recovered from PE women.

Topics: Adult; Cell Adhesion; Female; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Microfilament Proteins; Podocytes; Pre-Eclampsia; Pregnancy; Proteinuria; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Young Adult

Glucocorticoids are the primary therapy for nephrotic syndrome (NS), but have serious side effects and are ineffective in ~20-50% of patients. Thiazolidinediones have recently been suggested to be renoprotective, and to modulate podocyte glucocorticoid-mediated nuclear receptor signaling. We hypothesized that thiazolidinediones could enhance glucocorticoid efficacy in NS. We found that puromycin aminonucleoside-induced proteinuria in rats was significantly reduced by both high-dose glucocorticoids (79%) and pioglitazone (61%), but not low-dose glucocorticoids (25%). Remarkably, pioglitazone + low-dose glucocorticoids also reduced proteinuria (63%) comparably to high-dose glucocorticoids, whereas pioglitazone + high-dose glucocorticoids reduced proteinuria to almost control levels (97%). Molecular analysis revealed that both glucocorticoids and pioglitazone enhanced glomerular synaptopodin and nephrin expression, and reduced COX-2 expression, after injury. Furthermore, the glomerular phosphorylation of glucocorticoid receptor and Akt, but not PPARγ, correlated with treatment-induced reductions in proteinuria. Notably, clinical translation of these findings to a child with refractory NS by the addition of pioglitazone to the treatment correlated with marked reductions in both proteinuria (80%) and overall immunosuppression (64%). These findings together suggest that repurposing pioglitazone could potentially enhance the proteinuria-reducing effects of glucocorticoids during NS treatment.

Topics: Albuminuria; Animals; Creatinine; Cyclooxygenase 2; Drug Therapy, Combination; Glucocorticoids; Kidney Glomerulus; Male; Membrane Proteins; Microfilament Proteins; Nephrotic Syndrome; Phosphorylation; Pioglitazone; PPAR gamma; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Wistar; Signal Transduction; Thiazolidinediones; Urinalysis

Proteinuria is the hallmark of clinical manifestation of disease activity in lupus nephritis (LN) patients, which arises from direct or indirect podocyte injury. This study is to explore the relationship between intrarenal T cell infiltration and podocyte injury in lupus nephritis (LN), and to understand the potential mechanisms of podocyte injury induced by intrarenal T cells. Sixty renal biopsies from patients diagnosed with LN were included in the present study. Histological changes in LN patients were detected by light and electron microscopy. Podocyte-specific nephrin expression in renal tissues was detected by immunofluorescence. Infiltration of T cells (CD3+ cells), infiltration of macrophages (CD68+ cells) and the expression of osteopontin (OPN) in renal tissues were examined by immunohistochemical staining. Pearson or Spearman's tests were used to perform correlation analysis. Morphologic lesions of podocytes were more severe in LN patients than in normal control subjects. Compared with normal control subjects, nephrin expression was significantly decreased in LN patients. The expression level of nephrin was significantly lower in active LN patients than in the inactive group of patients (P < 0.05). Compared with normal control subjects, the number of infiltrated intrarenal T cells and macrophages was significantly increased in LN patients. T cells were mainly distributed in renal interstitium, with very few being in glomeruli, while macrophages were mainly located in glomeruli. The number of intrarenal infiltrated T cells and macrophages in active LN patients was more than that in the inactive group (P < 0.05). Compared with normal control subjects, OPN expression in LN patients was increased significantly. The expression level of OPN in active LN patients was significantly higher than that in the inactive group (P < 0.05). Podocyte-specific nephrin was negatively correlated with 24-hour proteinuria, intrarenal T cells infiltration and intrarenal OPN expression in LN patients (P < 0.001). Intrarenal macrophages had significantly positive correlation with intrarenal OPN expression (P < 0.001). The present study provides possible links between intrarenal T cells, OPN, macrophages with reduced podocyte-nephrin and podocytopathy in systemic lupus erythematosus. In addition, infiltration of macrophages in glomeruli induced by OPN that is induced by T cells may be a crucial mechanism for podocyte injury.

Topics: Adult; Biopsy; Female; Humans; Lupus Nephritis; Macrophages; Male; Membrane Proteins; Osteopontin; Podocytes; Proteinuria; T-Lymphocytes

Glomerular visceral epithelial cells (podocytes) play a critical role in the maintenance of glomerular permselectivity. Podocyte injury, manifesting as proteinuria, is the cause of many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA2γ KO mice have demonstrated an important role for iPLA2γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA2γ KO mice to better understand the role of iPLA2γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA2γ did not cause detectable albuminuria; however, it resulted in mitochondrial structural abnormalities and enhanced autophagy in podocytes as well as loss of podocytes in aging KO mice. Moreover, after induction of anti-glomerular basement membrane nephritis in young mice, iPLA2γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA2γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA2γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria.

Topics: Aging; Animals; Autophagy; Cells, Cultured; Endoplasmic Reticulum Stress; Glomerulonephritis; Group VI Phospholipases A2; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Phospholipases A2, Calcium-Independent; Podocytes; Proteinuria

To investigate the possibility of nephrinuria as a screening tool for the risk of pre-eclampsia (PE).. Prospective observational study.. A single university hospital. Changes in urinary nephrin:creatinine ratio (NCR, ng/mg) and protein:creatinine ratio (PCR, mg/mg) in pregnancy were determined. Significant proteinuria in pregnancy (SPIP) was defined as PCR>0.27. PE was diagnosed in women with both SPIP and hypertension.. 89 pregnant women in whom neither hypertension nor SPIP was present at enrolment, providing 31, 125 and 93 random urine samples during first, second and third trimesters, respectively.. PE developed in 14 of the 89 women. NCR increased with increasing PCR in 14 women with PE (correlation coefficient, 0.862; p<0.0001). In contrast, NCR did not change significantly despite significant increases in PCR in 75 women with normotensive pregnancies defined as neither SPIP nor hypertension, indicating that there was little increase in nephrinuria over the physiological range of proteinuria in pregnancy. Relative risk of later development of PE among asymptomatic second and third trimester women with NCR (ng/mg) >122 (95th centile value for 75 women with normotensive pregnancies) was 5.93 (95% CI 2.59 to 13.6; 60% (6/10) vs 10% (8/79)) and 13.5 (95% CI 3.31 to 55.0; 75% (6/8) vs 5.5% (2/36)), respectively, compared with women with NCR≤122 at that time.. Nephrinuria was unlikely to increase in normal pregnancy. A certain NCR cut-off may efficiently differentiate women at higher risk of PE.

Topics: Adult; Biomarkers; Creatinine; Feasibility Studies; Female; Humans; Hypertension, Pregnancy-Induced; Membrane Proteins; Parity; Pre-Eclampsia; Pregnancy; Pregnancy Trimesters; Prenatal Diagnosis; Prospective Studies; Proteinuria; Risk Assessment; Young Adult

The concentration of adenosine in the normal kidney increases markedly during renal hypoxia, ischemia, and inflammation. A recent study reported that an A3 adenosine receptor (A3AR) antagonist attenuated the progression of renal fibrosis. The adriamycin (ADX)-induced nephropathy model induces podocyte injury, which results in severe proteinuria and progressive glomerulosclerosis. In this study, we investigated the preventive effect of a highly selective A3AR antagonist (LJ1888) in ADX-induced nephropathy. Three groups of six-week-old Balb/c mice were treated with ADX (11 mg/kg) for four weeks and LJ1888 (10 mg/kg) for two weeks as following: 1) control; 2) ADX; and 3) ADX + LJ1888. ADX treatment decreased body weight without a change in water and food intake, but this was ameliorated by LJ1888 treatment. Interestingly, LJ1888 lowered plasma creatinine level, proteinuria, and albuminuria, which had increased during ADX treatment. Furthermore, LJ1888 inhibited urinary nephrin excretion as a podocyte injury marker, and urine 8-isoprostane and kidney lipid peroxide concentration, which are markers of oxidative stress, increased after injection of ADX. ADX also induced the activation of proinflammatory and profibrotic molecules such as TGF-β1, MCP-1, PAI-1, type IV collagen, NF-κB, NOX4, TLR4, TNFα, IL-1β, and IFN-γ, but they were remarkably suppressed after LJ1888 treatment. In conclusion, our results suggest that LJ1888 has a renoprotective effect in ADX-induced nephropathy, which might be associated with podocyte injury through oxidative stress. Therefore, LJ1888, a selective A3AR antagonist, could be considered as a potential therapeutic agent in renal glomerular diseases which include podocyte injury and proteinuria.

Topics: Actins; Adenosine; Adenosine A3 Receptor Antagonists; Albuminuria; Animals; Body Weight; Creatinine; Dinoprost; Disease Models, Animal; Doxorubicin; Immunohistochemistry; Kidney; Kidney Diseases; Lipid Peroxidation; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; NF-kappa B; Oxidative Stress; Plasminogen Activator Inhibitor 1; Proteinuria; Transforming Growth Factor beta1

Diabetic nephropathy (DN) is the major cause of end-stage renal disease worldwide. Podocytes are important for glomerular filtration barrier function and maintenance of size selectivity in protein filtration in the kidney. Podocyte damage is the basis of many glomerular diseases characterized by loss of interdigitating foot processes and decreased expression of components of the slit diaphragm. Nephrin, a podocyte-specific protein, is the main component of the slit diaphragm. Loss of nephrin is observed in human and rodent models of diabetic kidney disease. The long isoform of CIN85 (RukL) is a binding partner of nephrin that mediates nephrin endocytosis via ubiquitination in podocytes. Here we demonstrate that the loss of nephrin expression and the onset of proteinuria in diabetic mice correlate with an increased accumulation of ubiquitinated proteins and expression of CIN85/RukL in podocytes. CIN85/RukL deficiency preserved nephrin surface expression on the slit diaphragm and reduced proteinuria in diabetic mice, whereas overexpression of CIN85 in zebrafish induced severe edema and disruption of the filtration barrier. Thus, CIN85/RukL is involved in endocytosis of nephrin in podocytes under diabetic conditions, causing podocyte depletion and promoting proteinuria. CIN85/RukL expression therefore shows potential to be a novel target for antiproteinuric therapy in diabetes.

Topics: Adaptor Proteins, Signal Transducing; Animals; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Endocytosis; Genotype; Humans; Membrane Proteins; Mice; Microscopy, Electron, Transmission; Neoplasm Proteins; Nerve Tissue Proteins; Podocytes; Proteinuria

The present study was designed to examine the protective effect of notoginsenoside R1 (NR1) on podocytes in a rat model of streptozotocin (STZ)‑induced diabetic nephropathy (DN), and to explore the mechanism responsible for NR1-induced renal protection. Diabetes was induced by a single injection of STZ, and NR1 was administered daily at a dose of 5 mg/kg (low dose), 10 mg/kg (medium) and 20 mg/kg (high) for 16 weeks in Sprague-Dawley rats. Blood glucose levels, body weight and proteinuria were measured every 4 weeks, starting on the day that the rats received NR1. Furthermore, on the day of sacrifice, blood, urine and kidneys were collected in order to assess renal function according to general parameters. Pathological staining was performed to evaluate the renal protective effect of NR1, and the expression of the key slit diaphragm proteins, namely neprhin, podocin and desmin, were evaluated. In addition, the serum levels of inflammatory cytokines [tumor necrosis factor-α (TNF-α), tumor growth factor-β1 (TGF-β1), interleukin (IL)-1 and IL-6] as well as an anti-inflammatory cytokine (IL-10) were assessed, and the apoptosis of podocytes was quantified. Finally, the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and the involvement of nuclear factor-κB (NF-κB) inactivation was further analyzed. In this study, NR1 improved renal function by ameliorating histological alterations, increasing the expression of nephrin and podocin, decreasing the expression of desmin, and inhibiting both the inflammatory response as well as the apoptosis of podocytes. Furthermore, NR1 treatment increased the phosphorylation of both PI3K (p85) and Akt, indicating that activation of the PI3K/Akt signaling pathway was involved. Moreover, NR1 treatment decreased the phosphorylation of NF-κB (p65), suggesting the downregulation of NF-κB. This is the first study to the best of our knowledge, to clearly demonstrate that NR1 treatment ameliorates podocyte injury by inhibiting both inflammation and apoptosis through the PI3K/Akt signaling pathway.

Topics: Animals; Apoptosis; Blood Glucose; Cell Nucleus; Cytokines; Desmin; Diabetic Nephropathies; Ginsenosides; Inflammation; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Podocytes; Protective Agents; Proteinuria; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction

Podocytes are terminally differentiated epithelial cells in the kidney glomeruli that act as a key component of the glomerular filtration barrier. Although the inciting injury to the podocyte may vary between various glomerular diseases, the inevitable consequence of podocyte injury results in their loss, leading to progressive kidney disease. Here, we report that the expression of CCAAT/enhancer binding protein-α (C/EBP-α), a transcription factor known to interact with and activate PPAR-γ and NF-κB, is suppressed in the glomerular cells, particularly in podocytes, in human kidneys with focal segmental glomerulosclerosis. Genetic ablation of C/EBP-α in podocytes resulted in increased proteinuria, increased podocyte foot process effacement, and to decreased podocyte number in the setting of Adriamycin (ADR)-induced nephropathy. Overexpression of C/EBP-α in human podocytes in vitro led to an inhibition of MCP-1 and IL-6 expression in response to TNF-α and IL-1β treatments. Conversely, augmented production of MCP-1 and IL-6 was observed in the glomeruli of C/EBP-α knockout mice and was associated increased infiltration of macrophages in vivo. Together, our data suggest that C/EBP-α mediates anti-inflammatory effects in podocytes to confer protection against podocyte injury and loss that may contribute to worsening glomerulosclerosis.

Topics: Animals; CCAAT-Enhancer-Binding Protein-alpha; Cytokines; Doxorubicin; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Macrophages; Male; Membrane Proteins; Mesangial Cells; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Proteinuria

We investigated the relationship between proteinuria in L-NAME induced preeclampsia and the expression of nephrin and podocin, and the effect of low-molecular-weight-heparin (LMWH) on proteinuria in rats.. We detected nephrin and podocin expression of kidneys of pregnant rats after L-NAME and after LMWH intervening pregnant rats.. Glomerular nephrin expression in L-NAME induced preeclampsia significantly decreased, but not podocin. Nephrin was relatively increased after LMWH intervention and this was accompanied by a decrease in proteinuria.. We demonstrate that down-regulation of nephrin is involved in L-NAME induced proteinuria, and that LMWH reduces proteinuria by up-regulation of neprhin.

Topics: Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Heparin, Low-Molecular-Weight; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; NG-Nitroarginine Methyl Ester; Pre-Eclampsia; Pregnancy; Proteinuria; Random Allocation; Rats, Wistar

Proteinuria is not only a sign of kidney damage but is also involved in the progression of renal disease as an independent pathologic factor. Although patients with mutated type 1 cannabinoid receptors (CB1) polymorphism are associated with renal microvascular damage, the biologic role of CB1 signaling in proteinuria remains uncharacterized till now. Herein, we investigate whether CB1 participates in glomerular proteinuria in CB1 transgenic mice and treatment with CB1 agonist WIN55212-2 rat, neither of which are diabetic models. The CB1 transgenic mice and rats treated with CB1 agonist WIN55212-2 had higher kidney weight and urinary protein concentrations but not blood glucose levels compared with the wild-type group. A combination of laser-capture microsdissection, quantitative reverse transcription-polymerase chain reaction, immunoblotting and immunohistochemical validation revealed that CB1 transgenic mice and rats treated with CB1 agonist WIN55212-2 had higher vascular endothelial growth factor (VEGF) expression in renal glomeruli than that of the wild-type group. Geneticorpharmacological activation of CB1 by transgenic CB1 mice or treatment with WIN55212-2 reduced nephrin expression in the renal glomeruli compared with that of the wild-type group in the glomerular mesanglium. Taken together, CB1 transgenic mice and rats treated with CB1 agonist WIN55212-2 induced proteinuria with upregulation of CB1 resulting in impaired nephrin expression, by inducing excess VEGF reaction in the renal glomeruli. Genetic and pharmacological manipulation of CB1 signaling revealed VEGF-dependent nephrin depression of glomerulopathy. Controlling CB1 activity can be used an alternative strategy for sustaining renal function in the presence of CB1 activation.

Topics: Analgesics; Animals; Benzoxazines; Blood Glucose; Gene Expression Regulation; Glomerular Mesangium; Humans; Kidney Diseases; Male; Membrane Proteins; Mice; Mice, Transgenic; Morpholines; Naphthalenes; Organ Size; Proteinuria; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Signal Transduction; Vascular Endothelial Growth Factor A

The onset of diabetic nephropathy (DN) is highlighted by glomerular filtration barrier abnormalities. Identifying pathogenic factors and targetable pathways driving DN is crucial to developing novel therapies and improving the disease outcome. Semaphorin3a (sema3a) is a guidance protein secreted by podocytes. Excess sema3a disrupts the glomerular filtration barrier. Here, using immunohistochemistry, we show increased podocyte SEMA3A in renal biopsies from patients with advanced DN. Using inducible, podocyte-specific Sema3a gain-of-function (Sema3a(+)) mice made diabetic with streptozotocin, we demonstrate that sema3a is pathogenic in DN. Diabetic Sema3a(+) mice develop massive proteinuria, renal insufficiency, and extensive nodular glomerulosclerosis, mimicking advanced DN in humans. In diabetic mice, Sema3a(+) exacerbates laminin and collagen IV accumulation in Kimmelstiel-Wilson-like glomerular nodules and causes diffuse podocyte foot process effacement and F-actin collapse via nephrin, αvβ3 integrin, and MICAL1 interactions with plexinA1. MICAL1 knockdown and sema3a inhibition render podocytes not susceptible to sema3a-induced shape changes, indicating that MICAL1 mediates sema3a-induced podocyte F-actin collapse. Moreover, sema3a binding inhibition or podocyte-specific plexinA1 deletion markedly ameliorates albuminuria and abrogates renal insufficiency and the diabetic nodular glomerulosclerosis phenotype of diabetic Sema3a(+) mice. Collectively, these findings indicate that excess sema3a promotes severe diabetic nephropathy and identifies novel potential therapeutic targets for DN.

Topics: Actins; Animals; Chromones; Collagen Type IV; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Integrin alphaVbeta3; Laminin; Membrane Proteins; Mice; Mice, Knockout; Microfilament Proteins; Microtubule-Associated Proteins; Mixed Function Oxygenases; Nerve Tissue Proteins; Podocytes; Proteinuria; Receptors, Cell Surface; Renal Insufficiency; Semaphorin-3A; WT1 Proteins; Xanthones

Chronic renal fibrosis is the final common pathway of end stage renal disease caused by glomerular or tubular pathologies. Genetic background has a strong influence on the progression of chronic renal fibrosis. We recently found that Rowett black hooded rats were resistant to renal fibrosis. We aimed to investigate the role of sustained inflammation and oxidative/nitrative stress in renal fibrosis progression using this new model. Our previous data suggested the involvement of podocytes, thus we investigated renal fibrosis initiated by doxorubicin-induced (5 mg/kg) podocyte damage. Doxorubicin induced progressive glomerular sclerosis followed by increasing proteinuria and reduced bodyweight gain in fibrosis-sensitive, Charles Dawley rats during an 8-week long observation period. In comparison, the fibrosis-resistant, Rowett black hooded rats had longer survival, milder proteinuria and reduced tubular damage as assessed by neutrophil gelatinase-associated lipocalin (NGAL) excretion, reduced loss of the slit diaphragm protein, nephrin, less glomerulosclerosis, tubulointerstitial fibrosis and matrix deposition assessed by periodic acid-Schiff, Picro-Sirius-red staining and fibronectin immunostaining. Less fibrosis was associated with reduced profibrotic transforming growth factor-beta, (TGF-β1) connective tissue growth factor (CTGF), and collagen type I alpha 1 (COL-1a1) mRNA levels. Milder inflammation demonstrated by histology was confirmed by less monocyte chemotactic protein 1 (MCP-1) mRNA. As a consequence of less inflammation, less oxidative and nitrative stress was obvious by less neutrophil cytosolic factor 1 (p47phox) and NADPH oxidase-2 (p91phox) mRNA. Reduced oxidative enzyme expression was accompanied by less lipid peroxidation as demonstrated by 4-hydroxynonenal (HNE) and less protein nitrosylation demonstrated by nitrotyrosine (NT) immunohistochemistry and quantified by Western blot. Our results demonstrate that mediators of fibrosis, inflammation and oxidative/nitrative stress were suppressed in doxorubicin nephropathy in fibrosis-resistant Rowett black hooded rats underlying the importance of these pathomechanisms in the progression of renal fibrosis initiated by glomerular podocyte damage.

Topics: Aldehydes; Animals; Body Weight; Chemokine CCL2; Connective Tissue Growth Factor; Disease Progression; Disease Resistance; Dose-Response Relationship, Drug; Doxorubicin; Fibrosis; Kidney; Male; Membrane Proteins; Oxidative Stress; Proteinuria; Rats; Reactive Nitrogen Species; Species Specificity; Transforming Growth Factor beta1; Tyrosine

Defects in sialylation are known to have serious consequences on podocyte function leading to collapse of the glomerular filtration barrier and the development of proteinuria. However, the cellular processes underlying aberrant sialylation in renal disease are inadequately defined. We have shown in cultured human podocytes that puromycin aminonucleoside (PAN) downregulates enzymes involved in sialic acid metabolism and redox homeostasis and these can be rescued by co-treatment with free sialic acid. The aim of the current study was to ascertain whether sialic acid supplementation could improve renal function and attenuate desialylation in an in vivo model of proteinuria (PAN nephrosis) and to delineate the possible mechanisms involved. PAN nephrotic rats were supplemented with free sialic acid, its precursor N-acetyl mannosamine or the NADPH oxidase inhibitor apocynin. Glomeruli, urine, and sera were examined for evidence of kidney injury and therapeutic efficacy. Of the three treatment regimens, sialic acid had the broadest efficacy in attenuating PAN-induced injury. Proteinuria and urinary nephrin loss were reduced. Transmission electron microscopy revealed that podocyte ultrastructure, exhibited less severe foot process effacement. PAN-induced oxidative stress was ameliorated as evidenced by a reduction in glomerular NOX4 expression and a downregulation of urine xanthine oxidase levels. Sialylation dysfunction was improved as indicated by reduced urinary concentrations of free sialic acid, restored electrophoretic mobility of podocalyxin, and improved expression of a sialyltransferase. These data indicate that PAN induces alterations in the expression of enzymes involved in redox control and sialoglycoprotein metabolism, which can be ameliorated by sialic acid supplementation possibly via its properties as both an antioxidant and a substrate for sialylation.

Topics: Acetophenones; Animals; Dietary Supplements; Hexosamines; Kidney Glomerulus; Membrane Proteins; Microscopy, Electron, Transmission; N-Acetylneuraminic Acid; NADPH Oxidase 4; NADPH Oxidases; Nephrosis; Oxidative Stress; Podocytes; Proteinuria; Puromycin Aminonucleoside; Rats

Primary glomerulonephritis (PGN) is the most common reason inducing end stage renal disease in China, however, its pathogenesis remains unclear. The present study was designed to test the hypothesis that the formation and activation of NLRP3 (Nod-like receptor family pyrin domain containing 3) inflammasomes is an important initiating mechanism resulting in PGN.. Serum samples and frozen sections were collected from 38 cases with PGN, and renal tissues were obtained from 22 of them. NLRP3 inflammasomes were detected by RT-PCR and immunofluoresence methods. The relationship between NLRP3 and clinical/pathologic indexes was analyzed.. RT-PCR analyses demonstrated that the mRNA levels of NLRP3 and caspase-1 genes were elevated significantly in renal tissues of PGN patients compared to those from normal pericarcinoma tissues. Moreover, the increased level of NLRP3 mRNA was correlative with a decrease in nephrin mRNA level and an increase in desmin mRNA level, which indicates that NLRP3 participates in podocyte injury in PGN patients. Immunofluorescence analysis also showed the protein expressions of NLRP3 and caspase-1 were increased in the glomeruli of PGN patients. Neverthless, there was no obvious regularity was presented in further subgroup analysis according to pathological types. In addition, increased NLRP3 was associated with the deterioration of renal function and glomerulosclerosis. IL-1β, a product of NLRP3 inflammasome activation, had a significant correlation with proteinuria.. The formation and activation of NLRP3 inflammasomes in podocytes has been importantly implicated in the development of PGN-associated glomerular injury.

Topics: Adult; Carrier Proteins; Caspase 1; China; Desmin; Female; Glomerulosclerosis, Focal Segmental; Humans; Inflammasomes; Interleukin-1beta; Kidney; Kidney Function Tests; Male; Membrane Proteins; Middle Aged; NLR Family, Pyrin Domain-Containing 3 Protein; Podocytes; Proteinuria; Retrospective Studies; RNA, Messenger

The pathogenesis of proteinuria in Alport syndrome (AS) remains unclear. Vascular endothelial growth factor A (VEGFA) is a key regulator of the glomerular filtration barrier (GFB). This study explored the expression of VEGFA in the glomeruli and its accumulation in the glomerular basement membrane (GBM) and their relationship with podocyte injury and proteinuria in Alport syndrome (AS). Clinical data and renal tissues of control patients (11 cases) and AS patients (25 cases) were included. AS patients were further divided into 2 groups according to the quantities of their urinary protein: mild to moderate proteinuria group (proteinuria <50 mg/kg/d, 15 cases) and heavy proteinuria group (proteinuria ≥50 mg/kg/d, 10 cases). The expression and distribution of VEGFA and VEGF receptor 2 (VEGFR2) in the GFB, the phosphorylation of VEGFR2 (p-VEGFR2) and nephrin (p-nephrin), and the expression of synaptopodin and nephrin in the glomeruli were detected by immune electron microscopy and/or immunofluorescence, and their relationships to proteinuria in AS patients were analyzed. The accumulation of VEGFA in the GBM was increased in AS patients. The expression of VEGFA and the levels of p-VEGFR2 and p-nephrin in glomeruli were increased and were positively correlated with the degree of proteinuria in AS patients. The expression of synaptopodin and nephrin were decreased and were negatively correlated with the degree of proteinuria in AS patients. The over expressed VEGFA in the glomeruli and its accumulation in the GBM may activate the VEGFA-VEGFR2 and nephrin signaling pathways and lead to podocyte injury and occurrence of proteinuria in AS.

Topics: Adolescent; Biomarkers; Case-Control Studies; Child; Child, Preschool; Female; Glomerular Basement Membrane; Humans; Male; Membrane Proteins; Microfilament Proteins; Nephritis, Hereditary; Phosphorylation; Podocytes; Proteinuria; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur(-/-) mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR.

Topics: Animals; Down-Regulation; Gene Expression Regulation; Genetic Variation; Humans; Integrin alphaVbeta3; Membrane Proteins; Mice; Mice, Knockout; Podocytes; Promoter Regions, Genetic; Protein Binding; Proteinuria; Receptors, Urokinase Plasminogen Activator; WT1 Proteins

To determine the effect of Salvia przewalskii extract (SPE) from total phenolic acids on puromycin aminonucleoside (PAN)-induced rat podocyte injury.. The rats were divided into groups that were treated with either PAN only or PAN followed by tacrolimus or SPE. We evaluated the effects of SPE on podocyte injury 5, 10, 15 and 21 days following treatment.. (1) Proteinuria was observed starting on day 5 in all groups. The peak levels of proteinuria differed among the groups with tacrolimus and high-dose SPE, which significantly decreased proteinuria relative to the PAN and low- and medium-dose SPE groups. The proteinuria in each group decreased by day 15 and returned to a normal level by day 21. (2) H&E and PAS staining revealed no abnormality in glomerular morphology. With electron microscopy, we observed foot process effacement in the rats of all groups starting on day 5, but rats in the tacrolimus and high-dose SPE groups exhibited a lower degree. (3) IHC staining of nephrin and podocin revealed unaffected expression and better linear distributions in the high-dose SPE and tacrolimus groups. Western blot analysis confirmed that SPE could improve the expression of proteins. (4) The mRNA levels of nephrin and podocin in the tacrolimus and high-dose SPE groups were significantly higher than that in the others.. In our study, we first demonstrated the ability of SPE to reduce proteinuria, preserve the morphology and structure of podocytes and retain the levels of slit diaphragm proteins on PAN-induced rat podocytes injury.

Topics: Animals; Camphanes; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Panax notoginseng; Phytotherapy; Podocytes; Proteinuria; Puromycin; Rats, Sprague-Dawley; Saliva; Salvia miltiorrhiza; Tacrolimus

The aim of the present study was to investigate the mechanisms underlying the effects of prednisone on adriamycin-induced nephritic rat kidney damage via the focal adhesion kinase (FAK)/receptor activator of nuclear factor-κB ligand (RANKL)/mitogen‑activated protein kinase (MAPK) signaling pathway. An adriamycin‑induced nephritic rat model was established to investigate these mechanisms. A total of 30 healthy male Sprague‑Dawley rats were randomly assigned to the normal, model or prednisone group. Samples of urine were collected over the course of 24 h at days 7, 14, and 28, and renal cortex tissue samples were harvested at days 14, and 28 following nephritic rat model establishment. The total urinary protein content was measured by biuret colorimetry. Pathological changes in the kidney tissue samples were observed using an electron microscope. The mRNA expressions levels of FAK, RANKL, p38, extracellular signal‑regulated kinase (ERK), c‑Jun N‑terminal kinase (JNK), and nephrin were then quantified by reverse transcription‑quantitative polymerase chain reaction. In addition, the protein expressions levels of FAK, RANKL, p38, ERK, JNK, phosphorylated (p)‑FAK, p‑ERK, and p‑JNK were quantified by western blotting. As compared with the normal group, the protein expression levels of FAK, RANKL, p-FAK, p38 and p-ERK in the model group were increased. In the prednisone group, the protein expression levels of p-ERK decreased, as compared with the normal group. In the prednisone group, the urinary protein levels, the protein expression levels of FAK, RANKL, p38, p-FAK, p-p38 and the mRNA expression levels of FAK, p38, RANKL, ERK, JNK decreased, as compared with the model group. In the prednisone group, the mRNA and protein expression levels of nephrin and the serum expression levels of RANKL increased, the serum expression levels of osteoprotegerin (OPG) were decreased, as compared with the model group. No significant changes in the protein expression levels of JNK were observed among the groups. These results suggested that prednisone is able to protect podocytes from apoptosis, and reduce urinary protein levels by inhibiting the FAK/RANKL/MAPK signaling pathway in kidney tissue samples. Serum prednisone may induce osteoporosis via the OPG/RANK/RANKL signaling pathway.

Topics: Animals; Antibiotics, Antineoplastic; Doxorubicin; Drug Evaluation, Preclinical; Focal Adhesion Kinase 1; Gene Expression; Kidney; Male; MAP Kinase Signaling System; Membrane Proteins; Mitogen-Activated Protein Kinases; Nephritis; Osteoprotegerin; Prednisone; Proteinuria; RANK Ligand; Rats, Sprague-Dawley

To explore the combined detection of urine UmAlb and urinary nephrin (Unephrin), podocalyxin (UPCX) in podocyte of MKR mice with diabetic nephropathy.. Thirty 8 weeks old MKR mice were randomly divided into two groups as follows: negative control group, DN model group, and another 15 wild C57 mice were used as normal control. Mice in DN model group were received unilateral nephrectomy and high-fat diet feed for 2 months. The morphological structure changes of the podocytes were observed by transmission electron microscopes. The levels of FBG were detected by electrochemical detection method, The nephrin and PCX protein expression were measured by western blotting. The levels of UmAlb, Unephrin and UPCX were detected by ELISA.. The podocyte damage in the mice of DN model group increased significantly when compared with normal control. As compared with normal control, FBG in the model group increased significantly (P<0. 01), the expression level of nephrin and PCX in Renal Tissue and Unephrin, UPCX, and urine UmAlb were also increased significantly (P<0. 01).. The level of Unephrin and UPCX were positive correlated with the level of urine UmAlb, the loss of podocyte strcture protein might be one of the mechanism in leading proteinuria in diabetic nephropathy.

