nephrin and Glomerulosclerosis--Focal-Segmental

Recent advances show that human focal segmental glomerulosclerosis (FSGS) is a primary podocytopathy caused by podocyte-specific gene mutations including NPHS1, NPHS2, WT-1, LAMB2, CD2AP, TRPC6, ACTN4 and INF2. This review focuses on genes discovered in the investigation of complex FSGS pathomechanisms that may have implications for the current FSGS classification scheme. It also recounts recent recommendations for clinical management of FSGS based on translational studies and clinical trials. The advent of next-generation sequencing promises to provide nephrologists with rapid and novel approaches for the diagnosis and treatment of FSGS. A stratified and targeted approach based on the underlying molecular defects is evolving.

Topics: Actinin; Adalimumab; Adaptor Proteins, Signal Transducing; Adrenal Cortex Hormones; Anti-Inflammatory Agents; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Cytoskeletal Proteins; Formins; Glomerulosclerosis, Focal Segmental; High-Throughput Nucleotide Sequencing; Humans; Immunologic Factors; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Laminin; Membrane Proteins; Microfilament Proteins; Molecular Targeted Therapy; Mycophenolic Acid; Plasmapheresis; Rituximab; TRPC Cation Channels; TRPC6 Cation Channel; WT1 Proteins

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

Topics: Animals; Bone Morphogenetic Protein 2; Claudins; Diabetic Nephropathies; Diagnosis, Differential; Glomerulonephritis, IGA; Glomerulosclerosis, Focal Segmental; GTP-Binding Proteins; Humans; Kidney Diseases; Membrane Proteins; Nephrosis, Lipoid; Podocytes; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases

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

The nephrin gene NPHS1 was cloned in 1998. Studies in families with congenital nephrotic syndrome led to the identification of this critical component of the glomerular slit diaphragm. Studies such as the new one by Santín et al. are expanding our understanding of the spectrum of disease associated with NPHS1 mutations.

Topics: Glomerulosclerosis, Focal Segmental; Humans; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype

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

Focal and segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome in children and adults throughout the world. In the past 50 years, significant advances have been made in the identification and characterization of familial forms of nephrotic syndrome and FSGS. Resultant to these pursuits, several podocyte structural proteins such as nephrin, podocin, alpha-actinin 4 (ACTN4), and CD2-associated protein (CD2AP) have emerged to provide critical insight into the pathogenesis of hereditary nephrotic syndromes. The latest advance in familial FSGS has been the discovery of a mutant form of canonical transient receptor potential cation channel 6 (TRPC6), which causes an increase in calcium transients and essentially a gain of function in this cation channel located on the podocyte cell membrane. The TRP ion channel family is a diverse group of cation channels united by a common primary structure which contains six membrane-spanning domains, with both carboxy and amino termini located intracellularly. TRP channels are unique in their ability to activate independently of membrane depolarization. TRPC6 channels have been shown to be activated via phospholipase C stimulation. The mechanisms by which mutant TRPC6 causes an increase in intracellular calcium and leads to glomerulosclerosis are unknown. Mutant TRPC6 may affect critical interactions with the aforementioned podocyte structural proteins, leading to abnormalities in the slit diaphragm or podocyte foot processes. Mutant TRPC6 may also amplify injurious signals mediated by Ang II, a common final pathway of podocyte apoptosis in various mammalian species. Current evidence also suggests that blocking TRPC6 channels may be of therapeutic benefit in idiopathic FSGS, a disease with a generally poor prognosis. Preliminary experiments reveal the commonly used immunosuppressive agent FK-506 can inhibit TRPC6 activity in vivo. This creates the exciting possibility that blocking TRPC6 channels within the podocyte may translate into long-lasting clinical benefits in patients with FSGS.

Topics: Actinin; Adaptor Proteins, Signal Transducing; Animals; Channelopathies; Cytoskeletal Proteins; Genetic Diseases, Inborn; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Microfilament Proteins; Models, Biological; Mutation; Nephrotic Syndrome; Podocytes; TRPC Cation Channels; TRPC6 Cation Channel

Topics: Adaptor Proteins, Signal Transducing; Animals; Cytoskeletal Proteins; Glomerulosclerosis, Focal Segmental; Humans; Integrin alpha3beta1; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Nephrotic Syndrome; Podocytes; Sialoglycoproteins

Focal and segmental glomerulosclerosis (FSGS) is a pathologic entity that is a common and increasing cause of end-stage renal disease. Typical manifestations include proteinuria, hypertension, worsening renal insufficiency, and, frequently, renal failure. The etiology, however, remains unknown in a majority of patients. There is an estimated recurrence rate of 30% to 40% in renal transplant patients, suggesting that the pathogenesis is not solely a result of intrinsic kidney disease. Although some of its characteristics have been reported, the precise identification of a circulating factor associated with FSGS has not been made. Remarkable progress has been made in recent years regarding biologic mechanisms surrounding FSGS and proteinuria. Insight into the pathogenesis of FSGS has been gained through the study of hereditary forms of FSGS and nephrotic syndromes. Mutations in cytoskeletal proteins that affect podocyte structure have been the target until recently. Here we review the current understanding of this glomerular disease and areas for future concentration.

Topics: Adaptor Proteins, Signal Transducing; Biomarkers; Cytoskeletal Proteins; Disease Progression; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney Failure, Chronic; Membrane Proteins; Risk Factors; src Homology Domains; TRPC Cation Channels; TRPC6 Cation Channel

An increasing cause of end-stage renal disease is the pathological lesion focal and segmental glomerulosclerosis (FSGS). FSGS is characterized by proteinuria and frequently nephrotic syndrome with ensuing renal failure. The etiology remains unknown in the majority of individuals. The idiopathic form of FSGS is most common; however, secondary forms of FSGS do exist. There is a form of FSGS that is fulminant that frequently recurs after renal transplantation with an estimated frequency of approximately 30%, suggesting that the pathogenesis is not solely a result of intrinsic kidney disease. Recently, hereditary forms of the disease were recognized as well as those associated with other congenital syndromes. Known genetic causes of the hereditary form of this disease have been suggested to account for upwards of 18% of cases. This review will address recent discoveries of the genetic mechanisms of hereditary FSGS and the current interpretations of their interactions at the slit diaphragm.