Topics: Animals; Blotting, Western; Diabetic Nephropathies; Enzyme-Linked Immunosorbent Assay; Membrane Proteins; Mice; Podocytes; Proteinuria; Sialoglycoproteins

Proteinuria is a major feature of lupus nephritis (LN) and reflects podocyte injury. Analysis of podocyte biomarkers was performed attempting to identify if podocyte phenotype is distinct in pure membranous and proliferative LN. Expression of synaptopodin, Wilms tumor protein 1 (WT1), glomerular epithelial protein 1 (GLEPP1) and nephrin was evaluated in 52 LN biopsies by immunohistochemistry. Preserved synaptopodin expression was observed in only 10 (19.2%) of all biopsies while 42 (80.8%) had reduced expression. Both groups had comparable proteinuria at the time of biopsy (p = 0.22); however, in the mean follow-up of four years there was a tendency toward lower mean levels of proteinuria in patients with preserved synaptopodin staining (0.26±0.23 vs. 0.84±0.90 g/24 h, p = 0.05) compared with those with diminished expression. Thirty-nine (75%) biopsies were classified as proliferative and 13 (25%) as pure membranous. Comparison of podocyte biomarkers demonstrated a predominance of preserved staining of synaptopodin (69.2%), WT1 (69.2%), GLEPP1 (53.9%) and nephrin (60%) in the pure membranous group whereas only <10% of the proliferative showed preserved expression. Our data suggest that in proliferative forms there seems to occur structural podocyte damage, whereas in the pure membranous the predominant preserved pattern suggests a dysfunctional podocyte lesion that may account for the better long-term prognosis of proteinuria outcome.

Topics: Adult; Biomarkers; Biopsy; Cell Proliferation; Female; Glomerulonephritis, Membranoproliferative; Glomerulonephritis, Membranous; Humans; Immunohistochemistry; Lupus Nephritis; Male; Membrane Proteins; Microfilament Proteins; Middle Aged; Podocytes; Prognosis; Proteinuria; Receptor-Like Protein Tyrosine Phosphatases, Class 3; Time Factors; WT1 Proteins; Young Adult

Radix puerariae, a traditional Chinese herbal medication, has been used successfully to treat patients with early stage of diabetic nephropathy. However, the underlined mechanism of this renal protective effect has not been determined. In the current study, we investigated the effects and the mechanism of puerarin in Streptozotocin (STZ)-induced diabetic rats. We treated STZ-rats with either puerarin or losartan, an angiotensin II receptor blocker, as compared to those treated with vehicle. We found that both puerarin and losartan attenuated kidney hypertrophy, mesangial expansion, proteinuria, and podocyte foot process effacement in STZ rats. In addition, both puerarin and losartan increased expression of podocyte slit diaphragm proteins such as nephrin and podocin. Interestingly, we found that puerarin treatment induced a more pronounced suppression of oxidative stress production and S-nitrosylation of proteins in the diabetic kidneys as compared to losartan treatment. Furthermore, we found that matrix metalloproteinase-9 (MMP-9), which is known to be activated by oxidative stress and S-nitrosylation of proteins, was also suppressed more extensively by puerarin than losartan. In conclusion, these data provide for the first time the potential mechanism to support the use of puerarin in the treatment of early diabetic nephropathy.

Topics: Animals; Collagen Type IV; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Drug Evaluation, Preclinical; Enzyme Repression; Intracellular Signaling Peptides and Proteins; Isoflavones; Kidney; Losartan; Male; Matrix Metalloproteinase 9; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Wistar; Reactive Oxygen Species; Streptozocin

TGF-β-activated kinase 1 (TAK1) is a key intermediate in signal transduction induced by TGF-β or inflammatory cytokines, such as TNF-α and IL-1, which are potent inducers of podocyte injury responses that lead to proteinuria and glomerulosclerosis. Nevertheless, little is known about the physiologic and pathologic roles of TAK1 in podocytes. To examine the in vivo role of TAK1, we generated podocyte-specific Tak1 knockout mice (Nphs2-Cre(+):Tak1(fx/fx); Tak1(∆/∆)). Targeted deletion of Tak1 in podocytes resulted in perinatal lethality, with approximately 50% of animals dying soon after birth and 90% of animals dying within 1 week of birth. Tak1(∆/∆) mice developed proteinuria from P1 and exhibited delayed glomerulogenesis and reduced expression of Wilms' tumor suppressor 1 and nephrin in podocytes. Compared with Tak1(fx/fx) mice, Tak1(∆/∆) mice exhibited impaired formation of podocyte foot processes that caused disruption of the podocyte architecture with prominent foot process effacement. Intriguingly, Tak1(∆/∆) mice displayed increased expression of vascular endothelial growth factor within the glomerulus and abnormally enlarged glomerular capillaries. Furthermore, 4- and 7-week-old Tak1(∆/∆) mice with proteinuria had increased collagen deposition in the mesangium and the adjacent tubulointerstitial area. Thus, loss of Tak1 in podocytes is associated with the development of proteinuria and glomerulosclerosis. Taken together, our data show that TAK1 regulates the expression of Wilms' tumor suppressor 1, nephrin, and vascular endothelial growth factor and that TAK1 signaling has a crucial role in podocyte differentiation and attainment of normal glomerular microvasculature during kidney development and glomerular filtration barrier homeostasis.

Topics: Animals; Animals, Newborn; Capillaries; Cell Differentiation; Collagen; Female; Glomerular Filtration Barrier; Kidney Glomerulus; Male; MAP Kinase Kinase Kinases; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Pregnancy; Proteinuria; RNA, Messenger; Signal Transduction; Vascular Endothelial Growth Factor A; WT1 Proteins

The transcription factor Kruppel-like factor 4 (KLF4) has the ability, along with other factors, to reprogram somatic cells into induced pluripotent stem (iPS) cells. Here, we determined that KLF4 is expressed in kidney glomerular podocytes and is decreased in both animal models and humans exhibiting a proteinuric. Transient restoration of KLF4 expression in podocytes of diseased glomeruli in vivo, either by gene transfer or transgenic expression, resulted in a sustained increase in nephrin expression and a decrease in albuminuria. In mice harboring podocyte-specific deletion of Klf4, adriamycin-induced proteinuria was substantially exacerbated, although these animals displayed minimal phenotypical changes prior to adriamycin administration. KLF4 overexpression in cultured human podocytes increased expression of nephrin and other epithelial markers and reduced mesenchymal gene expression. DNA methylation profiling and bisulfite genomic sequencing revealed that KLF4 expression reduced methylation at the nephrin promoter and the promoters of other epithelial markers; however, methylation was increased at the promoters of genes encoding mesenchymal markers, suggesting selective epigenetic regulation of podocyte gene expression. Together, these results suggest that KLF4 epigenetically modulates podocyte phenotype and function and that the podocyte epigenome can be targeted for direct intervention and reduction of proteinuria.

Topics: Albumins; Animals; Diabetic Nephropathies; Disease Models, Animal; DNA Methylation; Doxorubicin; Epigenesis, Genetic; Gene Expression; Humans; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Permeability; Phenotype; Podocytes; Promoter Regions, Genetic; Proteinuria; Vimentin

Podocyte effacement and the reformation of foot processes and slit diaphragms can be induced within minutes experimentally. Therefore, it seems likely that the slit diaphragm proteins underlie orchestrated recycling mechanisms under the control of posttranslational modifiers. One of these modifiers, SUMO (small ubiquitin-like modifier), is an ubiquitin-like protein with a 20% corresponding identity to ubiquitin. Modification by SUMOs to proteins on lysine residues can block the ubiquitination of the same site leading to the stabilization of the target protein. Here we found in vitro and in vivo that nephrin is a substrate modified by SUMO proteins thereby increasing its steady-state level and expression at the plasma membrane. A conversion of lysines to arginines at positions 1114 and 1224 of the intracellular tail of murine nephrin led to decreased stability of nephrin, decreased expression at the plasma membrane, and decreased PI3K/AKT signaling. Furthermore, treatment of podocytes with the SUMOylation inhibitor ginkgolic acid led to reduced membrane expression of nephrin. Similarly, the conversion of lysine to arginine at position 1100 of human nephrin caused decreased stability and expression at the plasma membrane. As SUMOylation is a reversible process, our results suggest that SUMOylation participates in the tight orchestration of nephrin turnover at the slit diaphragm.

Topics: Animals; Arginine; Cell Membrane; HEK293 Cells; Humans; Kidney Glomerulus; Lysine; Male; Membrane Proteins; Mice; Podocytes; Proteinuria; Salicylates; Signal Transduction; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Transfection; Ubiquitin-Conjugating Enzymes

It is not clear how the podocyte damage manifests in different glomerulopathies. This study evaluated the podocyte-associated mRNA profiles in renal tissue and urine of patients with proliferative (PGs) or non-proliferative (NPGs) glomerulopathies.. Messenger RNA levels of nephrin, podocin, podocalyxin, synaptopodin, and alpha-actinin-4 were measured in the kidney tissue and urinary cells by real-time polymerase chain reaction. Podocyte-associated mRNAs were correlated with proteinuria and renal function, and the effect of immunosuppressive treatment of PGs and NPGs on urine mRNAs was assessed up to one year of follow up.. Podocyte-associated mRNAs were expressed consistently less in kidney tissue from patients with NPGs, and urinary podocyte mRNA levels were significantly higher in the PG group. After six months of immunosuppressive therapy, patients with PGs showed a significant reduction in the expression of podocin, podocalyxin, and alpha-actinin-4 compared with baseline (p<0.001). In the NPG group, alpha-actinin-4 levels decreased (p=0.008), and there was also a trend toward reduced podocalyxin mRNA (p=0.08). Urine podocyte-associated mRNAs correlated with the level of proteinuria at baseline and at six months, and there was a trend toward an inverse correlation between urinary mRNAs and kidney function at one year of follow up.. Podocyte-associated mRNAs were inhibited in kidney tissue concomitantly with their increase in urine in these patients with glomerulopathies. Different profiles of mRNA expression were seen, pointing to a higher degree of intra-renal podocytopenia in the NPGs and of podocyturia in the PGs. The immunosuppressive therapy effectively reduced the urinary levels of podocyte-associated mRNAs.

Topics: Actinin; Adult; Aged; Case-Control Studies; Cross-Sectional Studies; Female; Gene Expression Profiling; Genetic Markers; Glomerulonephritis; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Microfilament Proteins; Middle Aged; Podocytes; Predictive Value of Tests; Prospective Studies; Proteinuria; RNA, Messenger; Sialoglycoproteins; Time Factors; Treatment Outcome; Urinalysis; Young Adult

We investigated whether aliskiren, a direct renin inhibitor, provided protection in a model of diabetic nephropathy in mice and compared its protective effects to valsartan, an angiotensin II type 1 receptor blocker.. Hyperglycemia was induced with streptozotocin (STZ, 40 mg/kg/day × 5 days) injection in DBA/2J mice fed on a high fat diet. Mice were treated with either aliskiren (25 mg/kg/day) or valsartan (8 mg/kg/day) for 6 weeks.. Aliskiren and/or valsartan treatment significantly attenuated albuminuria, urinary nephrin excretion and glomerulosclerosis. Aliskiren and/or valsartan prevented reduction of podocin and WT1 protein abundance in diabetic mice. Aliskiren and/or valsartan significantly prevented increased expression of profibrotic growth factors (TGFβ, CTGF and PAI-1), proinflammatory cytokines (MCP-1, TNFα and IL-1β), endoplasmic reticulum (ER) stress markers (CHOP and XBP-1) and lipid accumulation in the kidney of diabetic animals. Aliskiren showed similar efficacy compared to valsartan therapy and dual treatment in some aspects has synergistic protective effects.. Our study indicates that aliskiren and/or valsartan protects against diabetic kidney disease through multiple mechanisms, including decreasing podocyte injury, activation of profibrotic growth factors and proinflammatory cytokines, ER stress and accumulation of lipids.

Topics: Albumins; Amides; Animals; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Endoplasmic Reticulum Stress; Fumarates; Inflammation; Lipid Metabolism; Male; Membrane Proteins; Mesangial Cells; Mice, Inbred DBA; Podocytes; Protective Agents; Proteinuria; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled; Renin-Angiotensin System; Tetrazoles; Valine; Valsartan

To investigate the effects of artemisinin against proteinuria and glomerular filtration barrier damage in rats with adriamycin-induced nephropathy, and the potential mechanism underpinned the action.. Forty adriamycin rats were randomly divided into two groups with the ratio of 1 : 3; the small-number group served as control group (n = 10), and the rats in the large-number group were treated with adriamycin to induce nephropathy; then they were further randomly assigned into 3 subgroups: benazepril group (n = 10), artemisinin group (n = 10), and adriamycin group (n = 10). The benazepril group and artemisinin group were treated with benazepril suspl (5.0 mg/kg daily) and artemisinin suspl (150 mg/kg daily) respectively after being modeled; those in the control group and adriamycin group were intragastrically administered an equivalent volume of distilled water every day. The treatment after model establishment lasted for a total of 4 weeks. The 24 h uric protein, blood biochemicals, renal pathological changes, renal ultrastrutural changes, Nephrin and Podocin proteins and gene expressions were measured by Coomassie brilliant blue assay, completely automatic biochemical analyzer, light microscope, electron microscopy, Western blot and reverse transcription polymerase chain reaction, respectively.. The rats in adriamycin group showed a significant increase in 24 h uric protein excretion, serum total cholesterol (TC), triglyceride (TG), blood urea nitrogen (BUN), serum creatinine (Scr) and decrease in albumin (Alb) (P < 0.05 or P < 0.01). Compared with adriamycin group, artemisinin could reduce uric protein excretion, decrease the serum TC, TG elevation, increase the serum Alb level, up-regulate the expressions of Nephrin and Podocin (P < 0.05 or P < 0.01), but no statistical significance effects on the levels of BUN, Scr in artemisinin group (P > 0.05). The renal pathological and ultrastrutural observation indicate that artemisinin could attenuate the severity of foot process effacement and fusion in the nephropathic rats.. Artemisinin might have an effect on the nephropathy in rats caused by adriamycin, which may be at least partly correlated with attenu- ation of the severity of foot process effacement and fusion, up-regulation of the expressions of Nephrin and Podocin in the glomeruli in the rats.

Topics: Animals; Artemisinins; Doxorubicin; Humans; Intracellular Signaling Peptides and Proteins; Kidney Diseases; Male; Membrane Proteins; Proteinuria; Rats

To investigate the effects of prednisone on the expressions of FAK and Pyk2 in the kidneys of rats with adriamycin-induced nephritis.. Thirty SD rats were randomized into normal control group, adriamycin-induced nephritic model group, and prednisone treatment group (n=10). Prednisone was administered at 10 mg/kg once daily in nephritic rats starting since the 7th day after adriamycin injection. Twenty-four-hour proteinuria was measured in the rats at different time points, and renal tissue histology was examined using transmission electron microscope. The expression levels of Pyk2, FAK and nephrin mRNA in the renal tissue were detected tested by RT-PCR, and the protein expressions of FAK, Pyk2, phosphorylated Pyk2 and phosphorylated FAK-Tyr397 were detected by Western blotting; immunohistochemistry was used for detecting nephrin protein expression in the kidney.. Compared with the normal control group, the rats with adriamycin-induced nephritis showed significantly increased proteinuria (P<0.01), which was obviously lowered by prednisone treatment (P<0.01). Transmission electron microscopy revealed extensive fusion of the foot processes of the podocytes in the model group. Prednisone treatment promoted nephrin expression in the kidney (P<0.05). Compared with the control group, the model and prednisone treated groups showed significantly lowered nephrin mRNA expression (P<0.01) but increased FAK mRNA expression (P<0.01), but prednisone-treated group had a higher nephrin mRNA expression than the model group (P<0.05). The model group exhibited significantly increased expressions of FAK total and phosphorylated proteins, P-FAK/FAK, and P-Pyk2/Pyk2 (P<0.01), which were all lowered in the treatment group (P<0.01). Correlation analysis suggested that the expressions of FAK mRNA, FAK, pFAK, Pyk2 mRNA and pPyk2/Pyk2 were positively correlated with proteinuria (r=0.819, 0.750, 0.838, 0.762, 0.934, respectively, P<0.01).. Adriamycin increases phosphorylated FAK and Pyk2 expressions to mediate kidney injury in rats. Prednisone inhibits Pyk2 and FAK activation, decreases proteinuria, and alleviates podocyte lesions to protect the glomerular filtration barrier.

Topics: Animals; Doxorubicin; Focal Adhesion Kinase 2; Kidney; Kidney Glomerulus; Membrane Proteins; Nephritis; Podocytes; Prednisone; Proteinuria; Rats; Rats, Sprague-Dawley; RNA, Messenger

Experiments determined whether the combination of endothelin A (ETA) receptor antagonist [ABT-627, atrasentan; (2R,3R,4S)-4-(1,3-benzodioxol-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid] and a thiazide diuretic (chlorthalidone) would be more effective at lowering blood pressure and reducing renal injury in a rodent model of metabolic syndrome compared with either treatment alone. Male Dahl salt-sensitive rats were fed a high-fat (36% fat), high-salt (4% NaCl) diet for 4 weeks. Separate groups of rats were then treated with vehicle (control), ABT-627 (ABT; 5 mg/kg per day, in drinking water), chlorthalidone (CLTD; 5 mg/kg per day, in drinking water), or both ABT plus CLTD. Mean arterial pressure (MAP) was recorded continuously by telemetry. After 4 weeks, both ABT and CLTD severely attenuated the development of hypertension, whereas the combination further reduced MAP compared with ABT alone. All treatments prevented proteinuria. CLTD and ABT plus CLTD significantly reduced nephrin (a podocyte injury marker) and kidney injury molecule-1 (a tubulointerstitial injury marker) excretion. ABT, with or without CLTD, significantly reduced plasma 8-oxo-2'-deoxyguanosine, a measure of DNA oxidation, whereas CLTD alone had no effect. All treatments suppressed the number of ED1(+) cells (macrophages) in the kidney. Plasma tumor necrosis factor receptors 1 and 2 were reduced only in the combined ABT and CLTD group. These results suggest that ABT and CLTD have antihypertensive and renal-protective effects in a model of metabolic syndrome that are maximally effective when both drugs are administered together. The findings support the hypothesis that combined ETA antagonist and diuretic treatment may provide therapeutic benefit for individuals with metabolic syndrome consuming a Western diet.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antihypertensive Agents; Arterial Pressure; Atrasentan; Cell Adhesion Molecules; Chlorthalidone; Deoxyguanosine; Disease Models, Animal; Diuretics; Drug Combinations; Endothelin A Receptor Antagonists; Endothelins; Hypertension; Inflammation; Kidney Diseases; Male; Membrane Proteins; Metabolic Syndrome; Oxidative Stress; Proteinuria; Pyrrolidines; Rats; Rats, Inbred Dahl; Rats, Sprague-Dawley; Receptor, Endothelin A; Sodium Chloride, Dietary

MAGUK Inverted 2 (MAGI-2) is a PTEN-interacting scaffold protein implicated in cancer on the basis of rare, recurrent genomic translocations and deletions in various tumors. In the renal glomerulus, MAGI-2 is exclusively expressed in podocytes, specialized cells forming part of the glomerular filter, where it interacts with the slit diaphragm protein nephrin. To further explore MAGI-2 function, we generated Magi-2-KO mice through homologous recombination by targeting an exon common to all three alternative splice variants. Magi-2 null mice presented with progressive proteinuria as early as 2 wk postnatally, which coincided with loss of nephrin expression in the glomeruli. Magi-2-null kidneys revealed diffuse podocyte foot process effacement and focal podocyte hypertrophy by 3 wk of age, as well as progressive podocyte loss. By 5.5 wk, coinciding with a near-complete loss of podocytes, Magi-2-null mice developed diffuse glomerular extracapillary epithelial cell proliferations, and died of renal failure by 3 mo of age. As confirmed by immunohistochemical analysis, the proliferative cell populations in glomerular lesions were exclusively composed of activated parietal epithelial cells (PECs). Our results reveal that MAGI-2 is required for the integrity of the kidney filter and podocyte survival. Moreover, we demonstrate that PECs can be activated to form glomerular lesions resembling a noninflammatory glomerulopathy with extensive extracapillary proliferation, sometimes resembling crescents, following rapid and severe podocyte loss.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Proliferation; Epithelial Cells; Guanylate Kinases; Immunohistochemistry; Inflammation; Kidney; Kidney Glomerulus; Membrane Proteins; Mice, Knockout; Neoplasm Proteins; Nerve Tissue Proteins; Podocytes; Proteinuria; Survival Analysis; Up-Regulation

The precise pathogenic mechanism and role of angiotensin II (Ang II) action in the development of proteinuria in minimal change nephrotic syndrome (MCNS) is uncertain.. The glomerular expressions of the slit diaphragm (SD) molecules nephrin, podocin and NEPH1 in rat puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, were analyzed. The effects of Ang II receptor blockade (ARB) (irbesartan 15 mg/kg body weight/day) on proteinuria and on the expression of the SD molecules were analyzed.. mRNA expressions of nephrin, podocin and NEPH1 were decreased to an undetectable level at 1 h. The staining of these SD molecules shifted to a discontinuous pattern, and their intensity was reduced. NEPH1 staining was reduced to an undetectable level on day 10. ARB treatment ameliorated the peak value of proteinuria (237.6 ± 97.0 vs. 359.0 ± 63.3 mg/day, p < 0.05), and prevented the decrease in the mRNA expression of the SD molecules (nephrin 66.96 %, podocin 60.40 %, NEPH1 77.87 % of normal level). The immunofluorescence staining of NEPH1 was restored by ARB. ARB treatment enhanced the expression of NEPH1 of normal rats.. Dysfunction of the SD molecules including NEPH1 is a crucial initiation event of PAN nephropathy. ARB treatment ameliorates proteinuria in PAN nephropathy by inhibiting the reduction of NEPH1 and nephrin. Ang II action regulates the expression of NEPH1 and nephrin in not only the pathological but also physiological state.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Biphenyl Compounds; Disease Models, Animal; Disease Progression; Female; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; Irbesartan; Kidney Glomerulus; Membrane Proteins; Nephrosis, Lipoid; Proteinuria; Puromycin Aminonucleoside; Rats, Wistar; Receptor, Angiotensin, Type 1; Tetrazoles; Time Factors

Although angiotensin (Ang) II blockade has become a standard antifibrotic therapy in kidney disease, the therapeutic efficacy of Ang II blockade is yet to be optimized. Considering the prognostic impact of proteinuria reduction, we hypothesized that titration of Ang II blockade for optimal anti-proteinuric effect would improve renoprotection. One day after induction of Thy 1.1 glomeruonephritis, rats were treated with increasing doses of the Ang II receptor blocker valsartan in drinking water. Six days after disease induction, the therapeutic effect on proteinuria, podocyte injury and glomerular fibrosis was evaluated. Increasing doses of valsartan resulted in increasing reduction of proteinuria. The maximally effective dose of valsartan was determined to be 1000 mg/l, which reduced proteinuria by 80% and maximally reduced glomerular matrix expansion, fibronectin, collagen I and collagen III staining and glomerular mRNAs for TGFß1, PAI-1, FN and collagen I. Notably, valsartan given at this dose prevented podocyte dysfunction by preserving expression of podocin and nephrin and the counter-regulating molecule B7-1 that is involved in podocyte injury. These results support the hypothesis that higher doses of valsartan are required to optimize proteinuria reduction and glomerulosclerosis amelioration. Further, the optimal dose of valsartan also provides an additional therapeutic effect by preventing podocyte dysfunction.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Dose-Response Relationship, Drug; Fibrosis; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Function Tests; Male; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Tetrazoles; Valine; Valsartan

Collapsing focal segmental glomerulosclerosis (cFSGS) is a progressive kidney disease characterized by glomerular collapse with epithelial hyperplasia. Here we used a transgenic mouse model of cFSGS with immunotoxin-induced podocyte-specific injury to determine the role for Notch signaling in its pathogenesis. The mice exhibited progressive loss of podocytes and severe proteinuria concomitant with histological features of cFSGS. Hyperplastic epithelium was negative for genetic podocyte tags, but positive for the parietal epithelial cell marker claudin-1, and expressed Notch1, Jagged1, and Hes1 mRNA and protein. Enhanced Notch mRNA expression induced by transforming growth factor-β1 in cultured parietal epithelial cells was associated with mesenchymal markers (α-smooth muscle actin, vimentin, and Snail1). Notch inhibition in vitro suppressed these phenotypic transcripts and Notch-dependent cell migration. Moreover, Notch inhibition in vivo significantly decreased parietal epithelial cell lesions but worsened proteinuria and histopathology in our cFSGS model. Thus, aberrant Notch1-mediated parietal epithelial cell migration with phenotypic changes appears to underlie the pathogenesis of cFSGS. Parietal epithelial cell hyperplasia may also represent an adaptive response to compensate for a disrupted filtration barrier with progressive podocyte loss.

Topics: Amyloid Precursor Protein Secretases; Animals; Antibodies, Monoclonal; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Calcium-Binding Proteins; Cell Line; Cell Movement; Cell Proliferation; Claudin-1; Dibenzazepines; Disease Models, Animal; Enzyme Inhibitors; Epithelial Cells; Exotoxins; Glomerulosclerosis, Focal Segmental; Homeodomain Proteins; Humans; Hyperplasia; Integrases; Intercellular Signaling Peptides and Proteins; Interleukin-2 Receptor alpha Subunit; Jagged-1 Protein; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Transgenic; Podocytes; Proteinuria; Receptor, Notch1; RNA, Untranslated; Serrate-Jagged Proteins; Time Factors; Transcription Factor HES-1; Transforming Growth Factor beta1

Minimal-change nephrotic syndrome (MCNS) is a kidney disease defined by selective proteinuria and hypoalbuminemia occurring in the absence of cellular glomerular infiltrates or immunoglobulin deposits. Recent observations suggest that nuclear factor κB (NF-κB) of podocyte is strongly associated with the development of proteinuria in MCNS. Dehydroxymethylepoxyquinomicin (DHMEQ) is a novel NF-κB inhibitor that potently inhibits DNA-binding activity of NF-κB, resulting in several therapeutic effects in various pathological conditions. We conducted this study to ask whether DHMEQ may ameliorate the nephrosis in mice induced by puromycin aminonucleoside (PAN), which is considered to be an animal model for MCNS.. Pretreatment with DHMEQ alleviated the proteinuria and reversed the serum abnormalities in mice nephrosis induced by 450 mg/kg of PAN. Increased serum interleukin-6 level in PAN-induced nephrosis was also completely suppressed by DHMEQ. Electron microscopic analyses of glo-meruli indicated that DHMEQ can inhibit the podocyte foot process effacement via blocking the translocation of podocyte NF-κB from cytoplasm to nucleus.. These results suggest that DHMEQ can be a potential therapeutic agent for MCNS.

Topics: Adenosine Deaminase; Albuminuria; Animals; Benzamides; Blood Proteins; Cholesterol; Cyclohexanones; Glycerolphosphate Dehydrogenase; Interleukin-6; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Nephrosis; NF-kappa B; Proteinuria; Puromycin Aminonucleoside; Rats; Serum Albumin

The protective effects of Rho kinase inhibitor fasudil against renal diseases have recently been reported. We compared the therapeutic effects of fasudil on the spontaneously hypercholesterolemic (SHC) rat, a model of chronic kidney disease (CKD) with proteinuria, with those of the angiotensin receptor blocker olmesartan (OL) by paying attention to the proteinuria and the macrophage phenotype. SHC rats were allocated to six treatment groups: a vehicle (Ve) group, a low-dose fasudil (FL) group, a high-dose fasudil (FH) group, an OL group, a combination of low-dose fasudil and OL (CL) group, and a combination of high-dose fasudil and OL (CH) group. Sprague-Dawley rats treated with vehicle served as a control (n = 7/each). The rats were treated for 24 wk. Compared with the Ve group, proteinuria was significantly decreased in the FH, OL, and CL groups, and it completely disappeared in the CH group. Glomerular stainings of nephrin and F-actin were focally impaired in the Ve group but were restored in the CH group. Western blotting showed that the CH group had significantly increased renal nephrin expression compared with the Ve group. Interstitial infiltration of macrophages was significantly increased in the Ve group, which was significantly attenuated in all treatment groups. The ratio of CD206 (M2 macrophage marker) to CD68 mRNA was significantly greater in the CH group than in the Ve group. These results indicate that fasudil with OL reduces proteinuria by protecting podocyte integrity and alters the interstitial macrophage density/phenotype, thereby exerting renoprotective effects against CKD.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Angiotensin II Type 1 Receptor Blockers; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Enzyme Inhibitors; Hypercholesterolemia; Imidazoles; Kidney Glomerulus; Lectins, C-Type; Macrophages; Male; Mannose Receptor; Mannose-Binding Lectins; Membrane Proteins; Microscopy, Electron; Phenotype; Proteinuria; Rats; Receptors, Cell Surface; Renal Insufficiency, Chronic; rho-Associated Kinases; RNA, Messenger; Tetrazoles

Semaphorin-3A (Sema3a), a guidance protein secreted by podocytes, is essential for normal kidney patterning and glomerular filtration barrier development. Here, we report that podocyte-specific Sema3a gain-of-function in adult mice leads to proteinuric glomerular disease involving the three layers of the glomerular filtration barrier. Reversibility of the glomerular phenotype upon removal of the transgene induction provided proof-of-principle of the cause-and-effect relationship between podocyte Sema3a excess and glomerular disease. Mechanistically, excess Sema3a induces dysregulation of nephrin, matrix metalloproteinase 9, and αvβ3 integrin in vivo. Sema3a cell-autonomously disrupts podocyte shape. We identified a novel direct interaction between the Sema3a signaling receptor plexinA1 and nephrin, linking extracellular Sema3a signals to the slit-diaphragm signaling complex. We conclude that Sema3a functions as an extracellular negative regulator of the structure and function of the glomerular filtration barrier in the adult kidney. Our findings demonstrate a crosstalk between Sema3a and nephrin signaling pathways that is functionally relevant both in vivo and in vitro.

Topics: Animals; Cell Shape; Down-Regulation; Glomerular Basement Membrane; Integrin alphaVbeta3; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Mice; Nerve Tissue Proteins; Podocytes; Protein Binding; Proteinuria; Receptors, Cell Surface; Semaphorin-3A; Signal Transduction

Prior study shows that maternal protein-restricted (LP) 16-wk-old offspring have pronounced reduction of nephron number and arterial hypertension associated with unchanged glomerular filtration rate, besides enhanced glomerular area, which may be related to glomerular hyperfiltration/overflow and which accounts for the glomerular filtration barrier breakdown and early glomerulosclerosis. In the current study, LP rats showed heavy proteinuria associated with podocyte simplification and foot process effacement. TGF-β1 glomerular expression was significantly enhanced in LP. Isolated LP glomeruli show a reduced level of miR-200a, miR-141, miR-429 and ZEB2 mRNA and upregulated collagen 1α1/2 mRNA expression. By western blot analyzes of whole kidney tissue, we found significant reduction of both podocin and nephrin and enhanced expression of mesenchymal protein markers such as desmin, collagen type I and fibronectin. From our present knowledge, these are the first data showing renal miRNA modulation in the protein restriction model of fetal programming. The fetal-programmed adult offspring showed pronounced structural glomerular disorders with an accentuated and advanced stage of fibrosis, which led us to state that the glomerular miR-200 family would be downregulated by TGF-β1 action inducing ZEB 2 expression that may subsequently cause glomeruli epithelial-to-mesenchymal transition.

Topics: Animals; Collagen; Desmin; Diet, Protein-Restricted; Epithelial-Mesenchymal Transition; Female; Fibronectins; Gene Expression Regulation, Developmental; Homeodomain Proteins; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; MicroRNAs; Podocytes; Pregnancy; Prenatal Nutritional Physiological Phenomena; Proteinuria; Rats; Transforming Growth Factor beta1

Tenc1 (also known as tensin2) is an integrin-associated focal adhesion molecule that is broadly expressed in mouse tissues including the liver, muscle, heart and kidney. A mouse strain carrying mutated Tenc1, the ICR-derived glomerulonephritis (ICGN) strain, develops severe nephrotic syndrome.. To elucidate the function of Tenc1 in the kidney, Tenc1(ICGN) was introduced into 2 genetic backgrounds, i.e. DBA/2J (D2) and C57BL/6J (B6), strains that are respectively susceptible and resistant to chronic kidney disease.. Biochemical and histological analysis revealed that homozygous Tenc1(ICGN) mice develop nephrotic syndrome on the D2 background (D2GN) but not on the B6 background (B6GN). Initially, abnormal assembly and maturation of glomerular basement membrane (GBM) were observed, and subsequently effacement of podocyte foot processes was noted in the kidneys of D2GN but not B6GN mice. These defects are likely to be involved in the integrin signaling pathway.. This study suggests that Tenc1 contributes to the maintenance of GBM structures and that the genetic background influences the severity of nephrotic syndrome.

Topics: Adaptor Proteins, Signal Transducing; Animals; Blotting, Western; Collagen Type IV; Cytoskeletal Proteins; Glomerular Basement Membrane; Glomerulonephritis; Integrin alpha3beta1; Kidney Glomerulus; Laminin; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Inbred ICR; Mice, Knockout; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Nephrotic Syndrome; Phosphoprotein Phosphatases; Podocytes; Proteinuria; Species Specificity; Tensins

The kidney's vital filtration function depends on the structural integrity of the glomerulus, the proximal portion of the nephron. Within the glomerulus, the architecturally complex podocyte forms the final cellular barrier to filtration. Injury to the podocyte results in a morphological change called foot process effacement, which is a ubiquitous feature of proteinuric diseases. The exact mechanism underlying foot process effacement is not known, but recently it has been proposed that this change might reflect activation of the Rac1 GTPase. To test this hypothesis, we generated a podocyte-specific, inducible transgenic mouse line that expressed constitutively active Rac1. When the Rac1 transgene was induced, we observed a rapid onset of proteinuria with focal foot process effacement. Using superresolution imaging, we verified that the induced transgene was expressed in damaged podocytes with altered foot process morphology. This work sheds new light on the complex balance of Rho GTPase signaling that is required for proper regulation of the podocyte cytoskeleton.

Topics: Actin Cytoskeleton; Animals; Cells, Cultured; Enzyme Activation; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Transgenic; Neuropeptides; Podocytes; Proteasome Endopeptidase Complex; Proteinuria; Proteolysis; rac1 GTP-Binding Protein

Recent studies have shown a beneficial effect of rapamycin in passive and active Heymann Nephritis (HN). However, the mechanisms underlying this beneficial effect have not been elucidated.. Passive Heymann Nephritis (PHN) was induced by a single intravenous infusion of anti-Fx1 in 12 Sprague-Dawley male rats. One week later, six of these rats were commenced on daily treatment with subcutaneous rapamycin 0.5 mgr/kg (PHN-Rapa). The remaining six rats were used as the proteinuric control group (PHN) while six more rats without PHN were given the rapamycin solvent and served as the healthy control group (HC). All rats were sacrificed at the end of the 7th week.. Rapamycin significantly reduced proteinuria during the autologous phase of PHN. Histological lesions were markedly improved by rapamycin. Immunofluorescence revealed attenuated deposits of autologous alloantibodies in treated rats. Untreated rats showed decreased glomerular content of both nephrin and podocin whereas rapamycin restored their expression.. Rapamycin monotherapy significantly improves proteinuria and histological lesions in experimental membranous nephropathy. This beneficial effect may be mediated by inhibition of the alloimmune response during the autologous phase of PHN and by restoration of the normal expression of the podocyte proteins nephrin and podocin.

Topics: Animals; Disease Models, Animal; Gene Expression Regulation; Glomerulonephritis, Membranous; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Proteinuria; Rats; Sirolimus

We used a rat model to assess the role of nephrin, podocin, and desmin in the pathogenesis of IgA nephropathy (IgAN). A rat IgAN model was established by administration of BSA, CCl(4), and lipopolysaccharide (LPS) and compared with healthy control rats. Urinary protein, urine red blood cells, and biochemical parameters were measured for 12 weeks. Renal morphology and ultrastructure were examined by light and electron microscopy. Immunofluorescence was used to assess IgA deposition in the glomeruli and to measure expression of nephrin, podocin, and desmin. Real-time quantitative PCR was used to measure expression of nephrin, podocin, and desmin mRNAs. IgAN rats developed proteinuria at week-6 and this worsened over time. Pathological changes were evident under light microscopy at week-8 and under electron microscopy at week-4. Immunofluorescence analysis showed deposition of IgA in the kidneys of IgAN rats, but not control rats. IgAN rats had increased expression of glomerular podocin, nephrin, and desmin mRNAs and proteins at week-4. The expression of nephrin, podocin and desmin proteins and the expression of podocin and desmin mRNAs preceded the increase in urinary protein. Taken together, our study of a rat model of IgAN indicates that changes in the expression and distribution of nephrin, podocin, and desmin precede and may cause foot process fusion and proteinuria.

Topics: Animals; Desmin; Disease Models, Animal; Disease Progression; Fluorescent Antibody Technique; Glomerulonephritis, IGA; Hematuria; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Microscopy, Electron, Transmission; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

Diabetic nephropathy (DN) is characterized by podocyte damage and increased phosphodiesterase-5 (PDE-5) activity-exacerbating nitric oxide (NO)-cyclic 3',5' guanosine monophosphate (cGMP) pathway dysfunction. It has been shown that PDE-5 inhibition ameliorates DN. The role of podocytes in this mechanism remains unclear. We investigated how selective PDE-5 inhibition influences podocyte damage in streptozotocin (STZ) diabetic rats.. Male Sprague-Dawley rats (250-300 g) were injected with STZ and divided into two groups: (i) STZ control (non-treated, STZ, n=6) and (ii) STZ+vardenafil treatment (10 mg/kg/day, STZ-Vard, n=8). Non-diabetic rats served as negative controls (Control, n=7). Following 8 weeks of treatment, immunohistochemical and molecular analysis of the kidneys were performed.. Diabetic rats had proteinuria, increased renal transforming growth factor (TGF)-β1 expression and podocyte damage when compared with controls. Vardenafil treatment resulted in preserved podocyte cGMP levels, less proteinuria, reduced renal TGF-β1 expression, desmin immunostaining in podocytes and restored both nephrin and podocin mRNA expression. Diabetes led to increased glomerular nitrotyrosine formation and renal neuronal nitric oxide synthase and endothelial nitric oxide synthase mRNA expression, but vardenafil did not influence these parameters.. Our data suggest that a dysfunctional NO-cGMP pathway exacerbates podocyte damage in diabetes. In conclusion, vardenafil treatment preserves podocyte function and reduces glomerular damage, which indicates therapeutic potential in patients with DN.

Topics: Animals; Blotting, Western; Cells, Cultured; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Imidazoles; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Nitric Oxide Synthase Type III; Oxidative Stress; Phosphodiesterase 5 Inhibitors; Piperazines; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfones; Transforming Growth Factor beta1; Triazines; Vardenafil Dihydrochloride

The present study employed a zinc-finger nuclease strategy to create heterozygous knockout (KO) rats for the transforming growth factor-β1 (Tgfb1) gene on the Dahl SS/Jr genetic background (TGF-β1(+/-) Dahl S). Intercrossing TGF-β1(+/-) rats did not produce any homozygous KO rats (66.4% +/-, 33.6% +/+), indicating that the mutation is embryonic lethal. Six-week-old wild-type (WT) littermates and TGF-β1(+/-) Dahl S rats were fed a 0.4% (low salt, LS) or 8% NaCl (high salt, HS) diet for 5 wk. Renal cortical expression of TGF-β1, urinary TGF-β1 excretion, proteinuria, glomerular injury and tubulointerstitial fibrosis, and systolic blood pressure were similar in WT and TGF-β1(+/-) Dahl S rats maintained on the LS diet. The expression and urinary excretion of TGF-β1 increased to a greater extent in WT than in TGF-β1(+/-)Dahl S rats fed an HS diet for 1 wk. Systolic blood pressure rose by the same extent to 235 ± 2 mmHg in WT and 239 ± 4 mmHg in TGF-β1(+/-) Dahl S rats fed a HS diet for 5 wk. However, urinary protein excretion was significantly lower in TGF-β1(+/-) Dahl S than in the WT animals. The degree of glomerular injury and renal cortical and outer medullary fibrosis was markedly less in TGF-β1(+/-) than in WT rats. These findings suggest that the loss of one copy of the TGF-β1 gene blunts the increase in renal TGF-β1 protein expression and slows the progression of proteinuria, glomerulosclerosis, and renal interstitial fibrosis in Dahl S rats fed an HS diet independently of changes in blood pressure.

Topics: Animals; Base Sequence; Fibrosis; Gene Expression Regulation; Gene Knockout Techniques; Hypertension; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Membrane Proteins; Molecular Sequence Data; Proteinuria; Rats; Rats, Inbred Dahl; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3

Mesenchymal stem cells (MSC) attenuate albuminuria and preserve normal renal histology in diabetic mice. However, the effects of MSC on glomerular podocyte injury remain uncertain. The aim of this study was to evaluate the effects of MSC on podocyte injury in streptozotocin (STZ)-induced diabetic rats. Thirty days after diabetes induction by STZ injection (65 mg/kg, intraperitoneally) in Sprague-Dawley rats, the diabetic rats received medium or 2 × 10(6) enhanced green fluorescent protein-labeled MSC via the renal artery. In vivo tracking of MSC was followed by immunofluorescence analysis. Diabetes-related physical and biochemical parameters were measured on day 60 after the MSC infusion. The expression of podocyte markers (nephrin and podocin), podocyte survival factors (VEGF and BMP-7), and the ultrastructural pathology of podocytes were also assessed. MSC were only detected in the glomeruli from the left kidney receiving MSC infusion. Compared with medium-treated diabetic rats, rats treated with MSC showed a suppressed increase in kidney weight, kidney to body weight index, creatinine clearance rate, and urinary albumin to creatinine ratio; however, the treatment had no effect on blood glucose or body weight levels. Furthermore, the MSC treatment reduced the loss of podocytes, effacement of foot processes, widening of foot processes, thickening of glomerular basal membrane (GBM), and loss of glomerular nephrin and podocin. Most important, MSC-injected kidneys expressed higher levels of BMP-7 but not of VEGF. Our results clearly demonstrated that intra-arterial administration of MSC prevented the development of albuminuria as well as any damage to or loss of podocytes, though there was no improvement in blood sugar levels. The protective effects of MSC may be mediated in part by increasing BMP-7 secretion.

Topics: Animals; Biomarkers; Blood Glucose; Bone Morphogenetic Protein 7; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gene Expression; Genes, Reporter; Green Fluorescent Proteins; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Streptozocin; Vascular Endothelial Growth Factor A

Glomerular podocyte molecules are involved in the pathogenesis of congenital nephrotic syndrome. However, their role in primary nephrotic syndrome is not clear. This study investigated the expression of nephrin, podocin and synaptopodin in primary nephrotic syndrome.. Eighty-seven patients with primary nephrotic syndrome including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN) and membranoproliferative glomerulonephritis Type I (MPGN) were included in the study. Glomerular expression of nephrin, podocin and synaptopodin was studied in renal biopsies by immunofluorescence and immunohistochemistry. Correlation of expression with clinical and biochemical parameters was performed.. The pattern of expression for all podocyte proteins in controls was uniform fine granular along the capillary walls towards the visceral epithelial cell aspect. Glomerular expression of nephrin was present in all renal biopsies and was similar to that in controls. Glomerular synaptopodin expression was seen in all MN and MPGN patients, while it was seen in 74 % (17/23) MCD and 93.5 % (29/31) FSGS. Reduced synaptopodin expression showed no correlation with clinical and biochemical factors. Podocin expression was present in 5/23 MCD (22 %), 3/31 FSGS (9.6 %), 13/17 MN (76.4 %) and 13/16 MPGN (81 %) patients. The reduced expression of podocin significantly correlated with the degree of proteinuria (p = 0.032). No correlation with age, gender and serum creatinine level was observed.. Reduction of glomerular podocin expression found in MCD and FSGS is related to the amount of proteinuria. Our findings suggest that alteration in podocyte phenotype may not be a primary event and may reflect the degree of podocyte injury in primary nephrotic syndrome.

Topics: Adolescent; Adult; Creatinine; Glomerulonephritis, Membranoproliferative; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Microfilament Proteins; Middle Aged; Nephrosis, Lipoid; Nephrotic Syndrome; Podocytes; Proteinuria

Nephrin, an important structural and signal molecule of podocyte slit-diaphragm (SD), has been suggested to contribute to the angiotensin II (Ang II)-induced podocyte injury. Caveolin-1 has been demonstrated to play a crucial role in signaling transduction. In the present study, we evaluated the role of caveolin-1 in Ang II-induced nephrin phosphorylation in podocytes. Wistar rats-receiving either Ang II (400 ng/kg/min) or normal saline (via subcutaneous osmotic mini-pumps, control) were administered either vehicle or telmisartan (3 mg/kg/min) for 14 or 28 days. Blood pressure, 24-hour urinary albumin and serum biochemical profile were measured at the end of the experimental period. Renal histomorphology was evaluated through light and electron microscopy. In vitro, cultured murine podocytes were exposed to Ang II (10(-6)M) pretreated with or without losartan (10(-5) M) for variable time periods. Nephrin and caveolin-1 expression and their phosphorylation were analyzed by Western-blotting and immunofluorescence. Caveolar membrane fractions were isolated by sucrose density gradient centrifugation, and then the distribution and interactions between Ang II type 1 receptor (AT1), nephrin, C-terminal Src kinase (Csk) and caveolin-1 were evaluated using Western-blotting and co-immunoprecipitation. Podocyte apoptosis was evaluated by cell nucleus staining with Hoechst-33342. Ang II-receiving rats displayed diminished phosphorylation of nephrin but enhanced glomerular/podocyte injury and proteinuria when compared to control rats. Under control conditions, podocyte displayed expression of caveolin-1 in abundance but only a low level of phospho moiety. Nonetheless, Ang II stimulated caveolin-1 phosphorylation without any change in total protein expression. Nephrin and caveolin-1 were co-localized in caveolae fractions. AT1 receptors and Csk were moved to caveolae fractions and had an interaction with caveolin-1 after the stimulation with Ang II. Transfection of caveolin-1 plasmid (pEGFPC3-cav-1) significantly increased Ang II-induced nephrin dephosphorylation and podocyte apoptosis. Furthermore, knockdown of caveolin-1 expression (using siRNA) inhibited nephrin dephosphorylation and prevented Ang II-induced podocyte apoptosis. These findings indicate that Ang II induces nephrin dephosphorylation and podocyte injury through a caveolin-1-dependent mechanism.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Apoptosis; Benzimidazoles; Blotting, Western; Caveolae; Caveolin 1; CSK Tyrosine-Protein Kinase; Immunoprecipitation; Losartan; Membrane Proteins; Mice; Phosphorylation; Plasmids; Podocytes; Protein-Tyrosine Kinases; Proteinuria; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Signal Transduction; src-Family Kinases; Transfection

Notch signaling in podocytes causes proteinuria and glomerulosclerosis in humans and rodents, but the underlying mechanism remains unknown. Here, we analyzed morphologic, molecular, and cellular events before the onset of proteinuria in newborn transgenic mice that express activated Notch in podocytes. Immunohistochemistry revealed a loss of the slit diaphragm protein nephrin exclusively in podocytes expressing activated Notch. Podocyte-specific deletion of Rbpj, which is essential for canonical Notch signaling, prevented this loss of nephrin. Overexpression of activated Notch decreased cell surface nephrin and increased cytoplasmic nephrin in transfected HEK293T cells; pharmacologic inhibition of dynamin, but not depletion of cholesterol, blocked these effects on nephrin, suggesting that Notch promotes dynamin-dependent, raft-independent endocytosis of nephrin. Supporting an association between Notch signaling and nephrin trafficking, electron microscopy revealed shortened podocyte foot processes and fewer slit diaphragms among the transgenic mice compared with controls. These data suggest that Notch signaling induces endocytosis of nephrin, thereby triggering the onset of proteinuria.

Topics: Animals; Animals, Newborn; beta-Cyclodextrins; Dynamins; Endocytosis; HEK293 Cells; Humans; Membrane Proteins; Mice; Mice, Transgenic; Podocytes; Proteinuria; Receptors, Notch; Signal Transduction

Diabetic glomerulosclerosis is the hardening of the renal glomeruli that can lead to kidney failure. In the early stage of glomerulosclerosis occur renal mesangial expansion and renal filtration dysfunction. Purple corn has been classified as a functional food and is rich in anthocyanins exerting potential disease-preventive activities. The in vitro study using human renal mesangial cells examined that anthocyanin-rich purple corn butanol fraction (PCB) can attenuate high glucose (HG)-promoted mesangial cell proliferation and matrix accumulation.. Cells were cultured for 3 days in media containing 33 mM glucose in the presence of 1-20 μg/mL PCB. In the in vivo animal study, db/db mice were treated with 10 mg/kg anthocyanin-rich polyphenolic extracts of purple corn (PCE) for 8 weeks.. HG enhanced mesangial production of the fibrosis biomarkers of collagen IV and connective tissue growth factor (CTGF), which was markedly attenuated by adding PCB. Such mesangial fibrosis entailed interleukin-8 activation via eliciting Tyk2-STAT signaling pathway. PCB dampened HG-promoted mesangial hyperplasia that appeared to be attributed to increased expression of platelet-derived growth factor. The 8-week administration of PCE lowered plasma glucose level of db/db mice and ameliorated severe albuminuria. Moreover, PCE lessened collagen fiber accumulation in kidney glomeruli and CTGF expression via retarding TGF-β signaling. Protein expressions of nephrin and podocin, key proteins for filtration barrier function of the glomerular capillary wall, were repressed by treating mice with PCE.. Purple corn may be a potent therapeutic agent for the treatment for diabetes-associated glomerulosclerosis accompanying proteinuria and kidney filtration dysfunction.

Topics: Albuminuria; Animals; Anthocyanins; Biomarkers; Blood Glucose; Cell Proliferation; Collagen Type IV; Connective Tissue Growth Factor; Diabetic Nephropathies; Fibrosis; Humans; Interleukin-8; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mesangial Cells; Mice; Plant Extracts; Platelet-Derived Growth Factor; Proteinuria; Signal Transduction; STAT Transcription Factors; Transforming Growth Factor beta; TYK2 Kinase; Zea mays

Varying degrees of metabolic abnormalities mediated by chronic inflammation are implicated in the chronic glomerular injuries associated with obesity. Interleukin (IL)-10, a pleiotropic cytokine, exerts anti-inflammatory effects in numerous biological settings. In the present study, we explored the biological benefits of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against the pathological renal characteristics observed in Zucker fatty rats (ZFRs). We injected an AAV vector, encoding rat IL-10 or enhanced green fluorescent protein (GFP) into male ZFRs at 5 weeks of age. Subsequently, the renal pathophysiological changes were analyzed. Persistent IL-10 expression significantly reduced the urinary protein excretion of ZFRs compared with GFP expression (47.1±11.6 mg per mg·creatinine versus 88.8±30.0 mg per mg·creatinine, P<0.01). The serum levels of IL-10 negatively correlated with the urinary protein in AAV-treated rats (r=-0.78, P<0.01). Renal hypertrophy, increased widths in the glomerular basement membrane, and the lack of uniformity and regularity of the foot process of the visceral glomerular epithelial cells of ZFRs were significantly blunted by IL-10 expression. IL-10 also abrogated the downregulation of glomerular nephrin observed in ZFRs treated with the GFP vector. Our findings provide insights into the potential benefit of the anti-inflammatory effects of IL-10 on the overall management of glomerulopathy induced by the metabolic disorders associated with obesity.

Topics: Animals; Dependovirus; Genetic Vectors; Interleukin-10; Kidney; Kidney Glomerulus; Male; Membrane Proteins; Obesity; Proteinuria; Rats; Rats, Zucker

Previous studies have shown that renin-angiotensin (Ang) system vaccines may be effective for the treatment of hypertension, but their efficacy for the prevention of renal disease is unclear. The aim of this study was to compare the effects of an Ang II type 1 (AT1) receptor vaccine with an Ang II receptor blocker (ARB) and a vasodilator on blood pressure (BP) and renal injury in the L-NAME nephropathy model. Male spontaneously hypertensive rats (SHRs) were divided into six groups and treated transiently with three injections of vehicle or AT1 receptor vaccine (0.1 mg) at age 4, 6 and 8 weeks, or continuously with candesartan cilexetil (0.1 mg kg(-1) per day) or hydralazine hydrochloride (5 mg kg(-1) per day), then administered NG-nitro-L-arginine methyl ester (L-NAME) from age 18 to 21 weeks to induce renal injury. Vaccination against the AT1 receptor caused a significant increase in AT1 receptor titers, and a sustained decrease in BP. L-NAME treatment resulted in a marked increase in proteinuria in the control groups, which was completely suppressed in the AT1 vaccine-treated group, and glomerular injury scores were also significantly decreased. Real-time RT-PCR and immunofluorescence studies revealed increased renin mRNA, and increased glomerular expression of nephrin. Comparable results were seen in rats treated continuously with the ARB candesartan, but not with hydralazine. These results suggest that transient AT1 vaccination is as effective as continuous treatment with ARB, not only for the attenuation of hypertension, but also for the prevention of L-NAME-induced nephropathy in SHR.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Biphenyl Compounds; Enzyme Inhibitors; Hydralazine; Kidney Diseases; Kidney Glomerulus; Male; Membrane Proteins; NG-Nitroarginine Methyl Ester; Proteinuria; Rats; Rats, Inbred SHR; Receptor, Angiotensin, Type 1; Tetrazoles; Vaccines

The mechanism of proteinuria in many common kidney diseases involves glomerular hemodynamic effects and local expression of angiogenic, fibrogenic, and vasoactive factors. Transforming growth factor (TGF)-β has been associated with many diseases involving proteinuria and renal fibrosis. TGF-β has been shown to induce podocyte dedifferentiation in vitro, but its in vivo effects on the glomerular filtration barrier are not well described. In this study, we used an adenovirus vector to transfer active TGF-β1 to the glomeruli of rat kidneys. Transient TGF-β1 overexpression induced significant proteinuria, podocyte foot process effacement, nephrin down-regulation, and nephrinuria. The expression of synaptopodin was also significantly down-regulated by TGF-β1. Increased glomerular expression of Snail, suggestive of an in vivo dedifferentiation process, was associated with a loss of podocyte epithelial markers. The expression of angiopoietin-1 and angiopoietin-2 was significantly increased in TGF-β1-transfected glomeruli, and TGF-β1 increased the expression of the angiopoietin receptor, Tie2, in podocyte cell culture. TGF-β1 down-regulated nephrin and synaptopodin expression in podocytes in cell culture; this effect was reversed by the blockade of both angiopoietin and Tie2 activities. These findings suggest that locally produced TGF-β1 can cause podocyte dedifferentiation marked by a loss of synaptopodin, nephrin, and foot process effacement, partly regulated by angiopoietins. This process represents a novel pathway that may explain proteinuria in a variety of common renal diseases.

Topics: Actins; Adenoviridae; Angiopoietin-1; Angiopoietin-2; Animals; Cell Dedifferentiation; Cells, Cultured; Down-Regulation; Female; Gene Transfer Techniques; Genetic Vectors; Glomerular Filtration Barrier; Kidney Glomerulus; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Snail Family Transcription Factors; Synaptophysin; Transcription Factors; Transforming Growth Factor beta1

Proteinuria is an important risk factor for the progression and prognosis of chronic kidney disease. Bufalin, a cardiotonic steroid, has been shown to posses a variety of biological activities including cardiotonic, anaesthetic and antineoplastic activities, and regulate the immune response. This study investigated the effects of bufalin against proteinuria and glomerular filtration barrier damage in rats with adriamycin (ADR)-induced nephropathy. We compared the blood and urine biochemical indices and the histologic and ultrastructure of the glomerulus in ADR rats with and without the intervention of bufalin or prednisone. The transcription, expression and distribution of the podocyte-associated molecules were compared utilising RT-PCR, western blotting and immunohistochemical staining. We found that bufalin reduced the urinary protein excretion and optimised the lipidaemia of the ADR rats. Bufalin alleviated the removal of podocyte foot processes and attenuated the changes in nephrin, podocin and integrin-linked kinase (ILK) stainings in the glomerulus of the ADR rats. Bufalin notably decreased the expression of nephrin and ILK but inhibited the down-regulation of podocin in protein levels on the renal cortex of the ADR rats. Additionally, bufalin inhibited the up-regulation of podocin and ILK in mRNA levels but did not affect nephrin mRNA levels. These results suggest that bufalin could alleviate ADR-induced proteinuria by protecting the glomerular filtration barrier and may be a novel potential therapeutic agent for proteinuria-associated kidney disease.

Topics: Animals; Bufanolides; Cardiotonic Agents; Doxorubicin; Gene Expression Regulation; Glomerular Filtration Barrier; Intracellular Signaling Peptides and Proteins; Kidney Diseases; Male; Membrane Proteins; Podocytes; Prednisone; Protein Serine-Threonine Kinases; Proteinuria; Rats; Rats, Sprague-Dawley

Multiple transforming growth factor (TGF)-β-induced fibrogenic signals have been described in vitro. To evaluate mechanisms in vivo, we used an adriamycin nephropathy model in 129x1/Svj mice that display massive proteinuria by days 5 to 7 and pathological findings similar to human focal segmental glomerulosclerosis by day 14. TGF-β mRNA expression increased after day 7 along with nuclear translocation of the TGF-β receptor-specific transcription factor Smad3. Inhibiting TGF-β prevented both pathological changes and type-I collagen and fibronectin mRNA expression, but proteinuria persisted. Renal Akt was phosphorylated in adriamycin-treated mice, suggesting PI3-kinase activation. Expression of mRNA for the p110γ isozyme of PI3-kinase was specifically increased and p110γ colocalized with nephrin by immunohistochemistry early in disease. Nephrin levels subsequently decreased. Inhibition of p110γ by AS605240 preserved nephrin expression and prevented proteinuria. In cultured podocytes, adriamycin stimulated p110γ expression. AS605240, but not a TGF-β receptor kinase inhibitor, prevented adriamycin-induced cytoskeletal disorganization and apoptosis, supporting a role for p110γ in podocyte injury. AS605240, at a dose that decreased proteinuria, prevented renal collagen mRNA expression in vivo but did not affect TGF-β-stimulated collagen induction in vitro. Thus, PI3-kinase p110γ mediates initial podocyte injury and proteinuria, both of which precede TGF-β-mediated glomerular scarring.

Topics: Animals; Apoptosis; Cells, Cultured; Class I Phosphatidylinositol 3-Kinases; Collagen Type I; Disease Models, Animal; Doxorubicin; Fibronectins; Fibrosis; Glomerulosclerosis, Focal Segmental; Immunohistochemistry; Kidney; Male; Membrane Proteins; Mice; Mice, 129 Strain; Phosphorylation; Podocytes; Protein Kinase Inhibitors; Proteinuria; Proto-Oncogene Proteins c-akt; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Smad3 Protein; Time Factors; Transforming Growth Factor beta; Up-Regulation

The aim of this study is to investigate the expression of nephrin, vascular endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-β1), and podocyte number in adriamycin (ADR)-induced nephropathy. A total of 60 male Sprague-Dawley rats were randomly divided into the control group and the ADR nephropathy group. The nephropathy was induced by tail-vein injection of ADR (4 mg/kg) twice at a 14-day interval. The expression levels of nephrin, VEGF, and TGF-β1 in glomeruli were assessed by immunohistochemistry and western blotting. The podocyte number was also evaluated after anti-Wilms' tumor-1 (WT1) immunohistochemical staining. In addition, the urinary protein content, biochemical parameters in serum samples and glomerular sclerosis index (SI) were compared between groups. In the ADR nephropathy group, the expression levels of nephrin was significantly decreased with the fusion of podocyte foot processes at 6 weeks after the first ADR injection, which was associated with a marked proteinuria. A decrease in podocyte number and an increase in SI with the overexpression of both VEGF and TGF-β1 were also observed in the glomeruli at 10 weeks after the first ADR injection. This was associated with focal segmental glomerulosclerosis (FSGS). The study data suggest that podocyte injury and decreased nephrin, as well as increased VEGF and TGF-β1, may contribute to the development of proteinuria and FSGS in ADR-induced nephropathy in rats.

Topics: Animals; Blotting, Western; Cell Count; Doxorubicin; Glomerular Basement Membrane; Glomerulosclerosis, Focal Segmental; Kidney Diseases; Male; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Rapamycin (Rapa) is an immunosuppressant used to prevent rejection in recipients of renal transplants. Its clinical use is limited by de novo onset or exacerbation of preexisting proteinuria. In the present study, Rapa administration was started 14 days after induction of murine nephrotoxic serum nephritis (NTS) to study glomerular effects of this mammalian target of rapamycin (mTOR) inhibitor. Glomeruli were laser-microdissected, and real-time PCR was performed to assess effects on glomerular cells and the expression of inflammatory cytokines. Immunohistochemical stainings were performed to confirm mRNA data on the protein level. Compared with nephritic control animals, Rapa-treated mice developed significantly increased albuminuria. This was accompanied by a more prominent glomerular infiltration by CD4(+) T cells and macrophages. Glomerular mRNA expression profiling revealed increased levels of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, and the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1β and their cognate macrophage-associated receptors CCR2 and CCR5 in the Rapa-treated animals. Furthermore, there were elevated glomerular transcription levels of the regulatory T cell phenotype transcription factor Foxp3. No differences in the glomerular expression of the podocyte marker nephrin or the endothelial cell marker CD31 were observed on the mRNA or protein level. In conclusion, our data indicate that Rapa-induced proteinuria in NTS is a result of the activation of the innate immune system rather than a direct toxicity to podocytes or glomerular endothelial cells.

Topics: Animals; Gene Expression Regulation; Immunity, Innate; Inflammation Mediators; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Nephritis; Platelet Endothelial Cell Adhesion Molecule-1; Proteinuria; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero-oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury.

Topics: Animals; Antigens, Differentiation; Disease Models, Animal; Humans; Kidney Glomerulus; Membrane Proteins; Mutation; Nephrotic Syndrome; Phosphorylation; Podocytes; Protein Binding; Proteinuria; Rats; Receptors, Immunologic; Tyrosine

The number of the chronic renal failure (CRF) patients is increasing explosively. Hypertension, proteinuria, inflammation, fibrosis, and oxidative stress are intertwined in a complicated manner that leads to the progression of CRF. However, the therapeutic strategies to delay its progression are limited. Since serine proteases are involved in many processes that contribute to these risk factors, we investigated the effects of a synthetic serine protease inhibitor, camostat mesilate (CM), on the progression of CRF in 5/6 nephrectomized (Nx) rats. Eighteen male Sprague-Dawley rats were divided into three groups: a sham-operated group (n = 6), a vehicle-treated Nx group (n = 6), and a CM-treated Nx group (n = 6). Following the 9-wk study period, both proteinuria and serum creatinine levels were substantially increased in the vehicle-treated Nx group, and treatment with CM significantly reduced proteinuria and serum creatinine levels. The levels of podocyte-associated proteins in glomeruli, such as nephrin and synaptopodin, were markedly decreased by 5/6 nephrectomy, and this was significantly ameliorated by CM. CM also suppressed the levels of inflammatory and fibrotic marker mRNAs including transforming growth factor-β1, TNF-α, collagen types I, III, and IV, and reduced glomerulosclerosis, glomerular hypertrophy, and interstitial fibrosis in histological studies. Furthermore, CM decreased the expression of NADPH oxidase component mRNAs, as well as reactive oxygen species generation and advanced oxidative protein product levels. Our present results strongly suggest the possibility that CM could be a useful therapeutic agent against the progression of CRF.

Topics: Animals; Creatinine; Disease Progression; Esters; Gabexate; Guanidines; Kidney; Kidney Failure, Chronic; Male; Membrane Proteins; Microfilament Proteins; Nephrectomy; Oxidative Stress; Proteinuria; Rats; Rats, Sprague-Dawley; Serine Proteinase Inhibitors; Treatment Outcome

Proteinuria and/or albuminuria are widely used for noninvasive assessment of kidney diseases. However, proteinuria is a nonspecific marker of diverse forms of kidney injury, physiologic processes and filtration of small proteins of monoclonal and other pathologic processes. The opportunity to develop new glomerular disease biomarkers follows the realization that the degree of podocyte depletion determines the degree of glomerulosclerosis, and if persistent, determines the progression to end-stage kidney disease (ESKD). Podocyte cell lineage-specific mRNAs can be recovered in urine pellets of model systems and in humans. In model systems, progressive glomerular disease is associated with decreased nephrin mRNA steady-state levels compared with podocin mRNA. Thus, the urine podocin:nephrin mRNA ratio (PNR) could serve as a useful progression biomarker. The use of podocyte-specific transcript ratios also circumvents many problems inherent to urine assays.. To test this hypothesis, the human diphtheria toxin receptor (hDTR) rat model of progression was used to evaluate potentially useful urine mRNA biomarkers. We compared histologic progression parameters (glomerulosclerosis score, interstitial fibrosis score and percent of podocyte depletion) with clinical biomarkers [serum creatinine, systolic blood pressure (BP), 24-h urine volume, 24-h urine protein excretion and the urine protein:creatinine ratio(PCR)] and with the novel urine mRNA biomarkers.. The PNR correlated with histologic outcome as well or better than routine clinical biomarkers and other urine mRNA biomarkers in the model system with high specificity and sensitivity, and a low coefficient of assay variation.. We concluded that the PNR, used in combination with proteinuria, will be worth testing for its clinical diagnostic and decision-making utility.