Topics: Genome, Human; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mutation; TRPV Cation Channels

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: Apoptosis; Chromosome Mapping; Chromosomes, Human, Pair 11; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Podocytes; TRPC Cation Channels; TRPC6 Cation Channel

Studies of Mendelian forms of focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome have provided new insights into the mechanism of these diseases. Congenital nephrotic syndrome and familial forms of FSGS form a spectrum of podocyte diseases of varying severity and age of onset. Mutations in both nephrin gene (NPHS1) alleles lead to congenital nephrosis, podocyte foot process efacement, and loss of slit-diaphragm structure. Mutations in both podocin gene (NPHS2) alleles lead to a wide range of human disease, from childhood-onset steroid-resistant FSGS and minimal change disease to adult-onset FSGS. Dominantly inherited mutations in ACTN4, the alpha-actinin-4 gene, can lead to a slowly progressive adult-onset form of FSGS. In addition, FSGS is observed as part of several rare multisystem inherited syndromes. Here we review recent progress in understanding the genetic basis of FSGS in humans.

Topics: Animals; Drug Resistance; Epithelial Cells; Genetic Predisposition to Disease; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Nephrosis; Nephrotic Syndrome; Point Mutation; Proteins; Steroids

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

Topics: Animals; Cell Count; Cytoskeleton; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Kidney Glomerulus; Membrane Microdomains; Membrane Proteins; Proteins

There is increasing recognition of the importance of genetic factors in the development of focal segmental glomerulosclerosis and related proteinuric disorders. Recently, four genes have been identified which, when defective, cause focal segmental glomerulosclerosis or nephrosis. All of these genes appear to be important in the maintenance of glomerular podocyte function. However, not all cases of familial nephrosis or proteinuria are explained by defects in these genes.

Topics: Actinin; Adaptor Proteins, Signal Transducing; Animals; Cytoskeletal Proteins; Disease Models, Animal; Glomerulosclerosis, Focal Segmental; Humans; Membrane Proteins; Mice; Microfilament Proteins; Mutation; Proteins

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

There are a large number of glomerular diseases that may be responsible for a nephrotic syndrome, the most frequent in childhood being minimal change disease. In the past few years, the molecular genetic basis of several conditions that may cause a nephrotic syndrome have been identified. Denys-Drash syndrome and Frasier syndrome are related diseases caused by mutations in the WT1 gene. Familial forms of idiopathic nephrotic syndrome with focal and segmental glomerular sclerosis/hyalinosis have been identified with an autosomal dominant or recessive mode of inheritance and linkage analysis have allowed to localize several genes on chromosomes 1, 11 and 17. The gene responsible for the Finnish type congenital nephrotic syndrome has been identified. This gene, named NPHS1, codes for nephrin, which is located at the slit diaphragm of the glomerular podocytes and is thought to play an essential role in the normal glomerular filtration barrier.

Topics: Disorders of Sex Development; Finland; Genes, Wilms Tumor; Genetic Linkage; Glomerulosclerosis, Focal Segmental; Humans; Male; Membrane Proteins; Nephrotic Syndrome; Proteins; Syndrome

Extra-capillary hypercellularity is a common finding in crescentic glomerulonephritis (GN) and focal segmental glomerulosclerosis (FSGS). In diabetic nephropathy (DN), extra-capillary hypercellularity is often observed as a finding of complications such as IgA nephropathy or microscopic polyangiitis superimposed on DN. However, in rare cases, epithelial cell proliferation may accompany DN. We experienced a case of nodular diabetic glomerulosclerosis with marked extra-capillary hypercellularity and revealed the origin of this atypical lesion using immunostainings.. A man in his 50 s was admitted to the hospital with nephrotic syndrome, and a renal biopsy was performed. Diffuse nodular lesions and extra-capillary hypercellularity were observed, but the results of serological examination or immunofluorescent assays did not implicate any other crescentic GN. Immunostaining for claudin-1 and nephrin was performed to identify the origin of the extra-capillary lesions. Given the clinical course and pathological findings, a diagnosis of DN-associated extra-capillary cell proliferation was made.. Extra-capillary hypercellularity, which resembles FSGS or crescentic GN, is a rare finding in DN and should therefore be treated with caution. In such cases, co-staining for claudin-1 and nephrin may facilitate the diagnosis of DN.

Topics: Cell Proliferation; Claudin-1; Diabetes Mellitus; Diabetic Nephropathies; Glomerulonephritis, IGA; Glomerulonephritis, Membranoproliferative; Glomerulosclerosis, Focal Segmental; Humans; Male; Membrane Proteins; Microscopic Polyangiitis; Middle Aged

Topics: Autoantibodies; Glomerulosclerosis, Focal Segmental; Humans; Kidney Transplantation; Membrane Proteins; Recurrence

Variants in. We generated a stable deletion in. A null allele of

Topics: Animals; Drosophila; Endocytosis; Endosomes; Glomerulosclerosis, Focal Segmental; Membrane Proteins; Mice; Nephrotic Syndrome; Podocytes

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

FSGS caused by mutations in. Live cell and quantitative imaging, fluorescent and surface biotinylation-based trafficking assays in cultured podocytes, and a new puromycin aminoglycoside nephropathy model of. Pathogenic

Topics: Animals; Cell Culture Techniques; Cytoplasmic Dyneins; Formins; Glomerulosclerosis, Focal Segmental; Membrane Proteins; Mice; Mutation; Podocytes; Protein Transport

Under healthy conditions, foot processes of neighbouring podocytes are interdigitating and connected by an electron-dense slit diaphragm. Besides slit diaphragm proteins, typical adherens junction proteins are also found to be expressed at this cell-cell junction. It is therefore considered as a highly specialized type of adherens junction. During podocyte injury, podocyte foot processes lose their characteristic 3D structure and the filtration slits typical meandering structure gets linearized. It is still under debate how this change of structure leads to the phenomenon of proteinuria. Using super-resolution 3D-structured illumination microscopy, we observed a spatially restricted up-regulation of the tight junction protein claudin-5 (CLDN5) in areas where podocyte processes of patients suffering from minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) as well as in murine nephrotoxic serum (NTS) nephritis and uninephrectomy DOCA-salt hypertension models, were locally injured. CLDN5/nephrin ratios in human glomerulopathies and NTS-treated mice were significantly higher compared to controls. In patients, the CLDN5/nephrin ratio is significantly correlated with the filtration slit density as a foot process effacement marker, confirming a direct association of local CLDN5 up-regulation in injured foot processes. Moreover, CLDN5 up-regulation was observed in some areas of high filtration slit density, suggesting that CLND5 up-regulation preceded the changes of foot processes. Therefore, CLDN5 could serve as a biomarker predicting early foot process effacement.

Topics: Adult; Aged; Aged, 80 and over; Animals; Claudin-5; Disease Models, Animal; Female; Glomerulosclerosis, Focal Segmental; Humans; Kidney Diseases; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Middle Aged; Podocytes

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

We have found that calcium calmodulin kinase IV is increased in T cells, podocytes, and mesangial cells from patients with systemic lupus erythematosus, as well as in lupus-prone mice, podocytes of patients with focal segmental glomerulosclerosis, and in mice injected with doxorubicin. We showed that this accounts for aberrant T cell function and glomerular damage. Using nanoparticles (nlg) loaded with a small drug inhibitor of calcium calmodulin kinase IV and tagged with antibodies directed to CD4 we have been able to show inhibition of autoimmunity and lupus nephritis. Also, using nlg tagged with antibodies to nephrin, we showed suppression of nephritis in lupus-prone mice and of glomerular damage in mice exposed to doxorubicin. We propose the development of approaches to deliver drugs to cells in a targeted and precise manner.

Topics: Animals; Antibiotics, Antineoplastic; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 4; CD4 Antigens; Disease Models, Animal; DNA Methylation; Doxorubicin; Drug Delivery Systems; Glomerulosclerosis, Focal Segmental; Humans; Lupus Erythematosus, Systemic; Lupus Nephritis; Membrane Proteins; Mice; Mice, Inbred MRL lpr; Molecular Targeted Therapy; Nanoparticles; Protein Kinase Inhibitors; Sulfonamides; T-Lymphocytes; T-Lymphocytes, Regulatory; Th17 Cells

This study aimed to investigate the effect of the Phellinus linteus (Mesima) decoction on podocyte injury in a rat model of focal and segmental glomerulosclerosis (FSGS) and evaluate the potential mechanisms.. FSGS resembling primary FSGS in humans was established in rats by uninephrectomy and the repeated injection of doxorubicin. The FSGS rats were randomly divided into the model group, low-dose group of P. linteus decoction (PLD-LD), medium-dose group of P. linteus decoction (PLD-MD), and high-dose group of P. linteus decoction (PLD-HD). Blood and urine analysis were performed after 12 weeks and the molecular indicators of renal function and the renal pathological changes were examined.. FSGS developed within 12 weeks in the test group and showed progressive proteinuria and segmental glomerular scarring. Urinary protein, serum creatinine, urea nitrogen, triglycerides and cholesterol were significantly reduced following the 12-week intervention with P.linteus decoction, especially in the PLD-LD group. Renal nephrin and podocin were markedly increased. Moreover, the pathological damage in the renal tissue was alleviated by the PLD-LD intervention.. The P. linteus decoction alleviated the podocyte injury in the FSGS rat model, thus minimizing the progression of glomerular sclerosis and improving renal function.

Topics: Animals; Disease Models, Animal; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Phellinus; Plant Extracts; Podocytes; Rats; Rats, Sprague-Dawley

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

Podocyte injury is a key event for progressive renal failure. We have previously established a mouse model of inducible podocyte injury (NEP25) that progressively develops glomerulosclerosis after immunotoxin injection. We performed polysome analysis of intact and injured podocytes utilizing the NEP25 and RiboTag transgenic mice, in which a hemagglutinin tag is attached to ribosomal protein L22 selectively in podocytes. Podocyte-specific polysomes were successfully obtained by immunoprecipitation with an antihemagglutinin antibody from glomerular homogenate and analyzed using a microarray. Compared with glomerular cells, 353 genes were highly expressed and enriched in podocytes; these included important podocyte genes and also heretofore uncharacterized genes, such as Dach1 and Foxd2. Podocyte injury by immunotoxin induced many genes to be upregulated, including inflammation-related genes despite no infiltration of inflammatory cells in the glomeruli. MafF and Egr-1, which structurally have the potential to antagonize MafB and WT1, respectively, were rapidly and markedly increased in injured podocytes before MafB and WT1 were decreased. We demonstrated that Maff and Egr1 knockdown increased the MafB targets Nphs2 and Ptpro and the WT1 targets Ptpro, Nxph3, and Sulf1, respectively. This indicates that upregulated MafF and Egr-1 may promote deterioration of podocytes by antagonizing MafB and WT1. Our systematic microarray study of the heretofore undescribed behavior of podocyte genes may open new insights into the understanding of podocyte pathophysiology.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Gene Expression Profiling; Genetic Predisposition to Disease; Glomerulosclerosis, Focal Segmental; Interleukin-2 Receptor alpha Subunit; Membrane Proteins; Mice, Transgenic; Oligonucleotide Array Sequence Analysis; Phenotype; Podocytes; Polyribosomes; Renal Insufficiency, Chronic; Transcriptome

Kidney injury due to focal segmental glomerulosclerosis (FSGS) is the most common primary glomerular disorder causing end-stage renal disease. Homozygous mutations in either glomerular basement membrane or slit diaphragm genes cause early renal failure. Heterozygous carriers develop renal symptoms late, if at all. In contrast to mutations in slit diaphragm genes, hetero- or hemizygous mutations in the X-chromosomal

Topics: Acute Kidney Injury; Adult; Child; Child, Preschool; Collagen Type IV; Female; Genetic Predisposition to Disease; Glomerulosclerosis, Focal Segmental; Hemizygote; Heterozygote; Humans; Infant; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mutation; Nephritis, Hereditary; Pedigree; Polymorphism, Single Nucleotide

Altered DNA methylation plays an important role in the onset and progression of kidney disease. However, little is known about how the changes arise in disease states. Here, we report that KAT5-mediated DNA damage repair is essential for the maintenance of kidney podocytes and is associated with DNA methylation status. Podocyte-specific KAT5-knockout mice develop severe albuminuria with increased DNA double-strand breaks (DSBs), increased DNA methylation of the nephrin promoter region, and decreased nephrin expression. Podocyte KAT5 expression is decreased, whereas DNA DSBs and DNA methylation are increased in diabetic nephropathy; moreover, KAT5 restoration by gene transfer attenuates albuminuria. Furthermore, KAT5 decreases DNA DSBs and DNA methylation at the same nephrin promoter region, which indicates that KAT5-mediated DNA repair may be related to DNA methylation status. These results suggest a concept in which an environment of DNA damage repair, which occurs with decreased KAT5, may affect DNA methylation status.