Topics: Animals; Biomarkers; Humans; Intracellular Signaling Peptides and Proteins; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Inbred F344; Real-Time Polymerase Chain Reaction; RNA, Messenger

Diphtheria toxin (DTx) receptor (DTR)-mediated conditional cell ablation in transgenic mice is a powerful tool to analyze cell function in vivo. Transgenic mice with cell-specific expression of the human DTR have been developed that allow conditional depletion of these cells in vivo through administration of the toxin. We have performed a careful analysis of mice after DTx injection and found an unexpected side effect. Treatment of wild-type C57BL/6 mice with DTx leads to a marked transient and completely reversible proteinuria, as a consequence of podocyte dysfunction that is morphologically characterized by foot process fusion and detachment from the glomerular basal membrane. In vitro analysis displayed that DTx-treated podocytes show diminished attachment to basal membrane proteins. Five to 9 days after DTx application the mice recover completely. Glomerular proteinuria is a hallmark of glomerular disease due to dysfunction of the filtration barrier. Rodents have been extensively used experimentally to better define the mechanisms of disease induction and progression. However, nongenetic mouse models of proteinuric glomerular damage are limited and display various shortcomings. We suggest DTx-induced transient kidney dysfunction as a new reversible model of experimental podocyte injury, which could be used as an additional approach to complement studies in human.

Topics: Analysis of Variance; Animals; Body Weight; Cell Adhesion; Cell Survival; Diphtheria Toxin; Disease Models, Animal; Female; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, SCID; Mice, Transgenic; Podocytes; Proteinuria

To investigate the injury effects of organic solvents on kidney, an animal model of Sprague-Dawley (SD) rats treated with mixed organic solvents via inhalation was generated and characterized. The mixed organic solvents consisted of gasoline, dimethylbenzene and formaldehyde (GDF) in the ratio of 2:2:1, and were used at 12,000 PPM to treat the rats twice a day, each for 3 hours. Proteinuria appeared in the rats after exposure for 5-6 weeks. The incidences of proteinuria in male and female rats after exposure for 12 weeks were 43.8% (7/16) and 25% (4/16), respectively. Urinary N-Acetyl-β-(D)-Glucosaminidase (NAG) activity was increased significantly after exposure for 4 weeks. Histological examination revealed remarkable injuries in the proximal renal tubules, including tubular epithelial cell detachment, cloud swelling and vacuole formation in the proximal tubular cells, as well as proliferation of parietal epithelium and tubular reflux in glomeruli. Ultrastructural examination found that brush border and cytoplasm of tubular epithelial cell were dropped, that tubular epithelial cells were partially disintegrated, and that the mitochondria of tubular epithelial cells were degenerated and lost. In addition to tubular lesions, glomerular damages were also observed, including segmental foot process fusion and loss of foot process covering on glomerular basement membrane (GBM). Immunofluorescence staining indicated that the expression of nephrin and podocin were both decreased after exposure of GDF. In contrast, increased expression of desmin, a marker of podocyte injury, was found in some areas of a glomerulus. TUNEL staining showed that GDF induced apoptosis in tubular cells and glomerular cells. These studies demonstrate that GDF can induce both severe proximal tubular damage and podocyte injury in rats, and the tubular lesions appear earlier than that of glomeruli.

Topics: Acetylglucosaminidase; Acute Kidney Injury; Animals; Apoptosis; Cytoskeleton; Female; Formaldehyde; Gasoline; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Kidney Tubules; Male; Membrane Proteins; Organ Specificity; Proteinuria; Rats; Rats, Sprague-Dawley; Solvents; Xylenes

Several studies in patients with primary aldosteronism (PA) have suggested that aldosterone (ALD) is directly contributing to albuminuria. However, there are limited data pertaining to the direct role of ALD in in vivo models in regard to the induction of renal injury and the involved mechanisms. In the present study, we established a high-dose ALD-infused rat model to evaluate urinary albumin excretion rate (UAER) and podocyte damage. Moreover, we studied the effect of eplerenone (EPL), telmisartan (TEL) and amlodipine (AML) on ALD-induced renal structural and functional changes.. Immunohistochemical and real-time PCR analyses, and TUNEL assays were performed to evaluate nephrin expression and podocyte injury.. ALD-receiving rats (ARR) showed a progressive increase in BP, UAER and proteinuria when compared with control rats (CR). Conversely, BP was significantly reduced in ALD + EPL (A/ERR)-, ALD + AML (A/ARR)- and ALD + TEL (A/TRR)-treated rats. However, UAER and proteinuria were decreased only in A/ERR and A/TRR, but not in A/ARR. Only EPL administration provided protection against ALD-induced podocyte apoptosis. Renal tissue of ARR revealed enhanced expression of nephrin protein and mRNA. This effect of ALD was inhibited by EPL, but not by TEL or AML. Conclusions. ALD induces direct glomerular injury independent of its haemodynamic effects; this effect of ALD is, at least in part, mediated through activation of the mineralocorticoid receptor.

Topics: Albuminuria; Aldosterone; Amlodipine; Angiotensin-Converting Enzyme Inhibitors; Animals; Apoptosis; Benzimidazoles; Benzoates; Blotting, Western; Calcium Channel Blockers; Eplerenone; Immunoenzyme Techniques; Kidney Glomerulus; Male; Membrane Proteins; Mineralocorticoid Receptor Antagonists; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spironolactone; Telmisartan

Angiotensin (ANG)-(1-7) constitutes an important functional end-product of the renin-angiotensin-aldosterone system that acts to balance the physiological actions of ANG II. In the kidney, ANG-(1-7) exerts beneficial effects by inhibiting growth-promoting pathways and reducing proteinuria. We examined whether a 2-wk treatment with a daily dose of ANG-(1-7) (0.6 mg·kg(-1)·day(-1)) exerts renoprotective effects in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). Body weight, glycemia, triglyceridemia, cholesterolemia, as well as plasma levels of Na+ and K+ were determined both at the beginning and at the end of the treatment. Also, the weekly evolution of arterial blood pressure, proteinuria, and creatinine clearance was evaluated. Renal fibrosis was determined by Masson's trichrome staining. Interleukin (IL)-6, tumor necrosis factor (TNF)-α, and nuclear factor-κB (NF-κB) levels were determined by immunohistochemistry and confirmed by Western blotting analysis. The levels of glomerular nephrin were assessed by immunofluorescence. Chronic administration of ANG-(1-7) normalized arterial pressure, reduced glycemia and triglyceridemia, improved proteinuria, and ameliorated structural alterations in the kidney of SHRSP as shown by a restoration of glomerular nephrin levels as detected by immunofluorescence. These results were accompanied with a decrease in both the immunostaining and abundance of IL-6, TNF-α, and NF-κB. In this context, the current study provides strong evidence for a protective role of ANG-(1-7) in the kidney.

Topics: Angiotensin I; Animals; Blood Pressure; Interleukin-6; Kidney; Male; Membrane Proteins; NF-kappa B; Peptide Fragments; Proteinuria; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sodium Chloride; Tumor Necrosis Factor-alpha

The slit diaphragm connecting the adjacent foot processes of glomerular epithelial cells (podocytes) is the final barrier of the glomerular capillary wall and serves to prevent proteinuria. Podocytes are understood to be terminally differentiated cells and share some common features with neurons. Neurexin is a presynaptic adhesion molecule that plays a role in synaptic differentiation. Although neurexin has been understood to be specifically expressed in neuronal tissues, we found that neurexin was expressed in several organs. Several forms of splice variants of neurexin-1α were detected in the cerebrum, but only one form of neurexin-1α was detected in glomeruli. Immunohistochemical study showed that neurexin restrictedly expressed in the podocytes in kidneys. Dual-labeling analyses showed that neurexin was colocalized with CD2AP, an intracellular component of the slit diaphragm. Immunoprecipitation assay using glomerular lysate showed that neurexin interacted with CD2AP and CASK. These observations indicated that neurexin localized at the slit diaphragm area. The staining intensity of neurexin in podocytes was clearly lowered, and their staining pattern shifted to a more discontinuous patchy pattern in the disease models showing severe proteinuria. The expression and localization of neurexin in these models altered more clearly and rapidly than that of other slit diaphragm components. We propose that neurexin is available as an early diagnostic marker to detect podocyte injury. Neurexin coincided with nephrin, a key molecule of the slit diaphragm detected in a presumptive podocyte of the developing glomeruli and in the glomeruli for which the slit diaphragm is repairing injury. These observations suggest that neurexin is involved in the formation of the slit diaphragm and the maintenance of its function.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animal Structures; Animals; Cerebrum; Cytoskeletal Proteins; Embryo, Mammalian; Female; Gene Expression; Glycoproteins; Guanylate Kinases; Kidney Glomerulus; Membrane Proteins; Molecular Sequence Data; Nephrotic Syndrome; Nerve Tissue Proteins; Neuropeptides; Podocytes; Protein Binding; Protein Isoforms; Proteinuria; Rats; Rats, Wistar; Receptors, Cell Surface; Specific Pathogen-Free Organisms

The mechanisms underlying the development of proteinuria in renal-transplant recipients converted from calcineurin inhibitors to sirolimus are still unknown.. This is a single-center cohort study. One hundred ten kidney transplant recipients converted from calcineurin inhibitors to sirolimus in the period from September 2000 to December 2005 were included in the study. All patients underwent a graft biopsy before conversion (T0) and a second protocol biopsy 2 years thereafter (T2), according to our standard clinical protocol. On the basis of the changes observed in proteinuria between T0 and T2 (median 70%), the patients were divided into two groups: group I (<70%) and group II (>70%). The authors blinded the sirolimus blood trough levels. We investigated in vivo the effects of sirolimus on nephrin, podocin, CD2ap, and actin protein expression. Slit diaphragm (SD)-associated protein expressions were evaluated in T0 and T2 biopsies. The same analysis was performed in cultured human podocytes treated with different doses of sirolimus (5, 10, 20, and 50 ng/mL).. The SD protein expression in group II T2 biopsies was significantly reduced compared with the T0 biopsies and with T2 group I biopsies. In addition, sirolimus blood trough levels directly and significantly correlated with the SD protein expression at T2 graft biopsies. Group II patients presented significantly higher sirolimus blood levels than group I. In vitro study confirmed that sirolimus effect on podocytes was dose dependent.. Our data suggest that sirolimus-induced proteinuria may be a dose-dependent effect of the drug on key podocyte structures.

Topics: Adaptor Proteins, Signal Transducing; Adult; Cell Line; Cohort Studies; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Kidney Transplantation; Membrane Proteins; Microscopy, Electron, Transmission; Middle Aged; Neprilysin; Podocytes; Proteinuria; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Time Factors; WT1 Proteins

Doxorubicin (DOX) is an anthracycline antibiotic utilized in antitumor therapy; however, its clinical use is frequently impeded by renal toxic effects. As peroxisome proliferator-activated receptor-α (PPAR-α) has renoprotective effects in drug-related kidney injuries, we tested its ability to inhibit DOX-induced renal injury. Although both male PPAR-α knockout mice and their wild-type littermates (pure 129/SvJ background) had significant proteinuria 4 weeks after DOX treatment, those with deletion of PPAR-α had more severe proteinuria. This was associated with more serious podocyte foot process effacement compared with wild-type mice. In contrast, the PPAR-α agonist fenofibrate effectively reduced proteinuria and attenuated DOX-induced podocyte foot process effacement. Consistently, glomerular nephrin expression was significantly lower in the knockout compared with wild-type mice following DOX treatment. Fenofibrate therapy significantly blunted the reduction in glomerular nephrin levels in DOX-treated wild-type mice. In cultured podocytes, DOX induced apoptosis, increased cleaved caspase-3 levels, and decreased Bcl-2 expression, all attenuated by pretreatment with fenofibrate. Thus, PPAR-α deficiency exacerbates DOX-related renal injury, in part, due to increased podocyte apoptosis.

Topics: Animals; Apoptosis; Caspase 3; Cell Line; Cytoprotection; Disease Models, Animal; Doxorubicin; Fenofibrate; Kidney Diseases; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, 129 Strain; Mice, Inbred BALB C; Mice, Knockout; Podocytes; PPAR alpha; Proteinuria; Proto-Oncogene Proteins c-bcl-2; Species Specificity; Time Factors; Transcription, Genetic

The MYH9 gene encodes a non-muscle myosin IIA heavy chain (NMMHC-IIA) expressed in podocytes. Heterozygous MYH9 mutations cause a set of overlapping syndromes characterized by variable degrees of deafness, morphologic abnormalities of platelets and focal segmental glomerulosclerosis (FSGS) with progressive renal dysfunction. Similar glomerular lesions are seen in a variety of nephropathies, including an idiopathic form of FSGS in children which recurs in renal allografts, implying a circulating factor that affects glomerular podocyte biology. It is unknown whether NMMHC-IIA is perturbed in the idiopathic form of FSGS. We describe a pediatric patient with typical idiopathic FSGS, in whom proteinuria recurred within hours of deceased donor renal transplantation but who responded to plasmapheresis. We demonstrate in vitro that plasmapheresis effluent from our patient rapidly decreased cultured podocyte levels of the phosphorylated myosin light chain (MLC) that mediates NMMHC-IIA binding to actin and induced dispersion of NMMHC-IIA from its usual position along actin stress fibers. FSGS plasma also caused dispersion of slit diaphragm proteins (nephrin and podocin) and vinculin-positive focal adhesion complexes. Our observations suggest that the putative circulating factor in idiopathic FSGS disrupts normal NMMHC-IIA function in podocytes and might contribute to the pathogenesis of recurrent FSGS in other children.

Topics: Adolescent; Apoptosis; Cell Line; Cell Size; Child; Female; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney Failure, Chronic; Kidney Transplantation; Membrane Proteins; Molecular Motor Proteins; Myosin Heavy Chains; Myosin Light Chains; Phosphorylation; Plasmapheresis; Podocytes; Protein Transport; Proteinuria; Recurrence; Stress Fibers; Time Factors; Treatment Outcome

The aim of the paper is to investigate the effects of adiponectin in diabetic nephropathy; we used an adenovirus to over-express adiponectin (Ad-Adipo) in streptozotocin (STZ)-induced diabetic rats. Animals were injected with either Ad-Adipo or control Ad-lacZ at 10 weeks after STZ treatment, and at two weeks postadenovirus injection, renal function was assessed. The degree of proteinuria was significantly reduced in Ad-Adipo rats compared with Ad-lacZ rats. Consistent with this, the mRNA expression levels of nephrin and transforming growth factor β (TGF-β) were significantly increased and decreased in the renal cortex of Ad-Adipo rats, respectively. Moreover, adiponectin over-expression in STZ rats decreased markers of endothelial dysfunction, a feature of diabetic nephropathy disease progression. Endothelin 1 (ET-1), plasminogen activator inhibitor 1 (PAI-1) and inducible nitric oxide synthase (iNOS) mRNA expression levels were significantly reduced in the renal cortex of Ad-Adipo rats, respectively. Concurrently, mRNA expression levels of endothelial nitric oxide synthase (eNOS), a positive regulator of endothelial function, were significantly increased in the renal cortex of Ad-Adipo rats. We have shown that chronic hyperadiponectinemia significantly alleviated the progression of proteinuria in early stage diabetic nephropathy. The mechanism whereby adiponectin decreases proteinuria involves an increase in nephrin expression, and an improvement of the endothelial dysfunction due to decreases in ET-1 and PAI-1, and an increase in eNOS expression in the renal cortex. Thus, over-expression of adiponectin has beneficial effects on early stage diabetic nephropathy.

Topics: Adenoviridae; Adiponectin; Animals; Body Weight; Diabetes Mellitus, Experimental; Endothelin-1; Endothelium; Gene Expression Regulation; HEK293 Cells; Humans; Kidney Cortex; Male; Membrane Proteins; Mice; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Plasminogen Activator Inhibitor 1; Proteinuria; Rats; Rats, Wistar; RNA, Messenger; Transduction, Genetic; Transforming Growth Factor beta

Multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) has been proved clinically effective in reducing proteinuria in chronic kidney disease in China. However, the mechanisms involved are still unclear. In this study we examined the effects of GTW at the different dosages on proteinuria and podocyte slit diaphragm (SD) dysfunction in anti-Thy1.1 glomerulonephritis (GN).. Rats with anti-Thy1.1 GN were divided into 2 groups, a GTW group and a vehicle group, and sacrificed at 30 min, on day 7, and on day 14 in Experiments 1, 2 and 3, respectively. The administration of GTW at the moderate and high doses was started 3 days before or at the same time of antibody injection till sacrifice. Proteinuria was determined in Experiments 1, 2, and 3. After sacrifice, the staining intensity of SD-associated key functional molecules including nephrin and podocin, podocyte structure, mesangial change, macrophage infiltration, and blood biochemical parameters were examined, respectively. Protein and mRNA expressions of nephrin and podocin in glomeruli were also investigated. Besides, liver histological characteristics were analyzed.. In Experiment 1, GTW pretreatment at the medium dose (75 mg/kg body weight) caused no influence on the induction of anti-Thy1.1 GN and the basal nephrin expression. In Experiment 2, the high dosage (100mg/kg body weight) of GTW ameliorated proteinuria, the distribution of nephrin and podocin, mesangial proliferation, and the activated macrophage accumulation, as compared with vehicle group (P<0.05). Additionally, it increased mRNA and protein expressions of nephrin and podocin in glomeruli on day 7, but had no influence on podocyte structure. In Experiment 3, the medium dosage (75 mg/kg body weight) of GTW improved proteinuria, the partial matrix expansion, and the distribution of nephrin and podocin on day 14, as compared with anti-Thy1.1 GN rats (P<0.05). GTW at the high or moderate dose did not affect hepatic function on day 7 and on day 14.. Podocyte SD dysfunction, such as the disordered distribution and down-regulation of nephrin and podocin expression, is critically involved in the pathogenesis of anti-Thy1.1 GN induced by mAb 1-22-3. The restoration of the distribution and expression of nephrin and podocin by GTW could be an important mechanism by which GTW ameliorates proteinuria and podocyte SD dysfunction.

Topics: Animals; Disease Models, Animal; Female; Glomerular Mesangium; Glomerulonephritis, Membranoproliferative; Intracellular Signaling Peptides and Proteins; Macrophage Activation; Membrane Proteins; Phytotherapy; Plant Extracts; Podocytes; Proteinuria; Rats; Rats, Wistar; RNA, Messenger; Thy-1 Antigens; Tripterygium

Immunosuppressants and inhibitors of the renin angiotensin system are major reagents to treat nephrotic syndrome but their clinical effects are not necessarily satisfactory. Injection of doxorubicin in several strains of mice causes nephrotic syndrome-like disorder. Zhou et al. report that PPAR-α expression is downregulated in murine doxorubicin nephropathy and a PPAR-α agonist, fenofibrate, partially ameliorates the disorder induced likely through stabilization of nephrin expression and suppression of apoptosis in podocytes, providing a new preventive strategy.

Topics: Animals; Apoptosis; Caspase 3; Cytoprotection; Disease Models, Animal; Doxorubicin; Fenofibrate; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Knockout; Podocytes; PPAR alpha; Proteinuria; Proto-Oncogene Proteins c-bcl-2; Time Factors; Transcription, Genetic

Congenital nephrotic syndrome of the Finnish type is a serious renal disease belonging to the Finnish disease heritage. It appears as substantial proteinuria, hypoproteinemia and edema in a newborn. Kidney transplantation is the only effective treatment. The cause of the disease is a mutation in the gene encoding the nephrin protein. Nephrin is produced by the epithelial cell (podocyte) of the glomerulus. It is expressed in the slit membrane connecting the pedicles of the podocyte. This finding has revolutionized the concept of glomerular filtration and set off active research on the pathogenetic mechanisms of proteinuria.

Topics: Edema; Finland; Genotype; Glomerular Filtration Rate; Humans; Hypoproteinemia; Infant, Newborn; Kidney Transplantation; Membrane Proteins; Mutation; Nephrotic Syndrome; Proteinuria

To evaluate severity of nephrinuria (NU) as a marker of podocyte dysfunction (PD) in patients with proteinuric forms of chronic glomerulonephritis (CGN) and to specify efficacy of this test for assessment of activity and prognosis of CGN.. We examined 74 CGN patients: 18 with inactive nephritis (group 1), 18--with subnephrotic proteinuria (group 2), 38--with nephrotic syndrome--NS (group 3). The control group consisted of 10 healthy subjects. Urinary excretion of nephrin was studied with indirect enzyme immunoassay. A response to immunosuppressive treatment (IST) was studied in 23 NS patients depending on a baseline NU level.. An NU level was higher in patients with proteinuric forms of CGN (groups 2 and 3) than in inactive disease and in healthy subjects, in NS patients significantly higher than in less severe proteinuria. NU was significantly higher in arterial hypertension, in persistent NS. Remission of NS was achieved within 6 months of treatment in 9 of 11 (82%) patients with a baseline NU level < 17 ng/ml. Eight from 12 (67%) patients with high NU did not respond to IST conducted for 9 months to 2 years. ROC-curve construction showed that NU assessment in NS patients has high informative value in assessment of prognosis and efficacy of treatment in 6 months to come.. The NU test in CGN patients is an informative diagnostic test allowing prognosis of a response to IST and assessment of PD severity.

Topics: Adolescent; Adult; Aged; Chronic Disease; Female; Glomerulonephritis; Humans; Male; Membrane Proteins; Middle Aged; Podocytes; Prognosis; Proteinuria; Remission, Spontaneous; Severity of Illness Index; Young Adult

Development of pharmacologic agents that protect podocytes from injury is a critical strategy for the treatment of kidney glomerular diseases. Retinoic acid reduces proteinuria and glomerulosclerosis in multiple animal models of kidney diseases. However, clinical studies are limited because of significant side effects of retinoic acid. Animal studies suggest that all trans retinoic acid (ATRA) attenuates proteinuria by protecting podocytes from injury. The physiological actions of ATRA are mediated by binding to all three isoforms of the nuclear retinoic acid receptors (RARs): RARα, RARβ, and RARγ. We have previously shown that ATRA exerts its renal protective effects mainly through the agonism of RARα. Here, we designed and synthesized a novel boron-containing derivative of the RARα-specific agonist Am580. This new derivative, BD4, binds to RARα receptor specifically and is predicted to have less toxicity based on its structure. We confirmed experimentally that BD4 binds to RARα with a higher affinity and exhibits less cellular toxicity than Am580 and ATRA. BD4 induces the expression of podocyte differentiation markers (synaptopodin, nephrin, and WT-1) in cultured podocytes. Finally, we confirmed that BD4 reduces proteinuria and improves kidney injury in HIV-1 transgenic mice, a model for HIV-associated nephropathy (HIVAN). Mice treated with BD4 did not develop any obvious toxicity or side effect. Our data suggest that BD4 is a novel RARα agonist, which could be used as a potential therapy for patients with kidney disease such as HIVAN.

Topics: AIDS-Associated Nephropathy; Animals; Benzoates; Benzopyrans; Binding, Competitive; Body Weight; Boron Compounds; Cell Differentiation; Cell Survival; Cells, Cultured; Female; Gene Expression Regulation; Kidney; Kidney Diseases; Male; Membrane Proteins; Mice; Mice, Transgenic; Microfilament Proteins; Molecular Structure; Podocytes; Proteinuria; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoids; Reverse Transcriptase Polymerase Chain Reaction; Tetrahydronaphthalenes; WT1 Proteins

The aim of this study is to investigate the effect of Shen-qi-di-huang decoction on reducing proteinuria and to discuss the mechanism of its action in Adriamycin (ADR)-induced nephropathy rats. The rats were randomly divided into three groups (n=12 each group): normal control (group A); ADR model control (group B); ADR + Shen-qi-di-huang decoction (group C). In group B and C, the rats were intravenously injected with ADR (6.5mg/kg). The rats in group C were orally administrated with Shen-qi-di-huang decoction after the injection of ADR. On day 7, 14, 28, 56 after ADR injection, 24h urine protein was detected. On day 28, 56 after ADR injection, ALB, ALT, serum creatinine (Scr) and BUN were examined. The morphological changes of the kidneys were observed by light microscope and electron microscope on day 28, 56 after ADR injection. The expression of nephrin was determined by immunohistochemistry and RT-PCR on day 28, 56 after ADR injection. Compared with group B, 24h urine protein and Scr decreased in group C on day 56 (P<0.05). The expression of nephrin determined by immunohistochemistry and RT-PCR increased in group C on day 28, 56 (P<0.05). The morphology observed by light microscope and electron microscope improved in group C on day 28, 56. Shen-qi-di-huang decoction decreases proteinuria, protects kidney function, and ameliorates histopathology in ADR-induced rats by preserving nephrin expression.

Topics: Animals; Creatinine; Doxorubicin; Drugs, Chinese Herbal; Immunohistochemistry; Kidney; Membrane Proteins; Nephrotic Syndrome; Proteinuria; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction

We recently reported that nephrin, a major slit-diaphragm protein, is phosphorylated at Y1204 and Y1228 in normal human glomeruli and that phosphorylation decreased significantly in minimal-change nephrosis. These results indicate that phosphorylation of nephrin is important for maintenance of normal podocyte morphology and function. On the other hand, phosphorylation of nephrin was reportedly increased in certain animal models of glomerular injury.. We performed immunofluorescent and immunoelectron staining of phosphorylated nephrin in human kidney biopsy specimens of membranous nephropathy (MN) to investigate whether phosphorylation of nephrin was altered in human MN and whether it correlated with MN staging.. Although aberrant localization of phosphorylated nephrin was detected using immunoelectron microscopy in stage I MN, a decrease in the immunofluorescent intensity of phosphorylated nephrin was not observed in stage I, and only a slight decrease was seen in stages II, III, and IV compared with controls. No significant correlation between nephrin phosphorylation and proteinuria was observed.. Nephrin phosphorylation was not significantly decreased in the early stage of MN.

Topics: Aged; Fluorescent Antibody Technique; Glomerulonephritis, Membranous; Humans; Kidney Glomerulus; Membrane Proteins; Microscopy, Immunoelectron; Middle Aged; Phosphorylation; Proteinuria; Tyrosine

Treatment with angiotensin II type 1 receptor blockers (ARBs) is the first-line therapy for hypertensive patients with diabetic nephropathy. However, emerging clinical evidence indicates that mineralocorticoid receptor (MR) blockers have blood pressure-independent antiproteinuric effects. We sought to determine whether treatment with an MR blocker, eplerenone, enhances the effects of an ARB, telmisartan, on podocyte injury and proteinuria in type 2 diabetic Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats. From 20 to 50 weeks old, diabetic OLETF rats showed higher systolic blood pressure (SBP) and urinary protein excretion (U(protein)V) than nondiabetic control Long-Evans-Tokushima-Otsuka rats. At 50 weeks old, OLETF rats also showed glomerular sclerosis and podocyte injury, whereas nephrin and podocin mRNA levels in isolated glomeruli were significantly decreased. Treatment with telmisartan (3 mg/kg/day p.o.) decreased SBP and U(protein)V, increased nephrin and podocin mRNA levels, and attenuated glomerular sclerosis and podocyte injury. Eplerenone (100 mg/kg/day p.o.) did not alter SBP but elicited similar changes in renal parameters. However, greater reductions in U(protein)V and podocyte injury and greater increases in nephrin and podocin mRNA levels were observed in the combination treatment group. Hydralazine (25 mg/kg/day p.o.) decreased SBP but did not alter any renal parameters. These data indicate that MR blockade enhances the SBP-independent antiproteinuric effect of an ARB through inhibiting podocyte injury in type 2 diabetic rats.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Benzoates; Diabetes Mellitus, Type 2; Drug Synergism; Eplerenone; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mineralocorticoid Receptor Antagonists; Podocytes; Proteinuria; Rats; Rats, Inbred OLETF; Spironolactone; Telmisartan

Mutations leading to nephrin loss result in massive proteinuria both in humans and mice. Early perinatal lethality of conventional nephrin knockout mice makes it impossible to determine the role of nephrin protein in the adult kidney and in extra-renal tissues. Herein, we studied whether podocyte-specific, doxycycline-inducible, rat nephrin expression can rescue nephrin-deficient mice from perinatal lethality. Fourteen littermates out of 72 lacked endogenous nephrin and expressed transgenic rat nephrin. Six of these rescued mice survived until 6 weeks of age, whereas the nephrin-deficient pups died before the age of 5 days. The rescued mice were smaller, developed proteinuria, and showed histological abnormalities in the kidney. Despite foot process effacement, slit diaphragms were observed. Importantly, the expression and localization of several proteins associated with the signaling capacity of nephrin or the regulation of the expression of nephrin were changed in the podocytes. Indeed, all rescued mice showed impaired locomotor activity and distinct histological abnormalities in the cerebellum, and the male mice were also infertile and showed genital malformations. These observations are consistent with normal nephrin expression in the testis and cerebellum. These observations indicate that podocyte-specific expression of rat nephrin can rescue nephrin-deficient mice from perinatal death, but is not sufficient for full complementation.

Topics: Animals; Behavior, Animal; Body Weight; Doxycycline; Fluorescent Antibody Technique; Gene Expression Regulation; Genotype; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Membrane Proteins; Mice; Mice, Transgenic; Organ Specificity; Perinatal Mortality; Phenotype; Podocytes; Proteinuria; Rats; RNA, Messenger; Transgenes

Membranous glomerulonephritis (MGN) is one of the most common causes of nephrotic syndrome in adults. NPHS1 encoding nephrin is a transmembrane protein of the immunoglobulin family. We clarified the relationship between NPHS1 gene polymorphisms and the susceptibility or progression of MGN.. We recruited a cohort of 132 biopsy-diagnosed MGN patients and 257 healthy subjects. Genotyping of three SNPs (rs401824, rs437168 and rs3814995) at chromosome positions 41034749 (5'UTR), 41026259(exon17) and 41034052 (exon 3) was performed using a Taqman SNP genotyping assay.. There was a significant difference in genotype frequency distribution of rs437168 polymorphism between MGN patients and controls. The results also showed that the frequency of the G allele was significantly higher in the patient group. Among the polymorphisms rs437168, rs401824 and rs3814995, no significant haplotype was shown in MGN patients. A stratified analysis revealed that a high disease progression in the AA genotype of rs401824 and GG genotype of rs437168 patients were associated with a low rate of remission.. The presence of the different genotypes of NPHS1 was associated with susceptibility of MGN and the remission of proteinuria during disease progression after the therapy.

Topics: 5' Untranslated Regions; Adult; Aged; Aged, 80 and over; Creatinine; Female; Gene Frequency; Genotype; Glomerulonephritis, Membranous; Haplotypes; Hematuria; Heterozygote; Homozygote; Humans; Immunosuppressive Agents; Male; Membrane Proteins; Middle Aged; Open Reading Frames; Polymorphism, Single Nucleotide; Proteinuria; Taiwan; Treatment Outcome

We sought to examine the pathogenic role of excessive VEGF-A expression in podocytes, since it has been reported that diabetic nephropathy and other glomerular diseases are associated with increased VEGF-A expression. The induction of podocyte-specific VEGF164 overexpression in adult transgenic mice led to proteinuria, glomerulomegaly, glomerular basement membrane thickening, mesangial expansion, loss of slit diaphragms, and podocyte effacement. When doxycycline-mediated VEGF164 was stopped, these abnormalities reversed. These findings were associated with reversible downregulation of metalloproteinase 9 and nephrin expression. Using transmission electron microscopy, we established that VEGF-A receptor-2 (VEGFR2) was expressed in podocytes and glomerular endothelial cells. We also found that VEGF164 induced VEGFR2 phosphorylation in podocytes. Further, we were able to co-immunoprecipitate VEGFR2 and nephrin using whole kidney lysates, confirming interaction in vivo. This implies that autocrine and paracrine VEGF-A signaling through VEGFR2 occurs in podocytes and may mediate the glomerular phenotype caused by VEGF164 overexpression. Thus, we suggest that podocyte VEGF164 overexpression in adult mice is sufficient to induce glomerular filtration barrier structural and functional abnormalities similar to those present in murine diabetic nephropathy.