Topics: Albuminuria; Animals; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; DNA Breaks, Double-Stranded; DNA Damage; DNA Methylation; DNA Repair; Glomerulosclerosis, Focal Segmental; Glucose; Humans; Kidney; Kidney Glomerulus; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lysine Acetyltransferase 5; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Promoter Regions, Genetic; Tamoxifen; Trans-Activators

Focal and Segmental Glomerulosclerosis (FSGS) is a severe glomerulopathy that frequently leads to end stage renal disease. Only a subset of patients responds to current therapies, making it important to identify alternative therapeutic options. The interleukin (IL)-1 receptor antagonist anakinra is beneficial in several diseases with renal involvement. Here, we evaluated the potential of anakinra for FSGS treatment. Molecular process models obtained from scientific literature data were used to build FSGS pathology and anakinra mechanism of action models by exploiting information on protein interactions. These molecular models were compared by statistical interference analysis and expert based molecular signature matching. Experimental validation was performed in Adriamycin- and lipopolysaccharide (LPS)-induced nephropathy mouse models. Interference analysis (containing 225 protein coding genes and 8 molecular process segments) of the FSGS molecular pathophysiology model with the drug mechanism of action of anakinra identified a statistically significant overlap with 43 shared molecular features that were enriched in pathways relevant in FSGS, such as plasminogen activating cascade, inflammation and apoptosis. Expert adjudication of molecular signature matching, focusing on molecular process segments did not suggest a high therapeutic potential of anakinra in FSGS. In line with this, experimental validation did not result in altered proteinuria or significant changes in expression of the FSGS-relevant genes COL1A1 and NPHS1. In summary, an integrated bioinformatic and experimental workflow showed that FSGS relevant molecular processes can be significantly affected by anakinra beyond the direct drug target IL-1 receptor type 1 (IL1R1) context but might not counteract central pathophysiology processes in FSGS. Anakinra is therefore not suggested for extended preclinical trials.

Topics: Animals; Collagen Type I; Disease Models, Animal; Doxorubicin; Drug Repositioning; Gene Expression; Glomerulosclerosis, Focal Segmental; Humans; Interleukin 1 Receptor Antagonist Protein; Membrane Proteins; Metabolic Networks and Pathways; Mice; Mice, Inbred BALB C; Podocytes

There are controversies whether Minimal Change Disease (MCD) and Focal and Segmental Glomerulosclerosis (FSGS) are distinct glomerular lesions or different manifestations within the same spectrum of diseases. The uPAR (urokinase-type plasminogen activator receptor) and some slit diaphragm proteins may be altered in FSGS glomeruli and may function as biomarkers of the disease in renal biopsies. Thus, this study aims to evaluate the diagnostic potential of uPAR and glomerular proteins for differentiation between MCD and FSGS in renal pediatric biopsy. Renal biopsies from 50 children between 2 and 18 years old were selected, with diagnosis of MCD (n = 29) and FSGS (n = 21). Control group consisted of pediatric autopsies (n = 15) from patients younger than 18 years old, with no evidences of renal dysfunction. In situ expressions of WT1, nephrin, podocin and uPAR were evaluated by immunoperoxidase technique. Renal biopsy of patients with MCD and FSGS expressed fewer WT1 (p≤0.0001, F = 19.35) and nephrin (p<0.0001; H = 21.54) than patients in the control group. FSGS patients expressed fewer podocin than control (p<0.0359, H = 6.655). FSGS cases expressed more uPAR than each of control and MCD (p = 0.0019; H = 12.57) and there was a positive and significant correlation between nephrin and podocin (p = 0.0026, rS = 0.6502) in these cases. Podocin had sensitivity of 73.3% and specificity of 86.7% (p = 0.0068) and uPAR had sensitivity of 78.9% and specificity of 73.3% (p = 0.0040) for diagnosis of FSGS patients. The main limitation of the study is the limited number of cases due to the difficulty in performing biopsy in pediatric patients. Podocin and uPAR are good markers for FSGS and differentiate these cases from MCD, reinforcing the theory of distinct glomerular diseases. These findings suggest that podocin and uPAR can be used as biomarkers in the routine analysis of renal biopsies in cases of podocytopathies when the lesion (sclerosis) is not sampled.

Topics: Adolescent; Autopsy; Biomarkers; Biopsy; Case-Control Studies; Child; Child, Preschool; Diagnosis, Differential; Female; Gene Expression; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Nephrosis, Lipoid; Predictive Value of Tests; Receptors, Urokinase Plasminogen Activator; WT1 Proteins

Podocytes are differentiated post-mitotic cells that cannot replace themselves after injury. Glomerular parietal epithelial cells are proposed to be podocyte progenitors. To test whether a subset of parietal epithelial cells transdifferentiate to a podocyte fate, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice, named PEC-PODO, were generated. Doxycycline administration permanently labeled parietal epithelial cells with tdTomato reporter (red), and upon doxycycline removal, the parietal epithelial cells (PECs) cannot label further. Despite the presence or absence of doxycycline, podocytes cannot label with tdTomato, but are constitutively labeled with an enhanced green fluorescent protein (EGFP) reporter (green). Only activation of the Nphs1-FLPo transgene by labeled parietal epithelial cells can generate a yellow color. At day 28 of experimental focal segmental glomerulosclerosis, podocyte density was 20% lower in 20% of glomeruli. At day 56 of experimental focal segmental glomerulosclerosis, podocyte density was 18% lower in 17% of glomeruli. TdTomato

Topics: Animals; Cell Transdifferentiation; Disease Models, Animal; Epithelial Cells; Genes, Reporter; Glomerulosclerosis, Focal Segmental; Humans; Intravital Microscopy; Luminescent Proteins; Membrane Proteins; Mice; Mice, Transgenic; Microscopy, Fluorescence; Podocytes; Red Fluorescent Protein