Topics: Age Factors; Animals; Autocrine Communication; Diabetic Nephropathies; Genotype; Glomerular Basement Membrane; Kidney Diseases; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Transgenic; Paracrine Communication; Phenotype; Phosphorylation; Podocytes; Protein Binding; Proteinuria; Signal Transduction; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Podocyte damage is the basis of many glomerular diseases with ultrastructural changes and decreased expression of components of the slit diaphragm such as nephrin and podocin. Under physiological conditions it is likely that the slit diaphragm underlies permanent renewal processes to indemnify its stability in response to changes in filtration pressure. This would require constant reorganization of the podocyte foot process and the renewal of slit diaphragm components. Thus far, the mechanisms underlying the turnover of slit diaphragm proteins are largely unknown. In this manuscript we examined a mechanism of nephrin endocytosis via CIN85/Ruk(L)-mediated ubiquitination. We can demonstrate that the loss of nephrin expression and onset of the proteinuria in CD2AP(-/-) mice correlates with an increased accumulation of ubiquitinated proteins and expression of CIN85/Ruk(L) in podocytes. In cultured murine podocytes CD2AP deficiency leads to an early ubiquitination of nephrin and podocin after stimulation with fibroblast growth factor-4. Binding assays with different CIN85/Ruk isoforms and mutants showed that nephrin and podocin are binding to the coiled-coil domain of CIN85/Ruk(L). We found that in the presence of CIN85/Ruk(L), which is involved in down-regulation of receptor-tyrosine kinases, nephrin is internalized after stimulation with fibroblast growth factor-4. Interestingly, coexpression of CIN85/Ruk(L) with CD2AP led to a decreased binding of CIN85/Ruk(L) to nephrin and podocin, which indicates a functional competition between CD2AP and CIN85/Ruk(L). Our results support a novel role for CIN85/Ruk(L) in slit diaphragm turnover and proteinuria.

Topics: Adaptor Proteins, Signal Transducing; Animals; Blotting, Western; Cell Line; Cells, Cultured; Cytoskeletal Proteins; Endocytosis; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factors; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Neoplasm Proteins; Nerve Tissue Proteins; Podocytes; Protein Binding; Proteinuria; RNA, Small Interfering; Ubiquitination

Several proteins constituting the slit diaphragm are considered important for maintaining capillary wall permselectivity. Early intervention with blockers of angiotensin II receptors (AR) and mineralocorticoid receptors (MR) is effective against proteinuria in models of chronic hypertensive and protein-induced renal damage. However, the effects of AR and/or MR blockers in a model of acute nephrotic syndrome remain unknown. The effects of AR and MR blockers were examined in puromycin aminonucleoside (PAN)-treated rats.. Six week old male Sprague-Dawley (SD) rats were injected with PAN or vehicle and assigned to groups as follows: vehicle (group C); PAN (group P); PAN followed 3 days later by administration of the MR blocker, eplerenone (group MR), and by the AR blocker, losartan (group AR). Blood pressure and urinary protein excretion were measured and all rats were killed for immunohistochemical investigation on day 14 after PAN administration.. Blood pressure did not change throughout the study period. Proteinuria was decreased in groups MR and AR compared with group P (on day 14 after PAN administration, respectively; group P vs AR, P < 0.01; group P vs MR, P < 0.05). Nephrin, podocin and podocalyxin staining was preserved in the glomeruli of groups MR and AR compared with group P.. The MR and AR blockers decreased proteinuria in the acute model of nephrotic syndrome with preserved expression of glomerular podocyte protein independently of blood pressure.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Blood Pressure; Disease Models, Animal; Eplerenone; Fluorescent Antibody Technique; Intracellular Signaling Peptides and Proteins; Losartan; Male; Membrane Proteins; Mineralocorticoid Receptor Antagonists; Nephrosis, Lipoid; Podocytes; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Sialoglycoproteins; Spironolactone; Time Factors

Idiopathic nephrotic syndrome comprises several podocyte diseases of unknown origin that affect the glomerular podocyte, which controls the permeability of the filtration barrier in the kidney to proteins. It is characterized by the daily loss of more than 3 g of protein in urine and the lack of inflammatory lesions or cell infiltration. We found that the abundance of c-mip (c-maf inducing protein) was increased in the podocytes of patients with various acquired idiopathic nephrotic syndromes in which the podocyte is the main target of injury. Mice engineered to have excessive c-mip in podocytes developed proteinuria without morphological alterations, inflammatory lesions, or cell infiltration. Excessive c-mip blocked podocyte signaling by preventing the interaction of the slit diaphragm transmembrane protein nephrin with the tyrosine kinase Fyn, thereby decreasing phosphorylation of nephrin in vitro and in vivo. Moreover, c-mip inhibited interactions between Fyn and the cytoskeletal regulator N-WASP (neural Wiskott-Aldrich syndrome protein) and between the adaptor protein Nck and nephrin, potentially accounting for cytoskeletal disorganization and the effacement of foot processes seen in idiopathic nephrotic syndromes. The intravenous injection of small interfering RNA targeting c-mip prevented lipopolysaccharide-induced proteinuria in mice. Together, these results identify c-mip as a key component in the molecular pathogenesis of acquired podocyte diseases.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins; Humans; Membrane Proteins; Mice; Mice, Transgenic; Phosphorylation; Podocytes; Protein Binding; Proteinuria; Proto-Oncogene Proteins c-fyn; RNA Interference; Signal Transduction; Wiskott-Aldrich Syndrome Protein, Neuronal

It is still unclear what happens in the glomerulus when proteinuria starts. Using puromycin aminonucleoside nephrosis (PAN) rats, we studied early ultrastructural and permeability changes in relation to the expression of the podocyte-associated molecules nephrin, α-actinin, dendrin, and plekhh2, the last two of which were only recently discovered in podocytes.. Using immune stainings, semiquantitative measurement was performed under the electron microscope. Permeability was assessed using isolated kidney perfusion with tracers. Possible effects of ACE inhibition were tested.. By day 2, some patchy foot process effacement, but no proteinuria, appeared. The amount of nephrin was reduced in both diseased and normal areas. The other proteins showed few changes, which were limited to diseased areas. By day 4, foot process effacement was complete and proteinuria appeared in parallel with signs of size barrier damage. Nephrin decreased further, while dendrin and plekhh2 also decreased but α-actinin remained unchanged. ACE inhibition had no significant protective effect.. PAN glomeruli already showed significant pathology by day 4, despite relatively mild proteinuria. This was preceded by altered nephrin expression, supporting its pivotal role in podocyte morphology. The novel proteins dendrin and plekhh2 were both reduced, suggesting roles in PAN, whereas α-actinin was unchanged.

Topics: Actinin; Animals; Kidney Diseases; Kidney Glomerulus; Male; Membrane Proteins; Nephrosis; Nerve Tissue Proteins; Permeability; Proteins; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley

Accumulation of plasma advanced oxidation protein products (AOPPs) promotes progression of proteinuria and glomerulosclerosis. To investigate the molecular basis of AOPPs-induced proteinuria, normal Sprague-Dawley rats were treated with AOPPs-modified rat serum albumin. The expression of glomerular podocyte slit diaphragm (PSD)-associated proteins, nephrin and podocin, was significantly decreased coincident with the onset of albuminuria in rats treated with AOPPs. Chronic inhibition of NADPH oxidase by apocynin prevented down-regulation of nephrin and podocin and decreased albuminuria in AOPPs-challenged rats. This suggested that accumulation of AOPPs promotes proteinuria, possibly via down-regulating the expression of PSD-associated proteins.

Topics: Animals; Blotting, Western; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mice; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Podocytes; Proteins; Proteinuria; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Serum Albumin

Edema in nephrotic syndrome results from renal retention of sodium and alteration of the permeability properties of capillaries. Nephrotic syndrome induced by puromycin aminonucleoside (PAN) in rats reproduces the biological and clinical signs of the human disease, and has been widely used to identify the cellular mechanisms of sodium retention. Unfortunately, mice do not develop nephrotic syndrome in response to PAN, and we still lack a good mouse model of the disease in which the genetic tools necessary for further characterizing the pathophysiological pathway could be used. Mouse resistance to PAN has been attributed to a defect in glomerular adenosine deaminase (ADA), which metabolizes PAN. We therefore attempted to develop a mouse line sensitive to PAN through induction of normal adenosine metabolism in their podocytes.. A mouse line expressing functional ADA under the control of the podocyte-specific podocin promoter was generated by transgenesis. The effect of PAN on urinary excretion of sodium and proteins was compared in rats and in mice over-expressing ADA and in littermates.. We confirmed that expression of ADA mRNAs was much lower in wild type mouse than in rat glomerulus. Transgenic mice expressed ADA specifically in the glomerulus, and their ADA activity was of the same order of magnitude as in rats. Nonetheless, ADA transgenic mice remained insensitive to PAN treatment in terms of both proteinuria and sodium retention.. Along with previous results, this study shows that adenosine deaminase is necessary but not sufficient to confer PAN sensitivity to podocytes. ADA transgenic mice could be used as a background strain for further transgenesis.

Topics: Adenosine Deaminase; Animals; Drug Resistance; Edema; Enzyme Induction; Genes, Synthetic; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Transgenic; Natriuresis; Nephrotic Syndrome; Podocytes; Promoter Regions, Genetic; Proteinuria; Puromycin Aminonucleoside; Rats; Recombinant Fusion Proteins; RNA, Messenger; Species Specificity

Tissue factor (TF) is a transmembrane protein that is essential for coagulation. TF is expressed on podocytes and its cytoplasmic domain has cell signalling functions in epithelial cells.. Mice lacking the cytoplasmic domain of TF (TF(CT-/-) mice) were used to study its role in physiological albuminuria and pathological proteinuria following induction of glomerulonephritis (GN).. Absence of the cytoplasmic domain of TF was associated with increased albuminuria, podocyte effacement, reduced podocyte numbers and increased spontaneous glomerular tumour necrosis factor α(TNFα) production under physiological conditions. In mice developing GN, absence of the cytoplasmic domain of TF resulted in increased proteinuria and enhanced renal TNFα production without altering other parameters of renal inflammation and injury. Studies in TF(CT-/-) chimeric mice (created by bone marrow transplantation) showed increased proteinuria and renal TNFα mRNA in GN was associated with absence of the cytoplasmic domain of TF in the kidney and was independent of the leucocyte phenotype.. These studies demonstrate that the cytoplasmic domain of TF contributes to renal albumin retention and its renal expression protects against proteinuria in leucocyte-mediated renal inflammation. Increased glomerular production of TNFα in the absence of cytoplasmic domain of TF may contribute to podocyte injury resulting in albuminuria and proteinuria.

Topics: Albuminuria; Animals; Cytoplasm; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Protein Structure, Tertiary; Proteinuria; Thromboplastin; Tumor Necrosis Factor-alpha

The capacity of renin-angiotensin system (RAS) inhibitors to delay progression of diabetic nephropathy depends on the time at which therapy is started. A multimodal intervention is required to afford renoprotection in overt diabetic nephropathy. Here we assessed the effects of maximal RAS inhibition by angiotensin-converting enzyme (ACE) inhibitor plus angiotensin II type 1 receptor blocker (ARB) in combination with statin in rats with overt diabetic nephropathy. Uninephrectomized rats made diabetic by streptozotocin were orally treated from 4 (when proteinuria and renal lesions had developed) to 8 mo with vehicle, lisinopril plus candesartan, lisinopril plus candesartan plus rosuvastatin, or rosuvastatin alone. Systolic blood pressure increased in diabetic rats and was significantly lowered by combined therapies. Dual RAS blockade significantly reduced proteinuria compared with vehicle. Addition of statin further lowered proteinuria to control levels. Glomerulosclerosis was ameliorated by RAS inhibitors or statin, and regression was achieved by the addition of statin. Loss of podocytes of diabetic rats was limited by ACE inhibitor plus ARB while normalized by the three drugs. Defective nephrin expression of diabetes was increased by dual RAS blockade or statin and restored by the triple therapy. Tubular damage, interstitial inflammation, and expression of the fibrotic markers transforming growth factor (TGF)-β1 and phosphorylated Smad 2/3 in tubuli were significantly reduced by the triple regimen. These data suggest a strategy to target proteinuria to try to achieve regression of renal disease in diabetic patients who do not fully benefit from RAS inhibition alone.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Cholesterol; Cholesterol Esters; Diabetic Nephropathies; Fibrosis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunohistochemistry; Kidney; Kidney Function Tests; Kidney Glomerulus; Lipid Metabolism; Male; Membrane Proteins; Nephritis, Interstitial; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System

The development of proteinuria has been observed in kidney-transplanted patients on m-TOR inhibitor (m-TORi) treatment. Recent studies suggest that m-TORi(s) may alter the behavior and integrity of glomerular podocytes. We analyzed renal biopsies from kidney-transplanted patients and evaluated the expression of nephrin, a critical component of the glomerular slit-diaphragm. In a group of patients on 'de novo' m-TORi-treatment, the expression of nephrin within glomeruli was significantly reduced in all cases compared to pretransplant donor biopsies. Biopsies from control transplant patients not treated with m-TORi(s) failed to present a loss of nephrin. In a group of patients subsequently converted to m-TORi-treatment, a protocol biopsy performed before introduction of m-TORi was also available. The expression of nephrin in the pre-m-TORi biopsies was similar to that observed in the pretransplant donor biopsies but was significantly reduced after introduction of m-TORi(s). Proteinuria increased after the m-TORi inititiation in this group. However, in some cases proteinuria remained normal despite reduction of nephrin. In vitro, sirolimus downregulated nephrin expression by human podocytes. Our results suggest that m-TORi(s) may affect nephrin expression in kidney-transplanted patients, consistently with the observation in vitro on cultured podocytes.

Topics: Adult; Aged; Cells, Cultured; Humans; Kidney Glomerulus; Kidney Transplantation; Membrane Proteins; Middle Aged; Podocytes; Proteinuria; Retrospective Studies; Sirolimus; TOR Serine-Threonine Kinases

The aim of the study was to present our experience in treating children with genetic forms of nephrotic syndrome and diagnosing these diseases. We retrospectively reviewed the clinical data, mutational analyses, histopathological features, treatment modalities, and outcome of 26 consecutive children (20 families) suffering from congenital and/or steroid-resistant nephrotic syndrome who were assessed by genetic analysis. Ten out of 26 children (38%) had congenital nephrotic syndrome, 4/26 (15%) had infantile nephrotic syndrome, 10/26 (38%) had late-onset nephrotic syndrome, and 2/26 (9%) had asymptomatic proteinuria. We detected a mutation in 21/26 (81%) patients and in 15/20 (75%) families. NPHS1 mutation analyses were positive in 4/20 (20%), NPHS2 mutations in 4/20 (20%), WT1 mutations in 4/20 (20%), and PLCE1 mutations in 3/20 (15%) families. NPHS1 and PLCE1 mutations were solely found in patients with the earliest onset. The majority of patients, especially those with early onset of nephrotic syndrome, had serious adverse events related to the nephrotic status, and 19/26 (73%) reached end-stage renal failure at a median age of 27 months. Genetic forms of nephrotic syndrome comprise a heterogeneous group of genetic mutations. The progression toward end-stage renal failure is the rule but is highly variable between patients.

Topics: Adolescent; Age of Onset; Belgium; Child; Child, Preschool; Denys-Drash Syndrome; Female; Frasier Syndrome; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Kidney Neoplasms; Male; Membrane Proteins; Nephrotic Syndrome; Phosphoinositide Phospholipase C; Proteinuria; Retrospective Studies; WT1 Proteins

SGK1 is critically important for mineralocorticoid/salt-induced glomerular injury. SGK1 inactivates GSK3, which downregulates Snail, a DNA-binding molecule repressing the transcription of nephrin, a protein critically important for the integrity of the glomerular slit membrane. PKB/SGK-dependent GSK regulation is disrupted in mice carrying a mutation, in which the serine in the SGK/PKB-phosphorylation consensus sequence is replaced by alanine. The present study explored whether PKB/SGK-dependent GSK3 regulation influences glomerular proteinuria. Gene-targeted knockin mice with mutated and thus PKB/SGK-resistant GSK3alpha,beta (gsk3(KI)) were compared with their wild-type littermates (gsk3(WT)). gsk3(KI) and gsk3(WT) mice were implanted with DOCA release pellets and offered 1% saline as drinking water for 21 days. Under standard diet, tap water intake and absence of DOCA, urinary flow rate, glomerular filtration rate, and urinary albumin excretion were significantly larger and blood pressure was significantly higher in gsk3(KI) than in gsk3(WT) mice. Within 18 days, DOCA/salt treatment significantly increased fluid intake and urinary flow rate, urinary protein and albumin excretion, and blood pressure in both genotypes but the respective values were significantly higher in gsk3(KI) than in gsk3(WT) mice. Plasma albumin concentration was significantly lower in gsk3(KI) than in gsk3(WT) mice. Proteinuria was abrogated by lowering of blood pressure with alpha(1)-blocker prazosin (1 microg/g body wt) in 8-mo-old mice. According to immunofluorescence, nephrin at 3 and 8 mo and podocin expression at 3 mo were significantly lower in gsk3(KI) than in gsk3(WT) mice. After 18 days, DOCA/salt treatment renal glomerular sclerosis and tubulointerstitial damage were significantly more pronounced in gsk3(KI) than in gsk3(WT) mice. The observations reveal that disruption of PKB/SGK-dependent regulation of GSK3 leads to glomerular injury with proteinuria, which may at least partially be secondary to enhanced blood pressure.

Topics: Animals; Blood Pressure; Desoxycorticosterone; Female; Gene Knock-In Techniques; Glomerular Filtration Rate; Glycogen Synthase Kinase 3; Immediate-Early Proteins; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Mutant Strains; Protein Serine-Threonine Kinases; Proteinuria; Proto-Oncogene Proteins c-akt

Pigment epithelium-derived factor (PEDF) is a glycoprotein with potent neuronal differentiating activity. We, along with others, have recently found that PEDF inhibits retinal hyperpermeability by counteracting the biological effects of vascular endothelial growth factor (VEGF). However, the protective role of PEDF against nephrotic syndrome (NS), a condition of hyperpermeability in the glomerular capillaries, remains to be elucidated. In this study, we investigated whether and how PEDF reduced proteinuria in rats with adriamycin (ADR)-induced nephropathy (ADN), an experimental model of NS.. ADN was induced by a single intravenous injection of doxorubicin hydrochloride (n = 12). Half the ADN rats were intravenously administrated human recombinant PEDF; the other half were given vehicle everyday for up to 14 days. Control rats (n = 6) received vehicle only.. In ADN, expression levels of PEDF in isolated glomeruli were significantly decreased, which were associated with a marked proteinuria and increased urinary excretion of nephrin, an index of podocyte damage. Loss of nephrin and decreased podocyte cell number and fusion of foot processes of podocytes with nuclear factor-kappa B (NF-kappaB) activation and VEGF overexpression were also observed in the glomeruli of rats with ADN. Intravenous administration of PEDF ameliorated all of these changes in ADN rats.. The present findings suggest that PEDF could reduce proteinuria by suppressing podocyte damage and decreased nephrin as well as increased VEGF expression in the glomeruli of ADN rats. Pharmacological up-regulation or substitution of PEDF may offer a promising therapeutic strategy for the treatment of nephrotic syndrome.

Topics: Animals; Disease Models, Animal; Doxorubicin; Eye Proteins; Humans; Injections, Intravenous; Kidney Glomerulus; Male; Membrane Proteins; Nephrotic Syndrome; Nerve Growth Factors; NF-kappa B; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Recombinant Proteins; Serpins; Vascular Endothelial Growth Factor A

Progressive proteinuria and glomerulosclerosis characterize chronic allograft nephropathy. However, the causes are not fully elucidated. Podocytes function to prevent proteinuria; injury to this glomerular cell leads to glomerulosclerosis. The potential role of podocytes in the failing transplanted kidney is unknown. A rat model of kidney transplantation, characterized by proteinuria and glomerulosclerosis, was utilized to examine the potential role of podocytes.. Archival tissue was examined from allografts (Dark Agouti kidneys transplanted into operationally tolerant Albino Surgery rats), isografts (Dark Agouti) and controls (Dark Agouti: age-matched or after unilateral nephrectomy). The number of podocytes (by WT-1 staining) as well as the podocyte proteins podocin, nephrin and synaptopodin (by immunostaining) were measured at days 0, 2, 6 and at 6 months after transplantation. Changes in these parameters were compared between groups and correlated with urinary protein excretion.. At 6 months, podocyte number was reduced in allografted kidneys, accompanied by a decrease in nephrin and synaptopodin, but not podocin staining. Remnant kidneys in the uninephrectomized rats also showed a decreased podocyte number but no change in podocyte protein staining. Podocyte loss in allografts was established on day 6, whereas a decrease in nephrin and synaptopodin was not evident until 6 months. In contrast, podocyte number and protein staining was decreased but not significantly so in remnant and isografted kidneys.. A decrease in the slit diaphragm proteins, nephrin and synaptopodin, is a component of chronic allograft pathology.

Topics: Animals; Cell Count; Chronic Disease; Creatinine; Disease Models, Animal; Glomerulonephritis; Kidney Transplantation; Male; Membrane Proteins; Microfilament Proteins; Podocytes; Proteinuria; Rats; Rats, Inbred Strains; Transplantation, Homologous

Recent studies have reported that statins have renoprotective effects, independent from lowering plasma cholesterol. In this study, we examined whether statins were beneficial in a murine model of HIV-associated nephropathy (HIVAN).. We used conditional transgenic mice that express one of the HIV-1 accessory genes, vpr, selectively in podocytes using podocin promoter and the Tet-on system. These mice develop aggressive collapsing focal segmental glomerular sclerosis with massive proteinuria and deterioration of renal function within 4 weeks following heminephrectomy and doxycycline administration. Fluvastatin was administrated simultaneously with doxycycline, and the effect was compared with untreated controls after 4 weeks.. Fluvastatin at 10 mg/kg/day significantly decreased urinary albumin excretion (87 versus 11 mg/day, P < 0.01) and glomerular sclerosis (2.4 versus 1.0, P < 0.01, assessed by semi-quantitative scoring: 0-4). Fluvastatin also decreased serum creatinine and total cholesterol, but these differences were not statistically significant (0.36 versus 0.32 mg/dl, P = 0.35; 492 versus 378 mg/dl, P = 0.11, respectively). Phenotypic changes in podocytes, as indicated by the downregulation of nephrin, Wilms' tumour 1 and synaptopodin, along with upregulation of proliferating cell nuclear antigen, were attenuated by fluvastatin, suggesting its protective effects against podocyte injuries. In cultured podocytes, angiotensin II treatment decreased nephrin expression to 13% of basal levels, which was reversed to 58% by adding fluvastatin.. In conclusion, fluvastatin was effective in treating experimental HIVAN. The beneficial effect of this drug might be caused, in part, by preserving nephrin expression in podocytes against angiotensin II-mediated injury.

Topics: AIDS-Associated Nephropathy; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Blotting, Western; Cells, Cultured; Disease Models, Animal; Fatty Acids, Monounsaturated; Fluorescent Antibody Technique; Fluvastatin; Genes, Wilms Tumor; Glomerulosclerosis, Focal Segmental; Immunoenzyme Techniques; Indoles; Kidney; Male; Membrane Proteins; Mice; Mice, Transgenic; Microfilament Proteins; Nephrectomy; Phenotype; Podocytes; Proteinuria

Topics: Actins; Albuminuria; Capillaries; Child; Edema; Humans; Hyperlipidemias; Incidence; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Nephrotic Syndrome; Phenotype; Proteinuria; Renal Circulation; RNA, Transfer, Leu

The relationship between vascular endothelial growth factor (VEGF) and the phosphorylated critical podocyte slit diaphragm molecule nephrin is not fully clarified. This study investigated the dynamic changes in VEGF expression and nephrin phosphorylation, and the effects of the antiproteinuric drugs prednisone and lisinopril on them in Adriamycin nephropathy rats.. Renal tissues from Adriamycin rats were collected at days 3, 7, 14, and 28. Distribution and expression of VEGF was revealed by immunohistochemistry, real-time PCR and Western blot. Phosphorylated nephrin was evaluated by immunoprecipitation.. A discontinuous redistribution of VEGF was displayed at day 3, followed by significant protein reduction at day 7 with persistent downregulation to day 28. Phosphorylated nephrin decreased evidently at day 14 and persisted to day 28. The reduction in VEGF and phosphorylated nephrin was not a result of podocyte loss. The intervention of prednisone and lisinopril evidently reduced proteinuria, effectively attenuated the severe lesions of podocyte foot processes, and restored the reduction in VEGF and nephrin phosphorylation. At day 28, the reduction in VEGF and phosphorylated nephrin was negatively correlated with proteinuria, whereas the phosphorylated nephrin was positively correlated with VEGF protein from day 7 to day 28.. The reduction in VEGF protein and nephrin phosphorylation was possibly involved in the proteinuria in Adriamycin rats, and there might be some relationship between VEGF and nephrin phosphorylation. The antiproteinuric effects of lisinopril and prednisone were achieved at least partially by restoring VEGF protein and nephrin phosphorylation.

Topics: Animals; Doxorubicin; Kidney Diseases; Male; Membrane Proteins; Phosphorylation; Proteinuria; Rats; Rats, Sprague-Dawley; Vascular Endothelial Growth Factor A

The renoprotective effect of cilnidipine ((+/-)-2-methoxyethyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate, CAS 132203-70-4), a L/N-type calcium channel antagonist, on puromycin aminonucleoside (PAN)-induced nephrosis was investigated in rats. In the Experiment I, rats were given an intravenous injection of PAN (70 mg/kg). Cilnidipine (3 mg/kg/day) and enalapril (CAS 75847-73-3, 5 mg/kg/day) were administered orally from 6 days after treatment with PAN (day 6) to day 26, and urinary analysis was performed on days 9, 15, 20 and 27. In the Experiment II, nephrosis was also induced by intravenous injection of PAN (70 or 100 mg/kg) in rats which were treated with cilnidipine and enalapril from days 6 to 10. Systolic blood pressure was measured on day 7 and urinary analysis was performed on day 10. On day 11, serum was collected and the kidneys were removed for immunofluorescence staining for nephrin and podocin proteins. In PAN-treated rats, the daily urinary protein excretion was dramatically elevated on day 5, reached a peak on day 9 and gradually returned to a normal level from days 15 to 27. Cilnidipine (3 mg/kg/ day) significantly suppressed the increase in proteinuria on day 9 and also improved the decrease in creatinine clearance without evident effect on the blood pressure. Furthermore, the elevations in serum total cholesterol and triglyceride tended to be suppressed by cilnidipine. The expression of nephrin and podocin proteins in PAN-treated rats showed the granular pattern in the glomeruli, while the intensity of staining seemed to be dependent on the urinary protein excretion level in the cilnidipine-treated rats. The results obtained in this study suggest a renoprotective effect of cilnidipine in PAN-induced nephrosis in rats.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antimetabolites, Antineoplastic; Blood Pressure; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Channels, N-Type; Creatinine; Dihydropyridines; Enalapril; Fluorescent Antibody Technique; Male; Membrane Proteins; Nephrosis; Protective Agents; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley

Focal segmental glomerulosclerosis (FSGS) is a progressive renal disease, and the glomerular visceral cell hyperplasia typically observed in cellular/collapsing FSGS is an important pathological factor in disease progression. However, the cellular features that promote FSGS currently remain obscure. To determine both the origin and phenotypic alterations in hyperplastic cells in cellular/collapsing FSGS, the present study used a previously described FSGS model in p21-deficient mice with visceral cell hyperplasia and identified the podocyte lineage by genetic tagging. The p21-deficient mice with nephropathy showed significantly higher urinary protein levels, extracapillary hyperplastic indices on day 5, and glomerular sclerosis indices on day 14 than wild-type controls. X-gal staining and immunohistochemistry for podocyte and parietal epithelial cell (PEC) markers revealed progressive podocytopenia with capillary collapse accompanied by PEC hyperplasia leading to FSGS. In our investigation, non-tagged cells expressed neither WT1 nor nestin. Ki-67, a proliferation marker, was rarely associated with podocytes but was expressed at high levels in PECs. Both terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy failed to show evidence of significant podocyte apoptosis on days 5 and 14. These findings suggest that extensive podocyte loss and simultaneous PEC hyperplasia is an actual pathology that may contribute to the progression of cellular/collapsing FSGS in this mouse model. Additionally, this is the first study to demonstrate the regulatory role of p21 in the PEC cell cycle.

Topics: Animals; Apoptosis; Cell Lineage; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Glomerular Filtration Rate; Glomerulosclerosis, Focal Segmental; Hyperplasia; In Situ Nick-End Labeling; Integrases; Ki-67 Antigen; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Knockout; Podocytes; Proteinuria; WT1 Proteins

Within the glomerulus, the scaffolding protein nephrin bridges the actin-rich foot processes that extend from adjacent podocytes to form the slit diaphragm. Mutations affecting a number of slit diaphragm proteins, including nephrin, cause glomerular disease through rearrangement of the actin cytoskeleton and disruption of the filtration barrier. We recently established that the Nck family of Src homology 2 (SH2)/SH3 cytoskeletal adaptor proteins can mediate nephrin-dependent actin reorganization. Formation of foot processes requires expression of Nck in developing podocytes, but it is unknown whether Nck maintains podocyte structure and function throughout life. Here, we used an inducible transgenic strategy to delete Nck expression in adult mouse podocytes and found that loss of Nck expression rapidly led to proteinuria, glomerulosclerosis, and altered morphology of foot processes. We also found that podocyte injury reduced phosphorylation of nephrin in adult kidneys. These data suggest that Nck is required to maintain adult podocytes and that phosphotyrosine-based interactions with nephrin may occur in foot processes of resting, mature podocytes.

Topics: Adaptor Proteins, Signal Transducing; Animals; Anti-Bacterial Agents; Cell Line; Disease Models, Animal; Doxycycline; Glomerular Filtration Rate; Glomerulonephritis; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Transgenic; Oncogene Proteins; Phosphorylation; Podocytes; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Tight Junctions

Elevated level of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with end-stage renal diseases. Hcy metabolizes in the body to produce hydrogen sulfide (H(2)S), and studies have demonstrated a protective role of H(2)S in end-stage organ failure. However, the role of H(2)S in HHcy-associated renal diseases is unclear. The present study was aimed to determine the role of H(2)S in HHcy-associated renal damage. Cystathionine-beta-synthase heterozygous (CBS+/-) and wild-type (WT, C57BL/6J) mice with two kidney (2-K) were used in this study and supplemented with or without NaHS (30 micromol/l, H(2)S donor) in the drinking water. To expedite the HHcy-associated glomerular damage, uninephrectomized (1-K) CBS(+/-) and 1-K WT mice were also used with or without NaHS supplementation. Plasma Hcy levels were elevated in CBS(+/-) 2-K and 1-K and WT 1-K mice along with increased proteinuria, whereas, plasma levels of H(2)S were attenuated in these groups compared with WT 2-K mice. Interestingly, H(2)S supplementation increased plasma H(2)S level and normalized the urinary protein secretion in the similar groups of animals as above. Increased activity of matrix metalloproteinase (MMP)-2 and -9 and apoptotic cells were observed in the renal cortical tissues of CBS(+/-) 2-K and 1-K and WT 1-K mice; however, H(2)S prevented apoptotic cell death and normalized increased MMP activities. Increased expression of desmin and downregulation of nephrin in the cortical tissue of CBS(+/-) 2-K and 1-K and WT 1-K mice were ameliorated with H(2)S supplementation. Additionally, in the kidney tissues of CBS(+/-) 2-K and 1-K and WT 1-K mice, increased superoxide (O(2)(*-)) production and reduced glutathione (GSH)-to-oxidized glutathione (GSSG) ratio were normalized with exogenous H(2)S supplementation. These results demonstrate that HHcy-associated renal damage is related to decreased endogenous H(2)S generation in the body. Additionally, here we demonstrate with evidence that H(2)S supplementation prevents HHcy-associated renal damage, in part, through its antioxidant properties.