Focal segmental glomerulosclerosis (FSGS) is still one of the common causes of refractory nephrotic syndrome. Nephrin, encoded by podocyte-specific NPHS1 gene, participated in the pathogenesis of FSGS. The sites of NPHS1 mutations in FSGS is not clarified very well. In this study, we investigated the specific mutations of NPHS1 gene in Chinese patients with sporadic FSGS.. A total of 309 patients with sporadic FSGS were collected and screened for NPHS1 mutations by second-generation sequencing. The variants were compared with those extracted from 2504 healthy controls in the 1000 Genomes Project. The possible pathogenic roles of missense variants were predicted by three different software. We also compared these candidate causal mutations with those summarized from the previous studies.. Thirty-two genetic mutations of NPHS1 gene were identified in FSGS patients, including 12 synonymous mutations, 17 missense mutations, 1 splicing mutation, and 2 intron mutations, of which c.G3315A (p.S1105S) was the most common variant (261/309). A novel missense mutation c.G2638 T (p.V880F) and a novel splicing mutation 35830957 C > T were identified in FSGS patients. The frequencies of the four synonymous mutations (c.C294T [p.I98I], c.C2223T [p.T741 T], c.C2289T [p.V763 V], c.G3315A [p.S1105S]) were much higher in FSGS patients than in controls. The frequencies of the four missense mutations (c.G349A [p.E117K], c.G1339A [p.E447K], c.G1802C [p.G601A], c.C2398T [p.R800C]) were much higher and one (c.A3230G [p.N1077S]) was lower in FSGS patients than in controls. Five missense mutations, c.C616A (p.P206T), c.G1802C (p.G601A), c.C2309T (p.P770L), c.G2869C (p.V957 L), and c.C3274T (p.R1092C), were predicted to be pathogenic mutations by software analysis.. NPHS1 gene mutations were quite common in sporadic FSGS patients. We strongly recommend mutation analysis of the NPHS1 gene in the clinical management of FSGS patients.

Topics: Adolescent; Adult; Aged; Asian People; Child; Female; Genetic Association Studies; Genetic Predisposition to Disease; Glomerulosclerosis, Focal Segmental; Humans; Introns; Male; Membrane Proteins; Middle Aged; Mutation; Nephrotic Syndrome; Young Adult

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

Lupus nephritis (LN) is a common and devastating complication caused by systemic lupus erythematosus. In this study, we evaluated the expression and mechanism of Fos-related antigen 2 (Fra-2) in LN. The results showed that Fra-2 was significantly increased in kidney biopsies of LN patients compared with healthy controls and other kidney disease in glomerular podocytes. The MRL/lpr mouse strain is a murine model of lupus, and it was used to study the mechanisms of Fra-2 in LN. The results showed that Fra-2 was expressed in the glomerular podocytes. We investigated the effects of inflammatory stimuli on Fra-2 protein expression in the glomerular podocytes, and found that interferon gamma was most effective at increasing Fra-2 protein expression. Knockdown of Fra-2 using siRNA enhanced the protein expression of nephrin. Therefore, Fra-2 may be a specific drug target for podocyte injury in LN.

Topics: Animals; Antiviral Agents; Fos-Related Antigen-2; Gene Knockdown Techniques; Glomerulonephritis, IGA; Glomerulonephritis, Membranous; Glomerulosclerosis, Focal Segmental; Humans; IgA Vasculitis; Interferon-gamma; Lupus Nephritis; Membrane Proteins; Mice; Mice, Inbred MRL lpr; Nephrosis, Lipoid; Podocytes

Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman's capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.

Topics: Adult Stem Cells; Animals; Cell Lineage; Cell Movement; Claudin-1; Cyclin-Dependent Kinase Inhibitor p57; Disease Models, Animal; Epithelial Cells; Female; Glomerulosclerosis, Focal Segmental; Intracellular Signaling Peptides and Proteins; Intravital Microscopy; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Transgenic; Microscopy, Fluorescence, Multiphoton; PAX8 Transcription Factor; Podocytes; Renin; Repressor Proteins; Stochastic Processes; WT1 Proteins

Transforming growth factor beta 1 (TGF-β1) plays a critical role in the pathogenesis of glomerulosclerosis. The purpose of this study was to examine the effects of inhibition of miR-155 on podocyte injury induced by TGF-β1 and to determine further molecular mediators involved in the effects of miR-155.. Conditionally immortalized podocytes were cultured in vitro and they were divided into four groups: control; TGF-β1 treatment; TGF-β1 with miR-155 knockdown [using antisense oligonucleotides against miR-155 (ASO-miR-155)] and TGF-β1 with negative control antisense oligonucleotides (ASO-NC). Real time RT-PCR and Western blot analysis were employed to determine the mRNA and protein expression of nephrin, desmin and caspase-9, respectively. Flow cytometry was used to examine the apoptotic rate of podocytes and DAPI fluorescent staining was used to determine apoptotic morphology. In addition, we examined the levels of miR-155, TGF-β1, nephrin, desmin and caspase-9 in glomerular tissues of nephropathy induced by intravenous injections of adriamycin in rats.. mRNA and protein expression of desmin and caspase-9 was increased in cultured TGF-β1-treated podocytes, whereas nephrin was decreased as compared with the control group. Importantly, miR-155 knockdown significantly attenuated upregulation of desmin and caspase-9, and alleviated impairment of nephrin induced by TGF-β1. Moreover, the number of apoptotic podocytes was increased after exposure to TGF-β1 and this was alleviated after miR-155 knockdown. Knocking down miR-155 also decreased an apoptosis rate of TGF-β1-treated podocytes. Note that negative control antisense oligonucleotides failed to alter an increase of the apoptosis rate in TGF-β1-treated podocytes. Consistent with in vitro results, expression of miR-155, TGF-β1, desmin and caspase-9 was increased and nephrin was decreased in glomerular tissues with nephropathy in vivo experiments.. TGF-β1 impairs the protein expression of nephrin and amplifies the protein expression of desmin and caspase -9 via miR-155 signal pathway. Inhibition of miR-155 alleviates these changes in podocytes-treated with TGF-β1 and attenuated apoptosis of podocytes. Our data suggest that miR-155 plays a role in mediating TGF-β1-induced podocyte injury via nephrin, desmin and caspase-9. Results of the current study also indicate that blocking miR-155 signal has a protective effect on podocyte injury. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of podocyte injury observed in glomerulosclerosis.

Topics: Animals; Apoptosis; Caspase 9; Cell Line, Transformed; Desmin; Doxorubicin; Gene Expression Regulation; Glomerulosclerosis, Focal Segmental; Male; Membrane Proteins; MicroRNAs; Oligoribonucleotides, Antisense; Podocytes; Rats; Rats, Sprague-Dawley; Signal Transduction; Transforming Growth Factor beta1

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

Topics: Adolescent; Adrenal Cortex Hormones; Adult; Albumins; Animals; CD40 Antigens; CD40 Ligand; Cell Membrane; Cell Membrane Permeability; Child; Child, Preschool; Cytotoxins; Epithelial Cells; Female; Gene Expression Regulation; Glomerulonephritis, Membranous; Glomerulosclerosis, Focal Segmental; Hemodialysis Solutions; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Kidney Transplantation; Male; Membrane Proteins; Mice; Nephrotic Syndrome; Plasma Exchange; Plasmapheresis; Rats

Steroid-resistant nephrotic syndrome (SRNS) is a genetically heterogeneous disorder for which more than 25 single-gene hereditary causes have been identified.. Whole exome sequencing was performed in a 3-year-old girl with SRNS. We analyzed the expression of Crb2 and slit diaphragm molecules in the patient's glomeruli, and compared it with that of controls or other nephrotic patients.. Whole-exome analysis identified novel compound heterozygous mutations in exons 10 and 12 of CRB2 (p.Trp1086ArgfsX64 and p.Asn1184Thr, each from different parents; Asn1184 within extracellular 15th EGF repeat domain). Renal pathology showed focal segmental glomerulosclerosis with effaced podocyte foot processes in a small area, with significantly decreased Crb2 expression. Molecules critical for slit diaphragm were well-expressed in this patient's podocytes. Crb2 expression was not altered in the other patients with congenital nephrotic syndrome with NPHS1 mutations.. These findings demonstrate that Crb2 abnormalities caused by these mutations are the mechanism of steroid-resistant NS. Although CRB2 mutations previously found in SRNS patients have been clustered within the extracellular tenth EGF-like domain of this protein, the present results expand the variation of CRB2 mutations that cause SRNS.

Topics: Anti-Inflammatory Agents; Carrier Proteins; Child, Preschool; Drug Resistance; Exome; Female; Gene Expression Regulation, Enzymologic; Glomerulosclerosis, Focal Segmental; HEK293 Cells; Humans; Kidney Glomerulus; Membrane Proteins; Mutation; Nephrotic Syndrome; Podocytes; Steroids

N-linked glycosylation, which is a post-translational modification process, plays an important role in protein folding, intracellular trafficking and membrane targeting, as well as in regulating the protein function. Recently, we identified a missense variant (p.T141L) in the short isoform 2 of the X-linked gene asparagine-linked glycosylation 13 (ALG13-is2), which segregated with focal segmental glomerulosclerosis and PCCD in a large Australian pedigree; however, any evidence of its pathogenicity was demonstrated. ALG13 gene encodes, through alternative splicing, 2 glycosyltransferase isoforms, which catalyse the second sugar addition of the highly conserved oligosaccharide precursor in the endoplasmic reticulum (ER). Mutations in the long isoform 1 were associated with epilepsy.. Here, we show a different expression of the 2 isoforms depending on the tissue. Specifically, the long isoform is highly expressed in lungs, ovaries, testes, cerebellum, cortex, retina, pituitary gland, and olfactory bulbs, while the short isoform is highly expressed in mouse podocytes and in human podocyte cell lines, at both mRNA and protein levels. The silencing of ALG13-is2 by specific siRNAs induces an altered N-linked glycosylation pattern of nephrin, as demonstrated by the presence of an additional immunostaining band of about 130 kD. In knock-down cells, immunofluorescence analysis shows perturbed organization of the cytoskeleton and altered localization of nephrin on the cellular membrane. We also demonstrated that the altered pattern of N-linked glycosylation induces an over-expression of binding immunoglobulin protein and calreticulin, suggesting ER stress.. These results provide preliminary evidence that ALG13-is2 could be an important modifier of renal filtration defects.

Topics: Animals; Gene Expression Profiling; Gene Knockdown Techniques; Glomerulosclerosis, Focal Segmental; Glycosylation; Humans; Isomerism; Membrane Proteins; Mice; N-Acetylglucosaminyltransferases; Podocytes; Protein Processing, Post-Translational; Tissue Distribution

Background /Aims: The underlying mechanisms leading to focal segmental glomerulosclerosis (FSGS) are lacking. In this report, we examined the role of protease-activated receptors (PARs) subtype PAR2 and its downstream signals in regulating the pathophysiological process of FSGS.. Nephropathy was induced by intravenous injections of adriamycin (ADR) in rats to study FSGS. Western Blot analysis and ELISA were employed to determine the protein expression levels of PAR2 and its downstream signal pathways as well as the levels of PICs.. In ADR rats, expression of PAR2, PKCε and PKA was amplified and this was accompanied with increases of pro-inflammatory cytokines (PICs) including IL-1β, IL-6 and TNF-α. Inhibition of PAR2 signal by systemic administration of FSLLRY-NH2 (FSL) attenuated amplification of PICs. Notably, FSL further influenced key molecular mediators during development of FSGS. i.e., it specifically restored the impaired nephrin and attenuated the exaggerated transforming growth factor beta 1 (TGF-β1), caspase-9 and desmin thereby improving worsened renal functions and glomerular injury. Consistent with this, in cultured podocytes FSL also largely restored downregulation of nephrin and attenuated amplifications of caspase-9 and desmin induced by TGF-β1.. Results of this study suggest that PAR2 plays an important role in mediating renal injury induced by glomerulosclerosis. Inhibition of PAR2 signal pathway has a protective effect on FSGS mainly via PIC and TGF-β1 mechanisms. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of FSGS observed in patients.