Topics: Animals; Antioxidants; Apoptosis; Cystathionine beta-Synthase; Desmin; Disease Models, Animal; Glutathione; Glutathione Disulfide; Homocysteine; Hydrogen Sulfide; Hyperhomocysteinemia; Kidney; Kidney Failure, Chronic; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nephrectomy; Oxidative Stress; Proteinuria; Sulfides; Superoxides

Increasing evidence implicates activation of NF-kappaB in a variety of glomerular diseases, but the mechanisms involved are unknown. Here, upregulation of NF-kappaB in the podocytes of transgenic mice resulted in glomerulosclerosis and proteinuria. Absence of the podocyte protein nephrin resulted in NF-kappaB activation, suggesting that nephrin negatively regulates the NF-kappaB pathway. Signal transduction assays supported a functional relationship between nephrin and NF-kappaB and suggested the involvement of atypical protein kinase C (aPKCzeta/lambda/iota) as an intermediary. We propose that disruption of the slit diaphragm leads to activation of NF-kappaB; subsequent upregulation of NF-kappaB-driven genes results in glomerular damage mediated by NF-kappaB-dependent pathways. In summary, nephrin may normally limit NF-kappaB activity in the podocyte, suggesting a mechanism by which it might discourage the evolution of glomerular disease.

Topics: Animals; Apoptosis Regulatory Proteins; Cell Line; Cytoplasm; Dogs; Gene Silencing; Glomerulonephritis; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Knockout; NF-kappa B; Podocytes; Protein Kinase C; Proteinuria; Transcription Factor RelA

Induction of heme oxygenase (HO)-1 is a key defense mechanism against oxidative stress. Compared with tubules, glomeruli are refractory to HO-1 upregulation in response to injury. This can be a disadvantage as it may be associated with insufficient production of cytoprotective heme-degradation metabolites. We, therefore, explored whether 1) targeted HO-1 expression can be achieved in glomeruli without altering their physiological integrity and 2) this expression reduces proteinuria in immune injury induced by an anti-glomerular basement membrane (GBM) antibody (Ab). We employed a 4.125-kb fragment of a mouse nephrin promoter downstream to which a FLAG-tagged hHO-1 cDNA sequence was inserted and subsequently generated transgenic mice from the FVB/N parental strain. There was a 16-fold higher transgene expression in the kidney than nonspecific background (liver) while the transprotein immunolocalized in glomerular epithelial cells (GEC). There was no change in urinary protein excretion, indicating that GEC-targeted HO-1 expression had no effect on glomerular protein permeability. Urinary protein excretion in transgenic mice with anti-GBM Ab injury (days 3 and 6) was significantly lower compared with wild-type controls. There was no significant change in renal expression levels of profibrotic (TGF-beta1) or anti-inflammatory (IL-10) cytokines in transgenic mice with anti-GBM Ab injury. These observations indicate that GEC-targeted HO-1 expression does not alter glomerular physiological integrity and reduces proteinuria in glomerular immune injury.

Topics: Animals; Autoantibodies; Disease Models, Animal; Epithelial Cells; Gene Expression Regulation, Enzymologic; Genetic Vectors; Glomerulonephritis; Heme Oxygenase-1; Interleukin-10; Kidney Glomerulus; Macrophages; Membrane Proteins; Mice; Mice, Transgenic; Permeability; Promoter Regions, Genetic; Proteinuria; Transforming Growth Factor beta1

Monocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes.. Expression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting.. In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin.. These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes.

Topics: Animals; Biopsy; Cells, Cultured; Chemokine CCL2; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Down-Regulation; Humans; In Vitro Techniques; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Phosphoproteins; Podocytes; Proteinuria; Recombinant Proteins; rho-Associated Kinases; RNA, Messenger; Zonula Occludens-1 Protein

A lot of mutations of podocin, a key protein of podocyte slit diaphragm (SD), have been found both in hereditary and sporadic focal segmental glomeruloscleorosis (FSGS). Nevertheless, the mechanisms of podocyte injury induced by mutant podocins are still unclear. A compound heterozygous podocin mutation was identified in our FSGS patient, leading to a truncated (podocin (V165X)) and a missense mutant protein (podocin (R168H)), respectively. Here, it was explored whether and how both mutant podocins induce podocyte injury in the in vitro cultured podocyte cell line. Our results showed that podocin (R168H) induced more significant podocyte apoptosis and expression changes in more podocyte molecules than podocin (V165X). Podocyte injury caused by the normal localized podocin(V165X) was effectively inhibited by TRPC6 knockdown. The abnormal retention of podocin(R168H) in endoplasmic reticulum (ER) resulted in the mis-localizations of other critical SD molecules nephrin, CD2AP and TRPC6, and significantly up-regulated ER stress markers Bip/grp78, p-PERK and caspase-12. These results implicated that podocin (R168H) and podocin (V165X) induced different degrees of podocyte injury, which might be resulted from different molecular mechanisms. Our findings provided some possible clues for further exploring the pharmacological targets to the proteinuria induced by different mutant podocins.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cells, Cultured; Cytoskeletal Proteins; Endoplasmic Reticulum Chaperone BiP; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Membrane Proteins; Mice; Mutation; Podocytes; Proteinuria; TRPC Cation Channels; TRPC6 Cation Channel

Slit diaphragm and/or podocyte's cytoskeleton alterations are related to proteinuria and nephrotic syndrome. In our population, focal and segmental glomerulosclerosis causing nephrotic syndrome is the more frequent biopsy demonstrated glomerulopathy. Our aim was search for alterations in some slit diaphragm-associated proteins in patients with nephrotic range proteinuria.. Renal tissue from 40 patients with nephrotic range proteinuria, 10 patients with non-nephrotic proteinuria, 3 with isolated hematuria, and 10 samples of normal renal tissue (deceased donors) were studied, by indirect immunofluorescence, for expression of nephrin, podocin, and alpha-actinin-4.. Expression of these proteins was lineal, homogeneous, in the glomerular capillary walls in normal renal tissue and in patients with isolated hematuria. In nephrotic proteinuria this normal appearance was altered and immunostaining showed a fine granular appearance. In 18 cases (45%) of patients with nephrotic proteinuria and 3 cases (30%) of patients with non-nephrotic proteinuria there was loss of at least one of these proteins (p = 0.49). These alterations were found in the diverse glomerulopathies more frequently causing nephrotic syndrome.. In nephrotic range proteinuria redistribution or loss of expression of slit diaphragm-associated proteins is very frequent. In many of our cases this fact could be more a consequence than a cause of proteinuria. These alterations can be also evidenced in patients with non-nephrotic proteinuria.

Topics: Actinin; Adolescent; Adult; Aged; Aged, 80 and over; Biopsy; Child; Child, Preschool; Female; Humans; Infant; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Middle Aged; Proteinuria; Young Adult

Extracts of Tripterygium wilfordii Hook F have been used to treat glomerulonephritis for more than 30 years in China with dramatic antiproteinuric effects. Triptolide, a diterpene triepoxide, is one of the major active components of these extracts. To clarify its antiproteinuric effects we induced podocyte injury by puromycin aminonucleoside. Triptolide effectively reduced the proteinuria induced by puromycin in nephrotic rats without reducing the glomerular filtration rate. The antiproteinuric effect was associated with improvement in the foot process effacement, a decrease in the podocyte injury marker desmin as well as the restoration of nephrin and podocin expression and distribution. In cultured mouse podocytes triptolide pretreatment prevented the puromycin-induced disruption of the actin cytoskeleton and microfilament-associated synaptopodin while protecting nephrin and podocin expression. Triptolide suppressed reactive oxygen species generation and p38 mitogen-activated protein kinase activation while restoring RhoA signaling activity. These results show that triptolide ameliorates puromycin aminonucleoside-mediated podocyte injury in vivo and in vitro.

Topics: Animals; Cells, Cultured; Cholesterol; Cytoskeleton; Desmin; Diterpenes; Epoxy Compounds; Glomerular Filtration Rate; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Membrane Proteins; Mice; Nephrosis; Phenanthrenes; Podocytes; Proteinuria; Puromycin Aminonucleoside; Rats; Reactive Oxygen Species; Serum Albumin; Triglycerides

Focal segmental glomerulosclerosis (FSGS) is a disease showing severe proteinuria, and the disease progresses to end-stage kidney failure in many cases. However, the pathogenic mechanism of FSGS is not well understood. The slit diaphragm (SD), which bridges the neighboring foot processes of glomerular epithelial cells, is understood to function as a barrier of the glomerular capillary wall. To investigate the role of SD dysfunction in the development of FSGS, we analyzed the expression of SD-associated molecules in rat adriamycin-induced nephropathy, a mimic of FSGS. The staining of the SD molecules nephrin, podocin, and NEPH1 had already shifted to a discontinuous dotlike pattern at the initiation phase of the disease, when neither proteinuria nor any morphological alterations were detected yet. The alteration of NEPH1 expression was the most evident among the molecules examined, and NEPH1 was dissociated from nephrin at the initiation phase. On day 28, when severe proteinuria was detected and sclerotic changes were already observed, alteration of the expressions of nephrin, podocin, and NEPH1 worsened, but no alteration in the expression of other SD-associated molecules or other podocyte molecules was detected. It is postulated that the dissociation of NEPH1 from nephrin initiates proteinuria and that the SD alteration restricted in these molecules plays a critical role in the development of sclerotic changes in FSGS.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibodies; Blotting, Western; Cloning, Molecular; Cytoskeletal Proteins; Doxorubicin; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Glomerulosclerosis, Focal Segmental; Integrins; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Kidney Tubules; Membrane Proteins; Molecular Sequence Data; Phosphoproteins; Podocytes; Protein Binding; Proteinuria; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Sialoglycoproteins; Zonula Occludens-1 Protein

Semaphorin3a was discovered as a secreted guidance protein that acts as a chemorepellent to migrating axons and endothelial cells. In the adult mouse kidney, it is expressed in podocytes and collecting tubules. Here, we show that exogenous semaphorin3a caused acute nephrotic range proteinuria associated with podocyte foot process effacement and fusion, endothelial cell damage, decreased vascular endothelial growth factor-A receptor expression, and downregulation of the slit-diaphragm proteins podocin, nephrin, and CD2-associated protein. When vascular endothelial growth factor 165 was administered at the same time as Semaphorin3a, no proteinuria or renal ultrastructural abnormalities occurred, suggesting that semaphorin3a effects may be mediated, in part, by downregulation of vascular endothelial growth factor receptor 2 signaling. Our findings indicate that a balance of semaphorin3a to vascular endothelial growth factor-A may be important for glomerular filtration barrier homeostasis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cytoskeletal Proteins; Down-Regulation; Glomerular Basement Membrane; Glomerular Filtration Rate; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred Strains; Permeability; Podocytes; Proteinuria; Recombinant Proteins; Semaphorin-3A; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Impaired glomerular endothelial integrity is pivotal in various renal diseases and depends on both the degree of glomerular endothelial injury and the effectiveness of glomerular endothelial repair. Glomerular endothelial repair is, in part, mediated by bone marrow-derived endothelial progenitor cells. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists have therapeutic actions independent of their insulin-sensitizing effects, including enhancement of endothelial progenitor cell function and differentiation. We evaluated the effect of PPAR-gamma agonist rosiglitazone (4 mg.kg(-1).day(-1)) on the course of anti-Thy1-glomerulonephritis in rats. Rosiglitazone limited the development of proteinuria and prevented plasma urea elevation (8.1 +/- 0.4 vs. 12.5 +/- 1.1 mmol/l, P = 0.002). Histologically, inflammatory cell influx was not affected, but rosiglitazone-treated rats did show fewer microaneurysmatic glomeruli on day 7 (26 +/- 3 vs. 41 +/- 5%, P = 0.01) and reduced activation of matrix production with reduced renal cortical transforming growth factor-beta, plasminogen activator inhibitor type 1, and fibronectin-1 mRNA expression. However, bone marrow-derived endothelial cell glomerular incorporation was not enhanced (3.1 +/- 0.4 vs. 3.6 +/- 0.3 cells/glomerular cross section; P = 0.31). Rosiglitazone treatment in nonnephritic rats did not influence proteinuria, urea, or renal histology. In conclusion, treatment with PPAR-gamma agonist rosiglitazone ameliorates the course of experimental glomerulonephritis in a nondiabetic model, but not through enhancing incorporation of bone marrow-derived endothelial cells in the glomerulus.

Topics: Aneurysm; Animals; Blood Pressure; Bone Marrow Transplantation; Cell Movement; Disease Models, Animal; Endothelial Cells; Extracellular Matrix; Fibronectins; Gene Expression; Glomerulonephritis, Membranous; Hypoglycemic Agents; Isoantibodies; Kidney Cortex; Kidney Glomerulus; Male; Membrane Proteins; Plasminogen Activator Inhibitor 1; PPAR gamma; Proteinuria; Rats; Rats, Inbred BN; Rosiglitazone; Stem Cells; Thiazolidinediones; Transforming Growth Factor beta; Urea

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte beta1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin beta1 (podocin-Cre beta1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre beta1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin beta1 activity in epithelial cells. To further explore whether integrin beta1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte beta1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.

Topics: Animals; Animals, Newborn; Antigens, CD; Basement Membrane; beta-Galactosidase; Carrier Proteins; Cell Cycle Proteins; Crosses, Genetic; DNA-Binding Proteins; Embryo, Mammalian; Endothelium; Epithelial Cells; Extracellular Matrix; Fluorescent Dyes; Gene Deletion; Genes, Reporter; Glomerulosclerosis, Focal Segmental; Indoles; Integrases; Integrin beta3; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Luminescent Proteins; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Mutant Strains; Mice, Transgenic; Nuclear Proteins; Podocytes; Protein Serine-Threonine Kinases; Proteinuria; Renal Insufficiency; Rhodamines; RNA Splicing Factors; Signal Transduction; Tetraspanin 29; Time Factors; Transgenes

Podocyte dysfunction plays an essential role in the pathogenesis of proteinuria and glomerulosclerosis. However, the mechanism underlying podocyte dysfunction in many common forms of chronic kidney diseases remains poorly understood. Here we tested the hypothesis that podocytes may undergo epithelial-to-mesenchymal transition after injury. Conditionally immortalized mouse podocytes were incubated with transforming growth factor (TGF)-beta1, a potent fibrogenic cytokine that is up-regulated in the diseased kidney. TGF-beta1 suppressed the slit diaphragm-associated protein P-cadherin, zonula occludens-1, and nephrin, a change consistent with loss of the epithelial feature. Meanwhile, TGF-beta1 induced the expression of the intermediate filament protein desmin and interstitial matrix components fibronectin and collagen I. Furthermore, TGF-beta1 promoted the expression and secretion of matrix metalloproteinase-9 by podocytes. Functionally, TGF-beta1 increased albumin permeability across podocyte monolayers, as demonstrated by a paracellular albumin influx assay. The expression of Snail, a key transcriptional factor that has been implicated in initiating epithelial-to-mesenchymal transition, was induced by TGF-beta1, and ectopic expression of Snail suppressed P-cadherin and nephrin in podocytes. In vivo, in addition to loss of nephrin and zonula occludens-1, mesenchymal markers such as desmin, fibroblast-specific protein-1, and matrix metalloproteinase-9 could be observed in glomerular podocytes of diabetic nephropathy. These results suggest that podocyte dedifferentiation and mesenchymal transition could be a potential pathway leading to their dysfunction, thereby playing a role in the genesis of proteinuria.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Cell Dedifferentiation; Cell Line; Desmin; Fluorescent Antibody Technique; Humans; Male; Matrix Metalloproteinase 9; Membrane Proteins; Mesoderm; Mice; Phosphoproteins; Podocytes; Proteinuria; Reverse Transcriptase Polymerase Chain Reaction; S100 Calcium-Binding Protein A4; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta1; Zonula Occludens-1 Protein

In in vitro studies, angiotensin (Ang) II has been demonstrated to promote podocyte apoptosis. The present study evaluates the effects of Ang II infusion in rats on podocyte nephrin expression and apoptosis and the molecular mechanisms involved in Ang II-induced proteinuria and mesangial expansion.. Sprague-Dawley rats were randomly assigned to receive either normal saline or Ang II (400 ng x kg(-1) x min(-1)) by means of a mini-osmotic pump for variable time periods. Systolic blood pressure and urinary protein and albumin excretion rate measurements were carried out on days 7, 14, 21, and 28. The animals were sacrificed on days 14 and 28 and evaluated for serum creatinine, renal pathological changes, podocyte apoptosis, renal nephrin mRNA, and protein expression.. The Ang II-infused rats developed hypertension and proteinuria. On day 14, the Ang II-infused rats showed narrowing of the slit diaphragm, an increase in podocyte nephrin mRNA and protein expression, and alterations in its distribution along the foot processes. On day 28, the Ang II-infused rats demonstrated the presence of apoptotic podocytes and decreased nephrin mRNA and protein expression. There was a negative correlation between nephrin expression and the numbers of apoptotic podocytes (r = -0.63, p < 0.05).. These results suggest that changes in nephrin expression may play a role in the pathogenesis of Ang II-induced podocyte apoptosis.

Topics: Angiotensin II; Animals; Apoptosis; Blood Pressure; Creatinine; Hypertension; In Situ Nick-End Labeling; Male; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; RNA, Messenger

Topics: Biopsy; Endothelium; Female; Humans; Kidney Glomerulus; Membrane Proteins; Podocytes; Pre-Eclampsia; Pregnancy; Proteinuria; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

Preeclampsia is a pregnancy-specific disorder characterized by hypertension and proteinuria. In other disease states, proteinuria has been linked to altered expressions of podocyte foot-process proteins, but this has not been studied in women with preeclampsia. We sought to test the hypothesis that proteinuria in preeclampsia is associated with dysregulated expression of the podocyte cytoskeleton and/or tight junction proteins.. Renal tissue was obtained from autopsy material from seven women who had severe preeclampsia during the second half of their pregnancies up to 48 h after delivery, and who subsequently died. As controls, we used autopsy material from two women who died accidentally during the second half of their otherwise normal pregnancies. Immunohistochemical stains for nephrin, synaptopodin and podocin were performed on representative sections prepared from paraffin-embedded material.. Expression of both nephrin and synaptopodin was markedly decreased in preeclamptic compared with control kidney sections. By contrast, both cases and controls demonstrated strong staining for podocin.. We conclude that down-regulation of nephrin and synaptopodin is associated with proteinuria in women with preeclampsia. Recent studies have demonstrated that soluble vascular endothelial growth factor receptor 1 (sFlt-1) levels are elevated in preeclampsia compared with normal pregnancy. Studies in mice have shown that sFlt-1 may play a role in inducing proteinuria by neutralizing vascular endothelial growth factor (VEGF) and suppressing nephrin. Proteinuria and elevations of sFlt-1 in preeclampsia are temporally related, further supporting a possible role of sFlt-1 in the dysregulation of podocyte foot-process proteins.

Topics: Adolescent; Adult; Animals; Antibodies; Biopsy; Case-Control Studies; Disease Models, Animal; Female; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred Strains; Microfilament Proteins; Pre-Eclampsia; Pregnancy; Proteinuria; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

Topics: Age of Onset; Amino Acid Sequence; Animals; Biopsy; Child; Chromosome Mapping; Chromosomes, Human, Pair 19; Female; Humans; Male; Membrane Proteins; Molecular Sequence Data; Nephrotic Syndrome; Pedigree; Proteinuria; Sequence Alignment; Sequence Homology, Amino Acid

Topics: Adenosine Triphosphate; Cell Differentiation; Endoplasmic Reticulum; Energy Metabolism; Humans; Integrin beta1; Membrane Proteins; Podocytes; Proteinuria

Topics: Adaptor Proteins, Signal Transducing; Angiotensin-Converting Enzyme Inhibitors; Cytoskeletal Proteins; Diaphragm; Humans; Kidney Glomerulus; Membrane Proteins; Nephrosis; Nephrotic Syndrome; Podocytes; Proteinuria

Filtrin (NEPH3/KIRREL2) is a recently characterized member of the nephrin-like proteins of the immunoglobulin superfamily, and it has been suggested to participate in the maintenance of the glomerular filtration barrier in the kidney. In this study, the gene and protein expression of filtrin were examined in patients with acquired proteinuric diseases.. Filtrin mRNA levels in renal biopsies were measured with quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in two sets of patients with proteinuria. The mRNA levels were normalized to the housekeeping gene GAPDH and also related to the podocyte-specific genes nephrin and podocin. Immunofluorescence microscopy was employed to explore changes in the glomerular distribution of filtrin.. Reduced glomerular expression of filtrin mRNA was observed in all studied diagnostic groups. In focal segmental glomerulosclerosis, the filtrin mRNA level was only one-tenth of the control samples (P approximately 5.0x10(-6)), and this finding was confirmed in a second set of samples. The ratios of filtrin to nephrin and podocin demonstrated a marked decrease in the expression of filtrin relative to the podocyte marker genes. However, no correlation between the expression of filtrin and the levels of serum creatinine and proteinuria was observed. Immunostaining showed changes in the expression pattern of filtrin in renal biopsies. Immunoelectron microscopic studies localized filtrin at the slit diaphragm of the podocyte foot processes.. Down-regulation of the filtrin gene and protein expression in the renal biopsies together with the localization to the inter-podocyte filtration slit imply a potential role for this molecule in the pathogenesis of proteinuric diseases.

Topics: Biopsy; Fluorescent Antibody Technique; Humans; Immunoglobulins; Kidney; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Microscopy, Immunoelectron; Proteinuria; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Distribution

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.

Topics: Actins; Animals; Apoptosis; B7-1 Antigen; Cells, Cultured; Cytoskeleton; Darbepoetin alfa; Desmin; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; In Situ Nick-End Labeling; Male; Membrane Proteins; Nephrotic Syndrome; Podocytes; Protective Agents; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Wistar; Receptor Protein-Tyrosine Kinases; Receptors, Erythropoietin; RNA, Messenger; Time Factors

Although angiotensin II (Ang II) type 1 receptor antagonist ameliorates proteinuria, its pharmacological mechanism and the differential roles of Ang II type 1 receptor (AT1R) and type 2 receptor (AT2R) are not well understood. We analyzed the effect of Ang II type 1 receptor antagonist on proteinuria caused by antibody against nephrin, a functional molecule of glomerular slit diaphragm and dysfunction of which is involved in the development of proteinuria in several glomerular diseases. We show here that AT1R antagonist ameliorated proteinuria by preventing a reduction in the functional molecules of the slit diaphragm. We also analyzed the role of AT1R- or AT2R-mediated actions on the expression of the slit diaphragm molecules in an in vivo study of normal rat and in an in vitro study of cultured podocytes. AT1R-mediated action hampered the mRNA expression of the slit diaphragm molecules, whereas AT2R-mediated action enhanced it. These findings indicate that Ang II receptor subtypes play opposite roles in regulating the barrier function of glomerular capillary wall and that the enhancement of AT2R stimulation may serve as a potential therapeutic strategy for proteinuria.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensin II Type 2 Receptor Blockers; Animals; Antibodies, Monoclonal; Capillaries; Female; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Phosphoproteins; Podocytes; Proteinuria; Rats; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Sialoglycoproteins; Zonula Occludens-1 Protein

Studies in experimental animals and younger women suggest a protective role for estrogen; however, clinical trials may not substantiate this effect in older females. Therefore, the present study assessed the outcome of ovariectomy in older mRen2. Lewis rats subjected to a high-salt diet for 4 wk. Intact or ovariectomized (OVX, 15 wk of age) mRen2. Lewis rats were aged to 60 wk and then placed on a high-salt (HS, 8% sodium chloride) diet for 4 wk. Systolic blood pressures were similar between groups [OVX 169 +/- 6 vs. Intact 182 +/- 7 mmHg; P = 0.22] after the 4-wk diet; however, proteinuria [OVX 0.8 +/- 0.2 vs. Intact 11.5 +/- 2.6 mg/mg creatinine; P < 0.002, n = 6], renal interstitial fibrosis, glomerular sclerosis, and tubular casts were lower in OVX vs. Intact rats. Kidney injury molecule-1 mRNA, a marker of tubular damage, was 53% lower in the OVX HS group. Independent from blood pressure, OVX HS rats exhibited significantly lower cardiac (24%) and renal (32%) hypertrophy as well as lower C-reactive protein (28%). Circulating insulin-like growth factor-I (IGF-I) levels were not different between the Intact and OVX groups; however, renal cortical IGF-I mRNA and protein were attenuated in OVX rats [P < 0.05, n = 6]. We conclude that ovariectomy in the older female mRen2. Lewis rat conveys protection against salt-dependent increase in renal injury.

Topics: Aging; Angiotensin I; Angiotensin II; Animals; Animals, Congenic; Blood Pressure; C-Reactive Protein; Cell Adhesion Molecules; Disease Models, Animal; Female; Fibrosis; Hypertension; Hypertrophy; Insulin-Like Growth Factor I; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Membrane Proteins; Ovariectomy; Peptide Fragments; Proteinuria; Rats; Rats, Inbred Lew; Renin; Renin-Angiotensin System; RNA, Messenger; Sodium Chloride, Dietary

Proteins are modified and folded within the endoplasmic reticulum (ER). When the influx of proteins exceeds the capacity of the ER to handle the load, the ER is "stressed" and protein biogenesis is affected. We have previously shown that the induction of ER stress by ATP depletion in podocytes leads to mislocalization of nephrin and subsequent injury of podocytes. The aim of the present study was to determine whether ER stress is associated with proteinuria in vivo and whether the immunosuppressant mizoribine may exert its antiproteinuric effect by restoring normal nephrin biogenesis. Induction of nephrotic-range proteinuria with puromycin aminonucleoside in mice increased expression of the ER stress marker GRP78 in podocytes, and led to the mislocalization of nephrin to the cytoplasm. In vitro, mizoribine, through a mechanism likely dependent on the inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) activity in podocytes, restored the intracellular energy balance by increasing levels of ATP and corrected the posttranslational processing of nephrin. Therefore, we speculate that mizoribine may induce remission of proteinuria, at least in part, by restoring the biogenesis of slit diaphragm proteins in injured podocytes. Further understanding of the ER microenvironment may lead to novel approaches to treat diseases in which abnormal handling of proteins plays a role in pathogenesis.

Topics: Adenosine Triphosphate; Animals; Cells, Cultured; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Energy Metabolism; Humans; Immunosuppressive Agents; IMP Dehydrogenase; Intracellular Membranes; Kidney Glomerulus; Male; Membrane Proteins; Membrane Transport Proteins; Mice; Nephrotic Syndrome; Podocytes; Protein Processing, Post-Translational; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Ribonucleosides; RNA, Messenger; Stress, Physiological

To investigate the effect of podocyte ultrastructure and changes of nephrin and the podocin expression on the pathogenesis of diabetic nephropathy (DN) in rats.. Twenty Wistar rats were divided into 2 groups: a normal control group and a DN model group.We determined the 24h proteinuria and other biochemical indexes, measured the ultrastructures of podocytes with electronic microscope, and detected the expression of nephrin and podocin with immunohistochemical technique and RT-PCR at the 4th and 8th weeks.. The 24h proteinuria increased in the DN group; the number of podocytes was significantly lower; and the foot process width (FPW) obviously increased in the DN group compared with the normal group (P<0.01). The protein and mRNA expressions of nephrin and podocin reduced in the DN group.There was a negative correlation between the proteinuria and the protein expression of nephrin and podocin (P<0.01).. The reduction in glomerular podocyte number,the increased FPW, and the down-regulated expression of nephrin and podocin appear at the early stage of DN and become more serious with the disease progression.The podocyte lesion not only is associated with the degree of proteinuria,but also correlates with the development of glomerulosclerosis and damage of renal function.

Topics: Animals; Diabetic Nephropathies; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Wistar

Changes in podocyte number or density have been suggested to play an important role in renal disease progression. Here, we investigated the temporal relationship between glomerular podocyte number and development of proteinuria and glomerulosclerosis in the male Munich Wistar Fromter (MWF) rat. We also assessed whether changes in podocyte number affect podocyte function and focused specifically on the slit diaphragm-associated protein nephrin. Age-matched Wistar rats were used as controls. Estimation of podocyte number per glomerulus was determined by digital morphometry of WT1-positive cells. MWF rats developed moderate hypertension, massive proteinuria, and glomerulosclerosis with age. Glomerular hypertrophy was already observed at 10 weeks of age and progressively increased thereafter. By contrast, mean podocyte number per glomerulus was lower than normal in young animals and further decreased with time. As a consequence, the capillary tuft volume per podocyte was more than threefold increased in older rats. Electron microscopy showed important changes in podocyte structure of MWF rats, with expansion of podocyte bodies surrounding glomerular filtration membrane. Glomerular nephrin expression was markedly altered in MWF rats and inversely correlated with both podocyte loss and proteinuria. Our findings suggest that reduction in podocyte number is an important determinant of podocyte dysfunction and progressive impairment of the glomerular permselectivity that lead to the development of massive proteinuria and ultimately to renal scarring.

Topics: Animals; Blotting, Western; Disease Models, Animal; Glomerulosclerosis, Focal Segmental; Hypertension; Immunohistochemistry; Male; Membrane Proteins; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Podocytes; Proteinuria; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

We report a Caucasian boy of Italian descent with congenital nephrotic syndrome of the Finnish type (NPHS1, CNF, MIM 256300) who developed recurrence of proteinuria and hypoalbuminemia on the seventh post-operative day following living related renal transplantation from his paternal aunt. The allograft biopsy was normal except for effacement of podocyte foot processes on electron microscopy. He was treated by the substitution of mycophenolate mofetil with cyclophosphamide for 12 weeks, in addition to cyclosporine, prednisone and daclizumab. His proteinuria resolved quickly following the initiation of cyclophosphamide treatment, and he remains in remission 4 years after receiving his transplant. His native and allograft kidneys were evaluated for nephrin expression by immunohistochemistry, DNA analysis for the NPHS1 mutation, serum for the presence of auto-antibodies to nephrin by both enzyme-linked immunosorbent assay (ELISA) and fetal glomeruli immunofluorescence assay, and serum for glomerular permeability to albumin (Palb) activity using a functional in vitro assay for Palb. Nephrin expression was completely absent in the native kidney, while it was decreased in the allograft compared with normal. DNA analysis of the NPHS1 gene revealed mutations 3248G>T and 3250delG in exon 24, causing G1083V and 1084Vfs, respectively, inherited from his father, and 3478C>T in exon 27, that leads to R1160X, inherited from his mother. Serum was negative for auto-antibodies to nephrin. Interestingly, the Palb activity was increased at the time of recurrence of proteinuria following transplantation (Palb 0.73+/-0.10) and remained elevated when retested more than 3 years later (Palb 0.54+/-0.09). This is the first report of increased Palb activity in recurrence of proteinuria following transplantation in NPHS1. We speculate the role of increased Palb activity in the recurrence of proteinuria following transplantation in NPHS1.