Topics: Animals; Caspase 9; Cell Line, Transformed; Cyclic AMP-Dependent Protein Kinases; Desmin; Doxorubicin; Gene Expression Regulation; Glomerulosclerosis, Focal Segmental; Interleukin-1beta; Interleukin-6; Male; Membrane Proteins; Mice; Oligopeptides; Podocytes; Protein Kinase C-epsilon; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Signal Transduction; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Renal transplantation is the optimal renal replacement therapy (RRT) in children, but some primary diseases can recur after transplantation, and recurrence accounts for a significant proportion of graft losses, being second only to acute rejection. The risk of disease recurrence is highest among patients with idiopathic focal segmental glomerulosclerosis (FSGS), presumably due to a circulating permeability factor. Less is clear about the genetic forms of FSGS, where the data regarding the frequency of recurrence are rather conflicting. We present a 12-year-old girl with rapidly progressive FSGS and end-stage renal disease in her native kidneys associated with heterozygous mutations in NPHS1 and in NPHS2, suffering from early post-transplant recurrence. On the basis of reviewed literature, and until further and more conclusive evidence considering pathogenicity is provided, we propose that FSGS patients with heterozygous mutations in NPHS1 or NPHS2 should be considered as having idiopathic FSGS, and post-transplant recurrence should be anticipated.

Topics: Child; Disease Progression; Female; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney Failure, Chronic; Kidney Transplantation; Membrane Proteins; Mutation; Recurrence

Mutations in the INF2 (inverted formin 2) gene, encoding a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause human focal segmental glomerulosclerosis (FSGS). INF2 interacts directly with certain other mammalian diaphanous formin proteins (mDia) that function as RhoA effector molecules. FSGS-causing INF2 mutations impair these interactions and disrupt the ability of INF2 to regulate Rho/Dia-mediated actin dynamics in vitro. However, the precise mechanisms by which INF2 regulates and INF2 mutations impair glomerular structure and function remain unknown. Here, we characterize an Inf2 R218Q point-mutant (knockin) mouse to help answer these questions. Knockin mice have no significant renal pathology or proteinuria at baseline despite diminished INF2 protein levels. INF2 mutant podocytes do show impaired reversal of protamine sulfate-induced foot process effacement by heparin sulfate perfusion. This is associated with persistent podocyte cytoplasmic aggregation, nephrin phosphorylation, and nephrin and podocin mislocalization, as well as impaired recovery of mDia membrane localization. These changes were partially mimicked in podocyte outgrowth cultures, in which podocytes from knockin mice show altered cellular protrusions compared to those from wild-type mice. Thus, in mice, normal INF2 function is not required for glomerular development but normal INF2 is required for regulation of the actin-based behaviors necessary for response to and/or recovery from injury.

Topics: Actins; Acute Kidney Injury; Animals; Cells, Cultured; Disease Models, Animal; Formins; Glomerulosclerosis, Focal Segmental; Heparin; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Microfilament Proteins; Microscopy, Electron, Transmission; Phenotype; Phosphorylation; Podocytes; Point Mutation; Protamines; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; 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

Recurrent disease occurs in around 30% of children transplanted for steroid-resistant nephrotic syndrome. Its precipitating risk factors have rarely been studied in the Middle East. The aim of our study was to determine what characterizes posttransplant recurrence of nephrotic syndrome in Syrian children.. We performed a retrospective analysis of 12 nephrotic children who received 1 renal allograft at the Kidney Hospital in Damascus from 2002 to 2013.. Native kidney biopsy results showed focal segmental glomerulosclerosis in 9 of 10 patients. Four patients had 1 or more sibling affected with nephrotic syndrome, and the remaining patients were labeled as having sporadic disease. Genetic screening for NPHS2, NPHS1, and Wilms tumor gene (WT1) mutations were done for 6 patients, and 1 novel homozygous NPHS2 mutation was identified in 1 patient. All patients received transplants from living donors. Four patients had recurrence of initial disease after transplant (overall recurrence rate of 33%). However, 1 patient showed complete and spontaneous remission 20 months after transplant; As expected, the patient with NPSH2 mutation had no recurrence. Patients with sporadic disease showed risk of recurrence 5 times higher than patients with familial disease (P = .24). Interestingly, all recurrent cases had received a kidney from a related donor and were initially classified as having sporadic disease. Although not statistically significant, the risk of recurrence from related donor grafts was 6.75 times higher than from unrelated donors (P = .16). To the best of our knowledge, this observation, the first of its kind, has never been investigated or pointed out in the literature.. Further research is needed to confidently determine whether living related donor grafts are associated with increased incidence of recurrence of nephrotic syndrome.

Topics: Adolescent; Child; Child, Preschool; Female; Genes, Wilms Tumor; Genetic Testing; Glomerulosclerosis, Focal Segmental; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Kidney Transplantation; Living Donors; Male; Membrane Proteins; Nephrotic Syndrome; Recurrence; Remission, Spontaneous; Retrospective Studies

Blockade of the renin-angiotensin system plays a key role in suppressing the progression of renal diseases. It has not been well established whether this therapy provides additional effects when combined with vitamin D or its analog in a model of adriamycin (ADR)-induced nephropathy.. We evaluated the effect of an angiotensin II subtype 1 receptor blocker (telmisartan) combined with a vitamin D analog (oxacalcitriol) on mice ADR-induced nephropathy (9.5 mg/kg single intravenous injection). We also tested immortalized murine podocytes to examine the effects on podocyte apoptosis.. Mice with ADR-induced nephropathy developed progressive albuminuria and glomerulosclerosis within 30 days accompanied by decreased expression of slit diaphragm (SD)-associated proteins (nephrin and podocin), reduced numbers of podocytes, and increased systolic blood pressure. Treatment with telmisartan or oxacalcitriol alone moderately ameliorated kidney injury. The combined treatment most effectively reduced the albuminuria and glomerulosclerosis. These effects were accompanied by the restoration of SD-associated proteins, reduction of podocyte apoptosis, and prevention of podocyte depletion in the glomeruli. Treatment with telmisartan, oxacalcitriol, and the combination therapy resulted in similar reductions in systolic blood pressure. In cultured murine podocytes, ADR stimulated the expression of Bax/Bcl-2 and apoptosis as determined by Hoechst 33342 staining. These changes were effectively inhibited by telmisartan or oxacalcitriol, but the combination treatment most effectively reduced these effects.. These data demonstrated that application of a renin-angiotensin system blocker plus a vitamin D analog effectively prevented renal injury in ADR-induced nephropathy. The observed amelioration of renal injury may be partly attributable to antiapoptotic effects in podocytes.