Topics: Albumins; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Autoantibodies; Capillary Permeability; Cyclophosphamide; Cyclosporine; Daclizumab; Humans; Hypoalbuminemia; Immunoglobulin G; Immunosuppressive Agents; Infant, Newborn; Kidney Glomerulus; Kidney Transplantation; Living Donors; Male; Membrane Proteins; Mutation; Mycophenolic Acid; Nephrectomy; Nephrotic Syndrome; Peritoneal Dialysis; Prednisone; Proteinuria; Recurrence

Pax2 is a transcription factor with important functions during kidney development . Ectopic expression of Pax2 in podocytes has been reported in various glomerular diseases , but the functional relevance remains unknown. We developed an inducible mouse model that allows activation of Pax2 specifically in podocytes. Persistent expression of Pax2 did not interfere with the initial differentiation of podocytes, but mice ectopically expressing PAX2 developed end-stage renal failure soon after birth. Similarly, activation of PAX2 in healthy adult animals resulted in renal disease within 3 weeks after podocyte-specific induction of a deleter Cre. PAX2 activation caused repression of the podocyte key regulator molecule Wt1 and consequently a dramatic reduction of nephrin expression. Recruitment of the groucho-related protein TLE4 may be involved in converting Pax2 into a transcriptional repressor of Wt1. Finally, treatment of mice with an angiotensin-converting enzyme (ACE) inhibitor normalized renal function and induced upregulation of the important structural molecule nephrin via a Wt1-independent pathway. Our data demonstrate the functional significance of PAX2 reexpression in mature podocytes for the development of glomerular diseases and suggest that reactivation of PAX genes in terminally differentiated cells leads to a more dedifferentiated phenotype.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Disease Models, Animal; Enalapril; Gene Expression Regulation; Kidney Failure, Chronic; Male; Membrane Proteins; Mice; PAX2 Transcription Factor; Podocytes; Proteinuria; WT1 Proteins

Nephrin, podocin, CD2AP, and alpha-actinin-4 are important podocyte proteins that help maintain the integrity of the slit diaphragm and prevent proteinuria. Studies have shown that angiotensin-converting enzyme inhibitors, glucocorticoids, and all-trans retinoic acid (ATRA) have antiproteinuric effects. However, it is still unclear whether these drugs, with different pharmacological mechanisms, lead to a reduction in proteinuria by changing the expression and distribution of these important podocyte proteins. In this study, changes in the expression and distribution of nephrin, podocin, CD2AP, and alpha-actinin-4 were dynamically detected in Adriamycin-induced nephrotic (ADR) rats treated with three different drugs: lisinopril, prednisone, and ATRA. Nephropathy was induced by an intravenous injection of Adriamycin. After Adriamycin injection, rats received lisinopril, prednisone, and ATRA treatment, respectively. Renal tissues were collected at Days 3, 7, 14, and 28. The distribution and the expression of messenger RNA and protein of nephrin, podocin, CD2AP, and alpha-actinin-4 were detected by indirect immunofluorescence, real-time polymerase chain reaction, and Western blotting, respectively. With the intervention of lisinopril, prednisone, and ATRA, changes in the expression of nephrin, podocin, and CD2AP were diverse, which was different from that detected in ADR rats. After lisinopril and prednisone intervention, podocin exhibited prominent earlier changes compared with those of nephrin and CD2AP, whereas CD2AP showed more prominent changes after ATRA intervention. There was no change in the expression of alpha-actinin-4 molecule. In summary, we conclude that the antiproteinuric effects of lisinopril, prednisone, and ATRA were achieved by changes in the expression and distribution of the important podocyte molecules nephrin, podocin, CD2AP, and alpha-actinin-4. The pattern in the change of podocyte molecules after lisinopril and prednisone intervention was similar, but the pattern in the change of podocyte molecules after ATRA intervention was different from that of lisinopril or prednisone intervention.

Topics: Actinin; Adaptor Proteins, Vesicular Transport; Angiotensin-Converting Enzyme Inhibitors; Animals; Antibiotics, Antineoplastic; Doxorubicin; Glucocorticoids; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Lisinopril; Male; Membrane Proteins; Microfilament Proteins; Nephrosis; Podocytes; Prednisone; Proteinuria; Random Allocation; Rats; Rats, Sprague-Dawley; Tretinoin

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that maintains the glomerular and peritubular capillary (PTC) network in the kidney. The soluble form of the VEGF receptor-1 (soluble fms-like tyrosine kinase 1 (sFlt-1)) is known to regulate VEGF activity by binding VEGF in the circulation. We hypothesized that VEGF may be beneficial for maintaining glomerular filtration barrier and vascular network in rats with progressive glomerulonephritis (GN). For blockade of VEGF activity in vivo, rats were transfected twice with plasmid DNA encoding the murine sFlt-1 gene into femoral muscle 3 days before and 2 weeks after the induction of antiglomerular basement membrane antibody-induced GN. Inhibition of VEGF with sFlt-1 resulted in massive urinary protein excretion, concomitantly with downregulated expression of nephrin in nephritic rats. Further, blockade of VEGF induced mild proteinuria in normal rats. Administration of sFlt-1 affected neither the infiltration of macrophages nor crescentic formation. In contrast, treatment of sFlt-1 accelerated the progression of glomerulosclerosis and interstitial fibrosis accompanied with renal dysfunction and PTC loss at day 56. VEGF may play a role in maintaining the podocyte function as well as renal vasculature, thereby protecting glomeruli and interstitium from progressive renal insults.

Topics: Animals; Disease Progression; Glomerulonephritis; Male; Membrane Proteins; Proteinuria; Rats; Rats, Inbred WKY; Time Factors; Vascular Endothelial Growth Factor A

The renoprotective potential of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist pioglitazone was explored in an immune model of progressive nephropathy, passive Heymann nephritis (PHN), compared with that of an angiotensin II receptor antagonist, taken as standard therapy for renoprotection. PHN rats received orally vehicle, pioglitazone (10 mg/kg twice daily), or candesartan (1 mg/kg twice daily) from months 2 to 8. Pioglitazone reduced proteinuria as effectively as candesartan and limited renal functional and structural changes. Kidneys from untreated PHN rats showed lower nephrin mRNA and protein than controls, both restored by pioglitazone. The effect was seen both early and late during the course of the disease. Whether the antiproteinuric effect of pioglitazone could be due to its effect on nephrin gene transcription also was investigated. HK-2 cells were transfected with plasmids that harbor the luciferase gene under portions (2-kb or 325-bp) of human nephrin gene promoter that contain putative peroxisome proliferator-responsive elements (PPRE) and incubated with pioglitazone (10 muM). Transcriptional activity of luciferase gene was highly increased by pioglitazone, with the strongest expression achieved with the 325-bp fragment. Increase in luciferase activity was prevented by bisphenol A diglycidyl ether, a PPAR-gamma synthetic antagonist. Electrophoretic mobility shift assay experiments showed a direct interaction of PPAR/retinoid X receptor heterodimers to PPRE present in the enhancer region of the nephrin promoter. In conclusion, pioglitazone exerts an antiproteinuric effect in immune-mediated glomerulonephritis as angiotensin II receptor antagonist does. Enhancement of nephrin gene transcription through specific PPRE in its promoter discloses a novel mechanism of renoprotection for PPAR-gamma agonists.

Topics: Angiotensin Receptor Antagonists; Animals; Benzimidazoles; Biphenyl Compounds; Gene Expression Regulation; Humans; Hypoglycemic Agents; Kidney Diseases; Male; Membrane Proteins; Pioglitazone; PPAR gamma; Proteinuria; Rats; Rats, Sprague-Dawley; Tetrazoles; Thiazolidinediones; Transcription, Genetic

Downregulation of nephrin in podocytes leads to development of proteinuria in human and experimental kidney diseases. However, little is understood about pathophysiologic substances that regulate nephrin expression. In this report, we established conditionally immortalized reporter podocytes REPON for sensitive, continuous monitoring of nephrin gene expression. A murine podocyte cell line harboring a temperature-sensitive simian virus 40 large T antigen was stably transfected with a gene encoding secreted alkaline phosphatase (SEAP) under the control of the 5.4 or 8.3 kb nephrin gene promoter. The established reporter cells REPON5.4 and REPON8.3 were exposed to various pathophysiologic substances, and culture media were subjected to SEAP assay to identify regulators of nephrin gene expression. Among the bioactive substances tested, three physiological ligands of nuclear receptors including all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, and dexamethasone significantly activated the nephrin gene promoter in a dose-dependent manner. These effects were observed in both REPON5.4 and REPON8.3 and were associated with upregulation of nephrin mRNA. The effects of these substances were synergistic, and the maximum effect was observed by combination of three agents. In contrast, inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha as well as phorbol ester significantly downregulated the activity of the nephrin promoter as well as nephrin gene expression. These results elucidated the bidirectional regulation of nephrin by distinct pathophysiologic substances and may provide molecular bases for explaining how proteinuria is induced under pathologic situations and why some ligands for nuclear receptors have the anti-proteinuric potential.

Topics: Alkaline Phosphatase; Animals; Calcitriol; Cells, Cultured; Dexamethasone; Dose-Response Relationship, Drug; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Gene Fusion; Genes, Reporter; Glomerular Filtration Rate; Interferon-gamma; Membrane Proteins; Mice; Mice, Transgenic; Podocytes; Proteinuria; Tretinoin

Synaptic vesicle protein 2B (SV2B) was identified by the subtraction hybridization technique as a molecule of which mRNA expression was decreased in puromycin aminonucleoside (PAN) nephropathy by glomerular cDNA subtraction assay. The expression of SV2B was detected in glomerular lysate with Western blot analysis. Dual-labeling immunofluorescence studies with glomerular cell markers demonstrated that SV2B is expressed in glomerular visceral epithelial cells (podocytes). The expression of SV2B is detected also in cultured podocyte and in human kidney section as podocytic pattern. The decrease of SV2B mRNA was already detected before the onset of proteinuria in PAN nephropathy. The mRNA expression of SV2B clearly is altered not only in PAN nephropathy but also in another proteinuric state that is caused by an antibody against nephrin, a functional molecule of the slit diaphragm. The decreased intensity in SV2B staining was already detected before the peak of proteinuria in both models with immunofluorescence study. A reduced amount of SV2B was detected in both models also with Western blot analysis. CD2AP, another functional molecule of the slit diaphragm, was observed in cytoplasm, including the processes area of the cultured podocyte, and when the podocyte was treated with small interfering RNA for SV2B, CD2AP staining at the process area was not detected. These results suggest that SV2B is a functional molecule of podocyte, and SV2B may play a role in the expression and proper localization of CD2AP.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibiotics, Antineoplastic; Blotting, Western; Cells, Cultured; Chlorocebus aethiops; COS Cells; Cytoplasm; Cytoskeletal Proteins; DNA, Complementary; Female; Fluorescent Antibody Technique; Glomerulonephritis; Humans; Kidney Glomerulus; Membrane Glycoproteins; Membrane Proteins; Nerve Tissue Proteins; Podocytes; Proteinuria; Puromycin Aminonucleoside; rab3A GTP-Binding Protein; Rats; Rats, Wistar; RNA, Messenger; RNA, Small Interfering; src Homology Domains; Subtraction Technique

To examine the effect of multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) on proteinuria and expression of slit diaphragm-associated molecules such as nephrin and podocin in glomerulonephritis induced by anti-Thy1.1 antibody (anti-Thy1 . 1 GN).. Anti-Thy1.1 GN was induced in rats by a single intravenous injection with 500 microg of anti-Thy1.1 mAb 1-22-3. Fourteen rats were randomly divided into 2 groups, the GTW-treated group and vehicle treated group, and sacrificed on day 14 in Experiment 1 or on day 7 in Experiment 2 after induction of Anti-Thy1.1 GN. Daily oral administration of GTW and vehicle as a control was started from 3 days before injection or at the same time of injection to the day of sacrifice in Experiment 1 or 2. Proteinuria was determined during 14 days in Experiment 1 or during 7 days in Experiment 2. From kidneys taken at sacrifice, glomerular morphological changes, glomerular macrophage infiltration, glomerular expression of nephrin and podocin, and its mRNA expression in renal tissue were examined.. In Experiment 1, proteinuria and mesangial matrix expansion were significantly attenuated by GTW treatment. No difference in staining intensity of nephrin and podocin in glomeruli was observed between GTW treated group and vehicle treated group on day 14. In Experiment 2, GTW treatment significantly ameliorated proteinuria, mesangial injury and activated macrophage infiltration in glomerulus. In addition, it significantly increased the expression of nephrin and podocin and its mRNA expression in glomeruli on day 7.. In anti-Thy1.1 GN, the reduced expression of nephrin and podocin may contribute to the development of mesangial injury and proteinuria. The findings suggest that GTW ameliorates not only proteinuria but also mesangial lesions in anti-Thy1 . 1 GN most likely by increasing the expression of nephrin and podocin.

Topics: Animals; Female; Fluorescent Antibody Technique; Glomerulonephritis, Membranoproliferative; Glycosides; Intracellular Signaling Peptides and Proteins; Isoantibodies; Membrane Proteins; Phytotherapy; Podocytes; Proteinuria; Rats; Reverse Transcriptase Polymerase Chain Reaction; Thy-1 Antigens; Tripterygium

The slit diaphragm plays a critical role in maintaining the barrier function of the glomerular capillary wall. The pathogenic mechanism of proteinuria in membranous nephropathy remains uncertain. This study was undertaken to analyze the pathogenic role of slit diaphragm in proteinuria in experimental membranous nephropathy.. The expression and the localization of slit diaphragm-associated molecules (nephrin, podocin, and CD2AP) and other podocyte-associated molecules (podocalyxin and alpha(3) integrin) in passive and active Heymann nephritis were analyzed by immunofluorescence and Western blot analysis. The interaction of slit diaphragm-associated molecules was investigated by the dual-labeling immunofluorescence method. The mRNA expression of these molecules was also analyzed.. Shifts in nephrin and podocin staining patterns, from linear to granular, were detected in the early stages of passive Heymann nephritis. These shifts were not parallel, and the dissociation of these molecules was detected by the dual-labeling immunofluorescence method in passive and active Heymann nephritis. Western blot analyses with sequentially solubilized materials indicated that the nephrin-rich fraction changed from being partly detergent-resistant to being predominantly detergent-soluble. This change did not occur with podocin. Nephrin excreted into urine was already detected in the early stages of passive Heymann nephritis. Decreased mRNA expression of nephrin and podocin was observed before the onset of proteinuria. By contrast, no extensive change in the expression of alpha(3) integrin was observed in this study.. Nephrin is dissociated from podocin and excreted into urine in the early stages of Heymann nephritis. The reduced expression of nephrin and podocin, along with their dissociation, may contribute to the development of proteinuria in Heymann nephritis.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Blotting, Western; Cytoskeletal Proteins; Female; Fluorescent Antibody Technique; Glomerulonephritis, Membranous; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Molecular Sequence Data; Proteins; Proteinuria; Rabbits; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Rats, Wistar; Sheep

Nephrotic syndrome (NS) is a clinical state characterized by massive proteinuria, hypoalbuminemia, and eventual edema formation. Although the mechanisms underlying this phenomenon are not yet fully clarified, it is well accepted that nephrin and podocin are involved in the development of proteinuria. The effects of early treatment with various antiproteinuric therapies on proteinuria and glomerular staining of nephrin and podocin in rats with experimental NS have not been previously studied. Proteinuria and glomerular nephrin and podocin immunofluorescence were examined in rat kidneys with adriamycin-induced NS and the effects of antiproteinuric drug therapies during 5 wk with enalapril, losartan, alone or in combination, omapatrilat, and mycophenolate mofetil on these parameters were assessed. Injection of adriamycin caused a significant increase in daily (from 21.8 +/- 1.4 to 983.1 +/- 45.8 mg/day, P < 0.01) and cumulative protein excretion (from negligible values to 22,490 +/- 931 mg, P < 0.001) during 5 wk. Early treatment with enalapril significantly decreased the daily (641.7 +/- 82.4 mg/day, P < 0.0023) and cumulative proteinuria (15,727 +/- 2,204 mg, P < 0.001). A similar effect, although to a lesser extent, was obtained after omapatrilat treatment: cumulative proteinuria was reduced to 18,706 +/- 1,042 mg, P < 0.001. In contrast, losartan treatment did not significantly influence the cumulative proteinuria that remained comparable (20,351 +/- 1,360 mg, P > 0.05) to that observed in untreated NS rats. Unexpectedly, when losartan was given in combination with enalapril, it abolished the beneficial effects of the latter. Pretreatment with mycophenolate mofetil exerted a moderate antiproteinuric effect, which appeared only during the last week of the experimental treatment. Nephrotic rats exhibited severe disruption of slit diaphragm structure as seen by rapid and profound loss of nephrin and podocin. Beneficial effects of enalapril, omapatrilat, and mycophenolate mofetil paralleled the preservation of nephrin, as determined immunohistochemically, and enabled prediction of significant antiproteinuric responses. Enalapril alone or in combination with losartan resulted in significant preservation of podocin. Pretreatment with enalapril, and to a lesser extent omapatrilat, is superior to losartan in reducing proteinuria in NS rats. A combination of ACE inhibitors with ANG II receptor blockers does not provide any advantageous antiproteinuric therapy in

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Doxorubicin; Enalaprilat; Enzyme Inhibitors; Fluorescent Antibody Technique; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Losartan; Male; Membrane Proteins; Mycophenolic Acid; Nephrotic Syndrome; Protein Synthesis Inhibitors; Proteinuria; Pyridines; Rats; Rats, Sprague-Dawley; Thiazepines

Sustained proteinuria is a major factor leading to kidney fibrosis and end-stage renal failure. Tubular epithelial cells are believed to play a crucial role in this process by producing mediators leading to fibrosis and inflammation. Congenital nephrotic syndrome of the Finnish type (NPHS1) is a genetic disease caused by mutations in a podocyte protein nephrin, which leads to constant heavy proteinuria from birth. In this work we studied the tubulointerstitial changes that occur in NPHS1 kidneys during infancy.. The pathologic lesions and expression of profibrotic and proinflammatory factors in nephrectomized NPHS1 kidneys were studied by immunohistochemistry, Western blotting, and cytokine antibody array. Oxidative stress in kidneys was assessed by measurement of gluthatione redox state.. The results indicated that (1) severe tubulointerstitial lesions developed in NPHS1 kidneys during infancy; (2) tubular epithelial cells did not show transition into myofibroblasts as studied by the expression of vimentin, alpha-smooth muscle actin (alpha-SMA), collagen, and matrix metalloproteinases 2 and 9 (MMP-2 and -9); (3) the most abundant chemokines in NPHS1 tissue were neutrophil activating protein-2 (NAP-2), macrophage inhibiting factor (MIF), and monocyte chemoattractant protein-1 (MCP-1); (4) monocyte/macrophage cells expressing CD14 antigen were the major inflammatory cells invading the interstitium; (5) the arteries and arterioles showed intimal hypertrophy, but the microvasculature in NPHS1 kidneys remained quite normal; and (6) excessive oxidative stress was evident in NPHS1 kidneys.. Heavy proteinuria in NPHS1 kidneys was associated with interstitial fibrosis, inflammation, and oxidative stress. The tubular epithelial cells, however, were resistant to proteinuria and did not show epithelial-mesenchymal transition.

Topics: Capillaries; Chemokines; Child, Preschool; Cytokines; Fibrosis; Humans; Infant; Kidney Tubules; Macrophages; Membrane Proteins; Monocytes; Nephritis; Nephrotic Syndrome; Oxidative Stress; Phenotype; Proteinuria

Podocytes are terminally differentiated and highly specialized epithelial cells. The factors governing podocyte differentiation are poorly understood. We tested the hypothesis that all-trans retinoic acid (ATRA), a vitamin A derivative, induces podocyte differentiation in vitro and in vivo.. We tested the effects of ATRA on podocytes. Primary rat, primary mouse, and immortalized mouse podocytes were exposed to ATRA (1, 5, 10, 20, 40, 50, 80, 160, and 200 micromol/L) or control (ethanol) for 72 hours. Cell morphology was examined by electron microscopy, the expression of podocyte specific proteins was measured by immunoflourescence and Western blot analysis, cell number and apoptosis were measured by 3-[4,5] dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst staining, respectively. To determine if ATRA alters podocyte differentiation in vivo, experimental injury was induced in C57BL6 mice using the antiglomerular antibody. Animals were given either daily intraperitoneal ATRA (16 mg/kg) or vehicle (corn oil). For end points, we measured proteinuria, podocyte-specific protein immunostaining, and proliferation [proliferating cell nuclear antigen (PCNA)] at days 5 and 14 (N= 5/group/time point).. ATRA induced podocyte process formation in vitro, and significantly increased the expression of nephrin and podocin. This coincided with a reduction in proliferation. ATRA also significantly prevented the decrease in staining for synaptopodin, nephrin, and podocin in experimental animals (P < 0.05 vs. control). This was accompanied by reduced proteinuria and decreased podocyte proliferation (P < 0.05 vs. control).. ATRA induces podocyte differentiation in vitro and in vivo and alters the expression of certain podocyte-specific proteins. Further studies are ongoing to delineate the mechanism of this effect.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Division; Cell Line, Transformed; Glomerulonephritis; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Proteinuria; Rats; Rats, Sprague-Dawley; Tretinoin

Nephrin, a product of the NPHS1 gene, is a component of the slit diaphragms that are found between glomerular foot processes and is a crucial element for glomerular filtration barrier. Recently, nephrin has been focused in a number of studies of proteinuria development including various types of acquired glomerular diseases including minimal change nephrotic syndrome and membranous nephropathy. However, the precise role of nephrin in such acquired glomerular diseases is still unknown. To analyse the role of nephrin further, two kinds of anti-nephrin antibodies were raised in the rabbits and applied to an experimental mouse model of chronic graft-versus-host disease, in which (C57BL/10 x DBA/2) F1 mice developed clinically apparent severe proteinuria with significant glomerular lesions 7 weeks after parental DBA/2 cell transfer. Antibody-sandwich ELISA detected anti-nephrin antibodies during week 2 to week 6, with the peak at week 2 or week 4. Colocalization of nephrin and IgG on week 4, week 6, and week 8 was revealed by confocal microscopic analysis, suggesting that in situ immune complex formation with nephrin in glomerular lesion. Taken together, it seems to be suggested nephrin and its autoantibody have a certain role in the development of glomerular lesion in our model mice.

Topics: Animals; Autoantibodies; Chronic Disease; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Glomerulonephritis; Graft vs Host Disease; Immune Complex Diseases; Lupus Nephritis; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Proteinuria; Rabbits

Podocyte slit diaphragm plays an important role in the control of glomerular permeability. We hypothesize that studying the gene expression profile of podocyte in urinary sediment may provide diagnostic and prognostic information on acquired proteinuric diseases.. We studied 28 patients who required kidney biopsy for acquired proteinuric diseases (diabetic glomerulosclerosis, 9 cases; IgA nephropathy, 10 cases; minimal change disease, 5 cases; membranous nephropathy, 5 cases). We also studied 10 cases of diabetic microalbuminuria and 9 healthy controls. The mRNA expressions of nephrin (NephRNA), podocin (PodRNA) and synaptopodin (SynRNA) in urinary sediment were measured by real time quantitative PCR. After recruitment, all patients were followed for at least 12 months.. There were significant differences in the NephRNA and PodRNA in the urinary sediment between diagnosis groups (p<0.005). On the other hand, SynRNA was only marginally significant between diagnosis groups (p<0.05). Although statistically significant, the degree of proteinuria had only modest correlations with the urinary expression of nephrin. After a median follow up for 23 months, there was a significant correlation between the rate of decline in renal function and NephRNA (r=0.559, p=0.001) and PodRNA (r=0.530, p=0.002), but not SynRNA (r=0.054, p=NS). The correlation remained statistically significant after multivariate analysis to adjust for the degree of proteinuria and initial renal function.. Urinary mRNA expression of podocyte markers, such as nephrin and podocin, are significantly different between proteinuric disease categories. Further, NephRNA and PodRNA correlated with the rate of decline in renal function. Our results suggest that urinary podocyte gene expression may be a useful non-invasive tool which provides additional information for the management of proteinuric diseases.

Topics: Adult; Biomarkers; Female; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Kidney Function Tests; Male; Membrane Proteins; Middle Aged; Podocytes; Proteinuria; RNA, Messenger

Albumin modified by Amadori-glucose adducts has been linked to the development of diabetic nephropathy through its ability, independent of hyperglycemia, to activate protein kinase C-beta (PKC-beta), up-regulate the transforming growth factor-beta (TGF-beta) system, and stimulate expression of extracellular matrix proteins in glomerular cells, and by the demonstration that reducing the burden of glycated albumin ameliorates renal structural and functional abnormalities in the db/db mouse.. To probe whether the salutary effects consequent to lowering glycated albumin, which include reduction of albuminuria, relate to an influence of the Amadori-modified protein on nephrin, the podocyte protein critical to regulation of protein excretion, and on the angiogenic vascular endothelial growth factor (VEGF), which induces microvascular permeability, diabetic db/db mice were treated with a small molecule that inhibits the nonenzymatic glycation of albumin.. Compared to nondiabetic db/m mice, diabetic controls exhibited increased urinary excretion of albumin and type IV collagen, elevated renal TGF-beta1 protein levels, reduced glomerular nephrin immunofluorescence and nephrin protein by immunoblotting, and increased glomerular VEGF immunostaining and renal VEGF protein content. Diabetic animals receiving test compound showed significant lowering of proteinuria, normalization of renal TGF-beta1 protein, and significant restoration of altered glomerular nephrin and VEGF expression.. The findings causally implicate the increased glycated albumin associated with the diabetic state in the abnormal renal nephrin and VEGF expression found in diabetes, thereby promoting proteinuria and glomerulosclerosis.

Topics: Albuminuria; Animals; Collagen Type IV; Diabetic Nephropathies; Fluorescent Antibody Technique; Glycated Serum Albumin; Glycation End Products, Advanced; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Mutant Strains; Proteinuria; Serum Albumin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Topics: Angiotensin II; Animals; Male; Membrane Proteins; Protein Biosynthesis; Proteins; Proteinuria; Rats; Rats, Sprague-Dawley; Vasoconstrictor Agents

To investigate the association between nephrin, podocin and alpha-actinin of the glomerular podocyte molecules, the morphometric change of podocyte foot process and the development of proteinuria.. Puromycin aminonucleoside (PAN) nephrosis was established. Immunofluorescence staining, image analysis and real time quantitative PCR were employed to study the distribution and quantitation of glomerular expression of nephrin, podocin and alpha-actinin. Morphometric methods were applied to evaluate the morphology change of podocyte foot processes under electron microscopy.. (1) Before the onset of proteinuria, 2 days after PAN injection, the podocyte foot process became swollen nephrin and podocin staining were changed into discontinuous pattern accompanied by the decrease of podocin staining intensity. The foot process became more swollen on day 5,and podocin intensity continued to decrease. Meanwhile, nephrin decreased significantly both in protein intensity and at mRNA level. (2) When heavy proteinuria [(130.8+/-30.7) mg/d, P=0.02] occurred, complete effacement of podocyte foot processes was revealed; both podocin and nephrin staining intensity decreased dramatically(P<0.01), and no linear staining could be observed; nephrin and podocin mRNA gained back. (3) During the recovery of proteinuria, the foot process morphology recovered stepwise; both the intensity of nephrin and podocin increased. When proteinuria disappeared, the podocyte foot process reappeared; podocin immunofluorescence intensity recovered whereas nephrin did not; the intensity of alpha-actinin increased significantly and the distribution changed too. (4) Podocyte foot process volume density correlated negatively with nephrin(r(nephrin)=-0.78, P=0.000 1) and podocin immunofluorescence intensity(r(podocin)=-0.76, P=0.000 1), respectively.. Before the onset of proteinuria, the first response of podocyte is the swollen foot process, the distribution change of nephrin and podocin and the decreased podocin intensity. There was close relationship between nephrin, podocin protein level and distribution pattern with the development of proteinuria and podocyte foot process effacement, whereas no major role was found for mRNA of nephrin and podocin. Our results suggest that proteinuria might occur after series of events of nephrin and podocin distribution change, their protein and mRNA expression level decrease, and morphology change of podocyte foot process.

Topics: Actinin; Animals; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Proteins; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; RNA, Messenger

Topics: Animals; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Proteins; Proteinuria

Nephrin, podocin and alpha-actinin are all involved in proteinuria, but it is unclear which molecular event plays a crucial role during the development of proteinuria. Immunofluorescence staining and real-time quantitative polymerase chain reaction were used to study the glomerular expression of these molecules in puromycin aminonucleoside (PAN) nephrosis. Morphometric methods were applied to evaluate the podocyte foot process (FP) morphology. Two days after PAN injection, nephrin and podocin staining became discontinuous, podocin intensity decreased and FP swelled. Nephrin protein and mRNA decreased at day 5. Both podocin and nephrin intensity decreased dramatically when heavy proteinuria occurred, but nephrin mRNA was regained. When proteinuria disappeared, podocin recovered whereas nephrin did not (P = 0.02); alpha-actinin intensity increased (P = 0.009) and the distribution changed. The podocyte FP volume density correlated negatively with nephrin (r = -0.78, P = 0.0001) and podocin immunofluorescence intensity (r = -0.76, P = 0.0001). We conclude that, before the onset of proteinuria, the first response was the nephrin and podocin distribution change, podocin protein decrease and swollen FP; the podocin quantitative change was earlier than nephrin. Podocin and nephrin distribution and the protein level was associated with proteinuria more closely than their mRNA level. The delayed alpha-actinin induction might be a reparative response.

Topics: Actinin; Animals; Antibiotics, Antineoplastic; Disease Models, Animal; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Nephrosis; Proteins; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Topics: Heterozygote; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mutation; Nephrotic Syndrome; Proteins; Proteinuria

Nephrin is an important constituent of the glomerular filtration barrier and alteration of its expression is associated with severe proteinuria. In this study we show that injection of an anti-Thy1.1 antibody in rats not only induces a mesangioproliferative glomerulonephritis associated with increased proteinuria, but also leads to a sustained increase of nephrin mRNA and protein expression in renal glomeruli over a time period of 29 days. In contrast, podocin and CD2AP, two proteins shown to interact with nephrin in the slit diaphragm, are acutely downregulated at days 3-7 and, thereafter, recovered again to normal levels after 29 days. Interestingly, immunofluorescence staining of kidney sections at day 10 of the disease shows a highly heterogeneous pattern, in that some podocytes show complete absence of nephrin, whereas others show highly accumulated staining for nephrin compared to control sections, which in total results in an increased level of nephrin per glomerulus. In summary, our data show that in the course of mesangioproliferative glomerulonephritis in rats, an upregulation of nephrin expression occurs with a concomitant transient downregulation of podocin and CD2AP which may account for a highly dysregulated filtration barrier and increased proteinuria.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Antibodies; Cell Shape; Glomerulonephritis; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Proteins; Proteinuria; Rats; Rats, Wistar; RNA, Messenger; Thy-1 Antigens

Blockade of the renin-angiotensin system has been established as a treatment for heart failure with hypertension and left ventricular hypertrophy, and for progressive kidney diseases. The present study was conducted to examine whether spironolactone, a mineralocorticoid receptor antagonist, alone or in combination with cilazapril, an angiotensin converting enzyme (ACE) inhibitor, ameliorates proteinuria and renal lesions in an immune-initiated progressive nephritis model. Wistar rats were uninephrectomized 7 days before injection of anti-Thy-1 monoclonal antibody 1-22-3 to induce progressive glomerulonephritis. The nephritic rats were untreated or treated with spironolactone (400 mg/kg body weight/day), cilazapril (1 mg/kg body weight/day), or both for 10 weeks. Proteinuria was increased in the untreated rats 1 week after nephritis induction and was maintained throughout the experiment. Compared with the untreated animals (212.9+/-49.2 mg/day), proteinuria was significantly reduced in the spironolactone-treated group (62.0+/-4.0 mg/day, p=0.0046) and the cilazapril-treated group (71.8+/-26.0 mg/day, p=0.0048) on day 70 after antibody injection. Further reduction of proteinuria (42.4+/-4.5 mg/day, p=0.0019 vs. the untreated group) and less renal cortex interstitial fibrotic change (fibrosis score: 142.0+/-18.4 vs. 80.3+/-18.5 in the untreated group, p=0.0123) were detected in the spironolactone plus cilazapril-treated group. Blood pressure did not differ among the three treatment groups. In conclusion, spironolactone ameliorates proteinuria to the same degree as cilazapril, and concomitant use of spironolactone and an ACE inhibitor further suppresses renal disease progression. These data suggest that concomitant treatment with spironolactone and an ACE inhibitor has beneficial effects on immune-initiated progressive kidney disease.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antibodies, Monoclonal; Blood Pressure; Cilazapril; Drug Therapy, Combination; Fibrosis; Glomerulonephritis, Membranoproliferative; Kidney; Male; Membrane Proteins; Mineralocorticoid Receptor Antagonists; Proteinuria; Rats; Rats, Wistar; Spironolactone; Thy-1 Antigens

The mouse, as opposed to the rat, is relatively resistant to the experimental nephrosis induced by puromycin aminonucleoside. The reason for this species specificity is not known. Apolipoprotein E (apoE)-deficient mice were used to determine whether hypercholesterolemia plays a role in inducing proteinuria.. Thirty-two mice were divided into normal and high cholesterol diet groups and then divided further into four subgroups: puromycin, puromycin+probucol, probucol and control. Urinary albumin of these mice was analyzed by nephelometry. The lipid peroxidation (LPO) end products malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were detected by immunohistochemistry, and the expression level of the glomerular slit diaphragm protein, nephrin, was studied by immunohistochemistry and real time RT-PCR.. Overt proteinuria was induced by puromycin only in the apoE knockout mice ingesting the high cholesterol diet. The staining intensities of MDA and 4-HNE were stronger in the glomeruli of proteinuric mice compared to glomeruli of non-proteinuric mice. When serum cholesterol levels were reduced by probucol, proteinuria decreased and fewer LPO end products were seen immunohistochemically. Three and eight days after puromycin injection the level of nephrin mRNA in the kidneys of proteinuric mice decreased in comparison to the controls. Puromycin-treated mice kidneys demonstrated a clearly reduced reactivity to the nephrin antibodies.. Hypercholesterolemia, possibly via LPO, is a prerequisite for puromycin-inducible glomerular damage in the mouse. Furthermore, nephrin protein and mRNA levels appear to be candidate markers of glomerular damage in the mouse.

Topics: Animals; Apolipoproteins E; Cholesterol, Dietary; Hypercholesterolemia; Immunohistochemistry; Lipid Peroxidation; Membrane Proteins; Mice; Mice, Knockout; Nephrosis; Protein Synthesis Inhibitors; Proteins; Proteinuria; Puromycin; RNA, Messenger

Nephrin and NEPH1, the gene products of NPHS1 and NEPH1, are podocyte membrane proteins of the Ig superfamily. Similar to the nephrin knockout, mice lacking NEPH1 show severe proteinuria leading to perinatal death. To identify the ligand of NEPH1, the extracellular domain of NEPH1 was fused to human IgG. This NEPH1-Ig fusion protein labeled the glomerular capillary wall of mouse kidneys in a staining pattern identical to NEPH1 and nephrin, prompting speculation that that NEPH1 might form homodimers and/or heterodimers with nephrin. In coimmunoprecipitation and pull-down assays, the NEPH1-Ig fusion protein precipitated wild-type NEPH1 from overexpressing HEK 293T cells. Truncational analysis revealed that the adhesive properties were not confined to a single Ig domain of NEPH1. Fusion proteins containing two Ig domains of NEPH1 were sufficient to immobilize NEPH1, but they failed to interact with control protein containing the phylogenetically related PKD repeats of polycystin-1. NEPH1 also precipitated nephrin, a protein with eight Ig domains and a fibronectin-like domain. Truncational analysis of nephrin revealed a very similar mode of interaction, i.e., two nephrin Ig domains fused to human IgG precipitated either nephrin or NEPH1, but not the control protein. Both NEPH1 and nephrin interactions were strictly dependent upon posttranslational glycosylation, and bacterially expressed protein failed to bind NEPH1. These findings demonstrate that the Ig domains of NEPH1 and nephrin form promiscuous homodimeric and heterodimeric interactions that may facilitate cis- and trans- homodimerizations and heterodimerizations of these molecules at the glomerular slit diaphragm.

Topics: Animals; Base Sequence; Cell Wall; Cells, Cultured; Dimerization; Kidney Glomerulus; Ligands; Membrane Proteins; Mice; Precipitin Tests; Proteins; Proteinuria

Mutations in the gene encoding alpha-actinin-4 (ACTN4), an actin crosslinking protein, are associated with a form of autosomal dominant focal segmental glomerulosclerosis (FSGS). To better study its progression, a transgenic mouse model was developed by expressing murine alpha-actinin-4 containing a mutation analogous to that affecting a human FSGS family in a podocyte-specific manner using the murine nephrin promoter. Consistent with human ACTN4-associated FSGS, which shows incomplete penetrance, a proportion of the transgenic mice exhibited significant albuminuria (8 of 18), while the overall average systolic BP was elevated in both proteinuric and non-proteinuric ACTN4-mutant mice. Immunofluorescence confirmed podocyte-specific expression of mutant alpha-actinin-4, and real-time RT-PCR revealed that HA-ACTN4 mRNA levels were higher in proteinuric versus non-proteinuric ACTN4-mutant mice. Only proteinuric mice exhibited histologic features consistent with human ACTN4-associated FSGS, including segmental sclerosis and tuft adhesion of some glomeruli, tubular dilatation, mesangial matrix expansion, as well as regions of podocyte vacuolization and foot process fusion. Consistent with such podocyte damage, proteinuric ACTN4-mutant kidneys exhibited significantly reduced mRNA and protein levels of the slit diaphragm component, nephrin. This newly developed mouse model of human ACTN4-associated FSGS suggests a cause-and-effect relationship between actin cytoskeleton dysregulation by mutant alpha-actinin-4 and the deterioration of the nephrin-supported slit diaphragm complex.

Topics: Actin Cytoskeleton; Actinin; Actins; Animals; Blood Pressure; Cloning, Molecular; COS Cells; Disease Models, Animal; DNA, Complementary; Gene Expression; Glomerulosclerosis, Focal Segmental; Humans; In Vitro Techniques; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Transgenic; Microfilament Proteins; Mutagenesis; Proteins; Proteinuria; Transgenes

A total of 179 children with sporadic nephrotic syndrome were screened for podocin mutations: 120 with steroid resistance, and 59 with steroid dependence/frequent relapses. Fourteen steroid-resistant patients presented homozygous mutations that were associated with early onset of proteinuria and variable renal lesions, including one case with mesangial C3 deposition. Single mutations of podocin were found in four steroid-resistant and in four steroid-dependent; five patients had the same mutation (P20L). Among these, two had steroid/cyclosporin resistance, two had steroid dependence, and one responded to cyclosporin. The common variant R229Q of podocin, recently associated with late-onset focal segmental glomerulosclerosis, had an overall allelic frequency of 4.2% versus 2.5% in controls. To further define the implication of R229Q, a familial case was characterized with two nephrotic siblings presenting the association of the R229Q with A297V mutation that were inherited from healthy mother and father, respectively. Immunohistochemistry with anti-podocin antibodies revealed markedly decreased expression of the protein in their kidneys. All carriers of heterozygous coding podocin mutation or R229Q were screened for nephrin mutation that was found in heterozygosity associated with R229Q in one patient. Finally, podocin loss of heterozygosity was excluded in one heterozygous child by characterizing cDNA from dissected glomeruli. These data outline the clinical features of sporadic nephrotic syndrome due to podocin mutations (homozygous and heterozygous) in a representative population with broad phenotype, including patients with good response to drugs. The pathogenetic implication of single podocin defects per se in proteinuria must be further investigated in view of the possibility that detection of a second mutation could have been missed. A suggested alternative is the involvement of other gene(s) or factor(s).

Topics: Actinin; Adolescent; Child; Child, Preschool; DNA Mutational Analysis; Exons; Female; Gene Frequency; Genetic Predisposition to Disease; Glomerulosclerosis, Focal Segmental; Heterozygote; Homozygote; Humans; Incidence; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Microfilament Proteins; Nephrotic Syndrome; Phenotype; Proteins; Proteinuria

Nephrin, the molecule responsible for congenital nephrotic syndrome of Finnish type, is crucial in maintaining the glomerular filtration barrier. Recently, its complete gene structure and common gene polymorphisms in its exons have been reported, although the functional and clinical significance of these polymorphisms has not yet been elucidated. We investigated a possible association of the NPHS1 polymorphisms with the development of Ig A nephropathy (IgAN), as well as the clinical and histologic manifestations in IgAN. A total of 464 Japanese subjects, including 267 patients with histologically proven IgAN and 197 healthy controls with normal urinalysis, were genotyped for the NPHS1 G349A, G2289A, and T3315C polymorphisms. The frequencies of the genotypes, alleles, and estimated haplotypes of NPHS1 polymorphisms were no different between patients with IgAN and the controls. Within the IgAN group, patients carrying at least one G allele of G349A tended to present with more proteinuria, lower renal function, and more severe histopathologic injury than those with the AA genotype, although the time from the first urinary abnormality to the renal biopsy was no different between both groups. The logistic regression analysis indicated that even after adjusting for the effect of proteinuria and hypertension the GG genotype of NPHS1 G349A was an independent risk factor for the deteriorated renal function at the time of diagnosis. This study suggests that the NPHS1 G349A polymorphism may be associated with heavy proteinuria and a decline in renal function in patients with IgAN.

Topics: Adolescent; Adult; Aged; Case-Control Studies; Child; DNA Primers; Female; Gene Frequency; Genotype; Glomerulonephritis; Glomerulonephritis, IGA; Humans; Male; Membrane Proteins; Middle Aged; Nephrotic Syndrome; Polymorphism, Genetic; Proteins; Proteinuria

Recent progress in defining the genetic basis of inherited glomerular diseases has provided a completely new understanding of the glomerular filter of the kidney and has helped illuminate the pathogenesis of acquired and inherited renal proteinuric diseases. Based on the findings of molecular genetics in nephrology we will discuss the current understanding of the glomerular filter and provide an idea how genetic testing in the future may help to guide therapy in patients suffering from nephrotic syndrome and progressive renal failure.

Topics: Adult; Animals; Child; Finland; Genetic Predisposition to Disease; Glomerular Filtration Rate; Glomerulosclerosis, Focal Segmental; Humans; Infant, Newborn; Kidney Diseases; Kidney Failure, Chronic; Kidney Glomerulus; Membrane Proteins; Mice; Mutation; Nephrotic Syndrome; Proteins; Proteinuria

For identifying potential diagnostic markers of proteinuric glomerulopathies, glomerular mRNA levels of molecules relevant for podocyte function (alpha-actinin-4, glomerular epithelial protein 1, Wilms tumor antigen 1, synaptopodin, dystroglycan, nephrin, podoplanin, and podocin) were determined by quantitative real-time RT-PCR from microdissected glomeruli. Biopsies from 83 patients with acquired proteinuric diseases were analyzed (minimal change disease [MCD; n = 13], benign nephrosclerosis [n = 16], membranous glomerulopathy [n = 31], focal and segmental glomerulosclerosis [FSGS; n = 9], and controls [n = 14]). Gene expression levels normalized to two different housekeeping transcripts (glyceraldehyde-3-phosphate-dehydrogenase and 18 S rRNA) did not allow a separation between proteinuric disease categories. However, a significant positive correlation between alpha-actinin-4, glomerular epithelial protein 1, synaptopodin, dystroglycan, Wilms tumor antigen 1, and nephrin was found in all analyzed glomeruli, whereas podocin mRNA expression did not correlate. Because varying amounts of housekeeper cDNA per glomerulus can confound expression ratios relevant for a subpopulation of cells, an "in silico" microdissection was performed using a podocyte-specific cDNA as a reference gene. Expression ratio of podocin to synaptopodin, the two genes with the most disparate expression, allowed a robust separation of FSGS from MCD and nephrosclerosis. Segregation of FSGS from MCD via this ratio was confirmed in an independent population of formaldehyde-fixed archival biopsies (MCD, n = 5; FSGS, n = 4) after glomerular laser capture microdissection. In addition, the expression marker was able to predict steroid responsiveness in diagnostically challenging cases of MCD versus FSGS (n = 6). As the above approach can be performed as an add-on diagnostic tool, these molecular diagnostic parameters could give novel information for the management of proteinuric diseases.

Topics: Actinin; Adult; Aged; Biomarkers; Cytoskeletal Proteins; Dystroglycans; Female; Glomerular Mesangium; Glomerulonephritis, Membranous; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Membrane Proteins; Microfilament Proteins; Middle Aged; Protein Tyrosine Phosphatases; Proteins; Proteinuria; Receptor-Like Protein Tyrosine Phosphatases, Class 3; RNA, Messenger; WT1 Proteins

The onset of proteinuria in passive Heymann nephritis, (PHN), a rat model of human membranous nephropathy (MN), is complement-dependent and is associated with altered podocyte slit diaphragm integrity and dissociation of nephrin from the actin cytoskeleton. These studies examined if complement is responsible for these podocyte changes.. PHN was induced with sheep anti-Fx1A. Controls were injected with normal sheep globulin. A third group was injected with anti-Fx1A and depleted of complement with cobra venom factor. Four days later, proteinuria was measured, slit diaphragm integrity was examined by electron microscopy, nephrin distribution was studied by immunofluorescence, and the glomerular content of nephrin and its association with actin were assessed by sequential extraction of isolated glomeruli and Western blotting.. Four days after immunization, seven out of eight PHN rats were proteinuric, whereas none of the complement depleted group had proteinuria despite similar levels of antibody deposition. Complement depletion preserved slit diaphragm morphology. Immunofluorescence microscopy with an antibody to the extracellular domain of nephrin showed a normal staining pattern in the rats depleted of complement and a shift to a more dispersed and clustered pattern in the PHN group. Western blot analysis of the glomerular extracts showed a significant reduction in the total amount of nephrin and in the fraction of actin-associated nephrin in the PHN group, whereas the amounts in the complement-depleted rats were similar to normal controls.. The onset of proteinuria in the PHN model of MN is coincident with complement-dependent alterations in the association of nephrin with the actin cytoskeleton and loss of podocyte slit diaphragm integrity.

Topics: Actins; Animals; Blotting, Western; Complement System Proteins; Disease Models, Animal; Fluorescent Antibody Technique; Glomerulonephritis; Glomerulonephritis, Membranous; Kidney; Membrane Proteins; Microscopy, Electron; Proteins; Proteinuria; Rats; Tissue Distribution

Topics: Biopsy; Epithelial Cells; Genes, Recessive; Humans; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Nephrotic Syndrome; Proteins; Proteinuria

Molecular components of the glomerular filtration mechanism play critical roles in renal diseases. Many of these components are produced during the final stages of differentiation of glomerular visceral epithelial cells, also known as podocytes. While basic domain leucine zipper (bZip) transcription factors of the Maf subfamily have been implicated in cellular differentiation processes, Kreisler (Krml1/MafB), the gene affected in the mouse kreisler (kr) mutation, is known for its role in hindbrain patterning. Here we show that mice homozygous for the kr(enu) mutation develop renal disease and that Kreisler is essential for cellular differentiation of podocytes. Consistent with abnormal podocyte differentiation, kr(enu) homozygotes show proteinuria, and fusion and effacement of podocyte foot processes, which are also observed in the nephrotic syndrome. Kreisler acts during the final stages of glomerular development-the transition between the capillary loop and mature stages-and downstream of the Pod1 basic domain helix-loop-helix transcription factor. The levels of Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome (NPHS2), and Nephrin, the gene mutated in congenital nephrotic syndrome of the Finnish type (NPHS1), are slightly reduced in kr(enu)/kr(enu) podocytes. However, these observations alone are unlikely to account for the aberrant podocyte foot process formation. Thus, Kreisler must regulate other unknown genes required for podocyte function and with possible roles in kidney disease.

Topics: Animals; Animals, Newborn; Avian Proteins; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; DNA-Binding Proteins; Epithelial Cells; Female; Homozygote; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Kidney Glomerulus; MafB Transcription Factor; Male; Membrane Proteins; Mice; Mice, Mutant Strains; Mutation; Oncogene Proteins; Proteins; Proteinuria; Survival Rate; Trans-Activators; Transcription Factors

The slit-diaphragm protein nephrin is an essential component of the glomerular filtration barrier. It is not clear whether renal injury in patients with acquired proteinuric diseases is associated with altered regulation of the nephrin gene or protein.. We examined expression patterns of nephrin protein and messenger RNA (mRNA) in renal biopsy specimens from patients with minimal lesion (n = 7), focal segmental glomerulosclerosis (FSGS; n = 14), or membranous nephropathy (MN; n = 7) and controls (n = 8) by immunohistochemistry, immunoelectron microscopy, in situ hybridization, and polymerase chain reaction (PCR) amplification of nephrin complementary DNA.. In normal kidney, nephrin staining showed a diffuse interrupted linear pattern along the glomerular basement membrane (GBM). Nephrin staining in minimal lesion specimens showed a finely granular pattern along the GBM and was positive in cell bodies of visceral glomerular epithelial cells. Nephrin staining was most disrupted in FSGS specimens. Immunoelectron microscopy showed that nephrin-specific gold particles were almost absent in effaced foot processes in proteinuric patients. An in situ hybridization study showed significantly decreased nephrin mRNA-expressing cells in cases of FSGS and MN compared with controls. Reverse-transcription PCR showed significantly lower levels of nephrin mRNA in cases of FSGS and MN than controls, but no significant difference between minimal lesion cases and controls. Relative levels of glomerular nephrin mRNA correlated inversely with percentage of glomeruli with sclerosis in proteinuric diseases.. These results suggest that nephrin-expression patterns in proteinuric diseases are different according to the specific glomerular disease or severity of glomerular damage.

Topics: Adolescent; Adult; Child; Child, Preschool; Female; Gene Expression Regulation; Humans; Immunohistochemistry; In Situ Hybridization; Kidney Diseases; Kidney Glomerulus; Male; Membrane Proteins; Microscopy, Immunoelectron; Middle Aged; Protein Biosynthesis; Proteins; Proteinuria; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Many factors contribute to the pathogenesis of glomerular proteinuria, but no exact molecular mechanisms are known to date. The recently reported protein nephrin, encoded by the NPHS1 gene, appears to be crucial for the integrity of the glomerular filtration barrier.. Immunohistochemistry was used to detect possible changes in glomerular nephrin, and a new proteinuria-associated protein expression was developed in various diagnostic groups of human kidney biopsies.. In normal control kidney, antibodies to intracellular and extracellular nephrin domain showed a typical podocyte pattern of reactivity, while the 18C7 antibody to a normally inaccessible proteinuria-associated epitope was negative. Instead, strong glomerular positivity by 18C7 was seen in membranous glomerulonephropathy, membranoproliferative glomerulonephritis, systemic lupus erythematosus and cryoglobulinemic nephritis, while with antibodies to either intracellular or extracellular nephrin domains, a down-regulation in nephrin expression pattern was shown.. Unmasking or de novo expression of distinct glomerular proteins may be an important feature reflecting the pathophysiological events in these diseases with altered glomerular permeability, while only mild changes in the slit diaphragm protein nephrin appear to take place.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Biopsy; Epitopes; Gene Expression; Humans; Immunoenzyme Techniques; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Molecular Sequence Data; Proteins; Proteinuria

Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) is caused by mutations in the NPHS1 gene. NPHS1 codes for nephrin, a cell adhesion protein located at the glomerular slit diaphragm. Renal transplantation is the only treatment option for most patients with NPHS1. We have previously described recurrence of severe proteinuria in grafts transplanted to children with NPHS1. Here we studied the pathophysiology of this proteinuria.. Clinical data, light and electron microscopic findings as well as the expression of nephrin in the proteinuric grafts were studied. The patients' sera were screened for antibodies against kidney glomerulus and nephrin molecule using indirect immunofluorescence and ELISA.. Fifteen episodes of recurrent nephrotic syndrome occurred in 13 (25%) of 51 grafts transplanted to 45 Finnish children with NPHS1. All nine patients with recurrence had a Fin-major/Fin-major genotype, which leads to absence of nephrin in the native kidney. Rescue therapy (cyclophosphamide) was successful in seven episodes, but six kidneys were lost due to this process. Antibodies reacting against glomerulus were found in eight, and high anti-nephrin antibody levels were detected in four of the nine patients. In electron microscopy, the fusion of the foot process and decreases in the detectable slit diaphragms in the podocyte pores were observed. The expression of nephrin mRNA was markedly reduced in two, and granular staining for nephrin was seen in three of five grafts.. Circulating anti-nephrin antibodies seem to have a pathogenic role in the development of heavy proteinuria in kidney grafts of NPHS1 patients with Fin-major/Fin-major genotype.

Topics: Autoantibodies; Child, Preschool; Humans; Infant; Kidney; Kidney Glomerulus; Kidney Transplantation; Membrane Proteins; Nephrotic Syndrome; Proteins; Proteinuria; Recurrence; RNA, Messenger; T-Lymphocytes

These studies examined the expression of the podocyte slit diaphragm protein nephrin and its association with actin at the onset of proteinuria in passive Heymann nephritis (PHN), a rat model of human membranous nephropathy. Four days after immunization, 58% of PHN rats had mild proteinuria. At that time, most slit diaphragms were still visible on electron microscopy; however, in those locations where the deposits encroached on the filtration slits, the slit diaphragms were either displaced or absent. On day 7, the PHN rats were severely proteinuric, and most slit diaphragms were either absent, displaced, or replaced by occluding-type junctions. Immunofluorescence microscopy with antibodies to the external and cytoplasmic domains of nephrin showed a progressive loss of staining and a change in the distribution of nephrin from an interrupted linear pattern in normal controls to a more dispersed and clustered pattern in PHN. In contrast, the intensity of staining for ZO-1 and CD2-associated protein (CD2AP), two other proteins that are located on the cytoplasmic face of the slit diaphragm, was undiminished. Immunogold electron microscopy confirmed the progressive disappearance of nephrin from podocyte foot processes and retention of CD2AP. Glomeruli and glomerular cell membranes were extracted sequentially with Triton X-100, followed by DNase I or potassium iodide to depolymerize actin. Western blot analysis of the extracts showed a progressive decline of total nephrin on days 4 and 7 of PHN as well as a reduction in the actin-associated fraction. These findings show that nephrin partly dissociates from actin at the onset of podocyte injury in PHN. This is accompanied by a progressive loss of nephrin from the podocyte foot processes and prominent changes in the morphology of the slit diaphragms. These events may underlie the loss of podocyte barrier function in membranous nephropathy.

Topics: Actins; Animals; Fluorescent Antibody Technique; Glomerulonephritis; Kidney Glomerulus; Male; Membrane Proteins; Microscopy, Electron; Microscopy, Immunoelectron; Proteins; Proteinuria; Rats; Rats, Sprague-Dawley; Solubility

High concentrations of alpha-fetoprotein (AFP) are used for prenatal diagnosis of the Finnish type of congenital nephrotic syndrome (NPHS1). We investigated the validity of this test. We retrospectively established fetal NPHS1 genotype and assessed renal pathology in 21 pregnancies that had been terminated because of raised concentrations of AFP in amniotic fluid. 12 fetuses were homozygous and nine were heterozygous (carriers) for NPHS1 mutations. Raised concentrations of AFP and similar proteinuric features in fetal kidneys were seen in both groups, indicating that these signs are unreliable for prenatal diagnosis of congenital nephrosis. We strongly recommend the use of mutation analysis of the NPHS1 gene to confirm the AFP results in prenatal diagnosis of NPHS1.

Topics: Alleles; alpha-Fetoproteins; Amniotic Fluid; Female; Finland; Genotype; Humans; Membrane Proteins; Mutation; Nephrosis; Pregnancy; Prenatal Diagnosis; Proteins; Proteinuria; Retrospective Studies; Statistics, Nonparametric

Mutations in the gene coding for the podocyte slit-pore membrane protein, nephrin, are responsible for the Finnish-type congenital nephrotic syndrome. The present study sought to examine whether nephrin expression may also be altered in experimental diabetes, and how such changes related to the development of proteinuria. In addition, the study also sought to examine nephrin expression in animals treated with different anti-proteinuric therapies.. Nephrin gene expression and localization were examined in rats with streptozotocin-induced diabetes at 6 months duration (proteinuric phase) and at 7 days (pre-proteinuric phase). In addition, the effects of anti-proteinuric drug therapies were also assessed in long-term diabetic rats, treated with either the angiotensin-converting enzyme inhibitor perindopril, or the blocker of advanced glycation end-product formation, aminoguanidine. Nephrin expression was determined using quantitative real-time PCR and in situ hybridization.. When compared with control animals, nephrin expression was reduced in the late proteinuric phase (45% reduction vs controls, P<0.01) but not in the early, pre-proteinuric phase of experimental diabetic nephropathy. While ACE inhibition and aminoguanidine both reduced proteinuria, only the former attenuated the diabetes-associated reduction in nephrin expression.. These findings suggests that reduction in nephrin may be a determinant of glomerular hyperpermeability in diabetic nephropathy. Attenuation of these changes with ACE inhibition suggest that this mechanism may contribute to the anti-proteinuric effects of this, but not all classes of drug which reduce urinary protein in diabetic nephropathy.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Base Sequence; Diabetes Mellitus, Experimental; Diabetic Nephropathies; DNA Primers; Guanidines; In Situ Hybridization; Male; Membrane Proteins; Mutation; Perindopril; Proteins; Proteinuria; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

A mouse model for congenital nephrotic syndrome (NPHS1) was generated by inactivating the nephrin gene (Nphs1) in embryonic stem cells by homologous recombination. The targeting construct contained the Escherichia coli lacZ gene as a reporter for the Nphs1 promoter. Mice homozygous for inactivated Nphs1 were born at an expected frequency of 25%. Although seemingly normal at birth, they immediately developed massive proteinuria and edema and died within 24 h. The kidneys of null mice exhibited enlarged Bowman's spaces, dilated tubuli, effacement of podocyte foot processes and absence of the slit diaphragm, essentially as found in human NPHS1 patients. In addition to expression in glomerular podocytes, the reporter gene was expressed in the brain and pancreas of (+/-) and (-/-) mice. In the brain, expression was localized to the ventricular zone of the fourth ventricle, the developing spinal cord, cerebellum, hippocampus and olfactory bulb. In the cerebellum, the expression was seen in radial glial cells. Neither anatomical nor morphological abnormalities were observed in the brains of null mice.

Topics: Alleles; Animals; Animals, Newborn; beta-Galactosidase; Blotting, Western; Brain; Edema; Electrophoresis, Polyacrylamide Gel; Gene Targeting; Genes, Reporter; Genotype; Homozygote; Immunohistochemistry; Kidney; Membrane Proteins; Mice; Microscopy, Electron; Models, Genetic; Organ Size; Pancreas; Promoter Regions, Genetic; Protein Biosynthesis; Proteins; Proteinuria; Time Factors

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site.

Topics: Adolescent; Adult; Aged; Blotting, Western; Cells, Cultured; Female; Fluorescent Antibody Technique; Gene Expression; Glomerulonephritis, Membranous; Humans; Kidney Glomerulus; Male; Membrane Proteins; Middle Aged; Nephrotic Syndrome; Proteins; Proteinuria; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.

Topics: Amino Acid Sequence; Animals; Base Sequence; DNA, Complementary; Gene Expression Profiling; Humans; Kidney; Membrane Proteins; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Proteins; Proteinuria

Nephrin is identified as a product of the gene mutated in a patient with congenital nephrotic syndrome of the Finnish type. However, its precise localization and function are not yet fully clarified.. To clone the rat homologue of nephrin, polymerase chain reaction (PCR) was employed. To elucidate the localization and expression of nephrin, immunohistological analysis with a specific antirat nephrin antibody, reverse transcription-PCR, and RNase protection assay were performed.. Amino acid sequences of rat and human nephrin are highly homologous (82.2% identity). The domain structure of nephrin is also highly conserved between rats and humans. The rat nephrin was detected only in kidney glomeruli along glomerular capillary walls, and its localization was always identical to that of the anti-slit diaphragm monoclonal antibody (mAb) 5-1-6-recognized antigen in normal matured and fetal rat glomeruli and in the glomeruli of proteinuric states. The nephrin staining pattern was clearly distinguished from that of zonula occludens-1 (ZO-1), alpha3-integrin, or podocalyxin. mRNA expression for nephrin was first detected in the fetal rat kidneys at 18.5 embryonic days. Nephrin mRNA expression decreased just after injection of mAb 5-1-6 (47.4%) or puromycin aminonucleoside (51.2%), and the staining pattern of nephrin shifted from a linear to a granular pattern in both proteinuric states.. Nephrin is localized in slit diaphragm in the matured glomeruli and is identical with mAb 5-1-6 antigen. Nephrin is involved in the development of proteinuria not only in mAb 5-1-6 nephropathy, but also in puromycin aminonucleoside nephropathy.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Base Sequence; Cloning, Molecular; Fetus; Humans; Kidney Glomerulus; Membrane Proteins; Molecular Sequence Data; Organ Specificity; Proteins; Proteinuria; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Topics: Animals; Antibodies, Monoclonal; Humans; Membrane Proteins; Nephrotic Syndrome; Proteins; Proteinuria; Rats

Congenital nephrotic syndrome (NPHS1) is a rare disease inherited as an autosomally recessive trait. The NPHS1 gene mutated in NPHS1 children has recently been identified. The gene codes for nephrin, a cell-surface protein of podocytes. Two mutations, named Fin-major and Fin-minor, have been found in over 90% of the Finnish patients. In this study, we correlated the NPHS1 gene mutations to the clinical features and renal findings in 46 Finnish NPHS1 children.. Clinical data were collected from patient files, and kidney histology and electron microscopy samples were re-evaluated. The expression of nephrin was studied using immunohistochemistry, Western blotting, and in situ hybridization.. Nephrotic syndrome was detected in most patients within days after birth regardless of the genotype detected. No difference could be found in neonatal, renal, cardiac, or neurological features in patients with different mutations. Nephrin was not expressed in kidneys with Fin-major or Fin-minor mutations, while another slit diaphragm-associated protein, ZO-1, stained normally. In electron microscopy, podocyte fusion and podocyte filtration slits of various sizes were detected. The slit diaphragms, however, were missing. In contrast to this, a nephrotic infant with Fin-major/R743C genotype expressed nephrin in kidney had normal slit diaphragms and responded to therapy with an angiotensin-converting enzyme inhibitor and indomethacin.. The most common NPHS1 gene mutations, Fin-major and Fin-minor, both lead to an absence of nephrin and podocyte slit diaphragms, as well as a clinically severe form of NPHS1, the Finnish type of congenital nephrotic syndrome.

Topics: Blotting, Western; Finland; Gene Expression; Genes, Recessive; Genotype; Humans; Hypoproteinemia; In Situ Hybridization; Infant, Newborn; Kidney; Membrane Proteins; Microscopy, Electron; Mutation, Missense; Nephrotic Syndrome; Phosphoproteins; Proteins; Proteinuria; RNA, Messenger; Zonula Occludens-1 Protein

Nephrin is a novel transmembrane protein of kidney glomerular podocytes, which appears crucially important for the maintenance of the glomerular filtration barrier. According to its predicted structure, nephrin has additional roles in cell-cell adhesion and/or signal transduction. We have previously cloned the rat homologue of nephrin and described its alternatively spliced transcripts alpha and beta. In this study we examined the alterations in expression and regulation of particularly the major alternatively spliced nephrin-alpha giving rise to a variant lacking the membrane spanning domain in the puromycin nephrosis of the rat. A down-regulation of up to 78% was observed of the full length mRNA after 10 d of PAN treatment. The expression changes of nephrin-alpha followed closely the expression of the full length mRNA. Interestingly, we also found nephrin protein in urine at the peak proteinuria samples of this model. These results suggest that soluble nephrin variants may be important markers for proteinuric diseases.

Topics: Alternative Splicing; Animals; Blotting, Western; Kidney Glomerulus; Male; Membrane Proteins; Nephrotic Syndrome; Protein Structure, Tertiary; Proteins; Proteinuria; Puromycin; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Structure-Activity Relationship

mAb 5-1-6 identifies an antigen on rat podocyte slit-diaphragms and induces severe proteinuria when injected into rats. Nephrin, an Ig-like transmembrane protein that is mutated in congenital nephrotic syndrome of the Finnish type, has been localized to the slit-diaphragm on human podocytes. Here we document that the mAb 5-1-6 antigen is rat nephrin. After incubation of rat glomeruli with this mAb, the antibody/antigen complex was chemically cross-linked, extracted, and immunoprecipitated, prior to Western analysis. By mass spectrometry and 2D gel electrophoresis, we identified several peptides with complete identity to human nephrin. In addition, the 185-kDa protein immunoprecipitated by mAb 5-1-6 from rat glomerular extracts reacts with a rabbit anti-mouse nephrin antibody. Finally, nephrin and the mAb 5-1-6 antigen have identical glomerular localization patterns on immunofluorescence of rat kidney. These results demonstrate that the nephritogenic mAb 5-1-6 identifies the extracellular domain of nephrin, thereby documenting the importance of the slit-diaphragm and its component, nephrin, in the regulation of glomerular permselectivity.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens; Cross-Linking Reagents; Electrophoresis, Gel, Two-Dimensional; Fluorescent Antibody Technique; Humans; Kidney Glomerulus; Mass Spectrometry; Membrane Proteins; Molecular Sequence Data; Nephrotic Syndrome; Precipitin Tests; Proteins; Proteinuria; Rats; Rats, Sprague-Dawley; Sequence Alignment; Succinimides