Topics: Albuminuria; Angiotensin II Type 1 Receptor Blockers; Animals; Antibiotics, Antineoplastic; Apoptosis; Benzimidazoles; Benzoates; Calcitriol; Doxorubicin; Drug Therapy, Combination; Female; Glomerulosclerosis, Focal Segmental; Intracellular Signaling Peptides and Proteins; Kidney Diseases; Membrane Proteins; Mice; Mice, Inbred BALB C; Podocytes; Telmisartan

The Wilms' tumor suppressor WT1 is a key regulator of podocyte function that is mutated in Denys-Drash and Frasier syndromes. Here we have used an integrative approach employing ChIP, exon array, and genetic analyses in mice to address general and isoform-specific functions of WT1 in podocyte differentiation. Analysis of ChIP-Seq data showed that almost half of the podocyte-specific genes are direct targets of WT1. Bioinformatic analysis further identified coactivator FOXC1-binding sites in proximity to WT1-bound regions, thus supporting coordinated action of these transcription factors in regulating podocyte-specific genes. Transcriptional profiling of mice lacking the WT1 alternative splice isoform (+KTS) had a more restrictive set of genes whose expression depends on these alternatively spliced isoforms. One of these genes encodes the membrane-associated guanylate kinase MAGI2, a protein that localizes to the base of the slit diaphragm. Using functional analysis in mice, we further show that MAGI2α is essential for proper localization of nephrin and the assembly of the slit diaphragm complex. Finally, a dramatic reduction of MAGI2 was found in an LPS mouse model of glomerular injury and in genetic cases of human disease. Thus, our study highlights the central role of WT1 in podocyte differentiation, identifies that WT1 has a central role in podocyte differentiation, and identifies MAGI2α as the crucial isoform in slit diaphragm assembly, suggesting a causative role of this gene in the etiology of glomerular disorders.

Topics: Adaptor Proteins, Signal Transducing; Alternative Splicing; Animals; Binding Sites; Cell Differentiation; Down-Regulation; Exons; Female; Forkhead Transcription Factors; Glomerulonephritis, Membranoproliferative; Glomerulosclerosis, Focal Segmental; Guanylate Kinases; Humans; Lipopolysaccharides; Membrane Proteins; Mice; Mutation; Oligonucleotide Array Sequence Analysis; Podocytes; Promoter Regions, Genetic; Protein Isoforms; Repressor Proteins; Transcription, Genetic; WT1 Proteins

Individuals with TRPC6 mutations have variable phenotypes, ranging from healthy carrier to focal segmental glomerulosclerosis (FSGS) leading to renal failure. Here, we describe a family where six members had a novel TRPC6 p.R68W (c.202C>T) mutation, two of whom had renal failure from FSGS, and one had proteinuria. One healthy carrier donated a kidney to her sister. Both donor and recipient had no proteinuria at 20 years posttransplant. Two synonymous NPHS1 polymorphisms, rs2285450 (c.294C>T) and rs437168 (c.2289C>T) segregated with renal failure in this family. These variants had higher allele frequencies in 97 unrelated patients with nephrotic syndrome or FSGS compared to 224 controls. Using patch-clamp experiments in HEK293 and podocytes, we showed that the p.R68W mutation increased TRPC6 current amplitudes, which may be explained by enhanced TRPC6 surface expression. Additionally, while wild-type nephrin suppressed TRPC6 currents, this ability was lost in the presence of NPHS1 c.294C>T polymorphism. When cells were transfected according to combined TRPC6 and NPHS1 genotypes in the family, those representing the donor had lower TRPC6 currents than cells representing the recipient, suggesting that interactions between TRPC6 and NPHS1 variants could possibly account for the variable penetrance of TRPC6 mutations and the absence of recurrence in the graft.

Topics: Adolescent; Adult; Aged; Animals; Blotting, Western; Case-Control Studies; Child; Child, Preschool; Female; Follow-Up Studies; Gene Frequency; Genotype; Glomerular Filtration Rate; Glomerulosclerosis, Focal Segmental; Graft Rejection; Graft Survival; HEK293 Cells; Humans; Infant; Kidney Function Tests; Kidney Transplantation; Male; Membrane Proteins; Mice; Mice, Knockout; Middle Aged; Mutation; Pedigree; Phenotype; Podocytes; Polymorphism, Single Nucleotide; Postoperative Complications; Prognosis; Recurrence; Risk Factors; TRPC Cation Channels; TRPC6 Cation Channel; Young Adult

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

Podocyte depletion is a characteristic feature of progressive renal failure. We hypothesize that studying the podocyte mRNA level in urinary sediment may provide diagnostic and prognostic information in adult nephrotic syndrome.. We studied 25 patients with minimal change nephropathy (MCN), 25 with focal segmental glomerulosclerosis (FSGS), and 17 healthy controls. The mRNA levels of nephrin, podocin, and synaptopodin in urinary sediment were quantified.. There were significant differences in the urinary sediment nephrin and podocin, but not synaptopodin, mRNA levels between diagnosis groups. Post-hoc analysis further showed that urinary nephrin mRNA levels of the MCN group were lower than those in the control and FSGS groups, although the difference between MCN and FSGS groups did not reach statistical significance. The degree of proteinuria inversely correlated with urinary nephrin mRNA levels in the MCN (r = -0.526, p = 0.007) as well as in the FSGS group (r = -0.521, p = 0.008). For the FSGS group, the rate of renal function decline significantly correlated with baseline urinary synaptopodin mRNA levels (r = -0.496, p = 0.012).. Urinary nephrin and podocin mRNA levels were reduced in patients with MCN and probably FSGS, and the magnitude of reduction correlated with the degree of proteinuria. Urinary synaptopodin mRNA levels correlated with the subsequent rate of renal function decline in patients with FSGS. Our result indicates that urine sediment podocyte mRNA levels provide novel insights in the pathophysiology of nephrotic syndrome and could be useful for risk stratification.

Topics: Adult; Aged; Female; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Microfilament Proteins; Middle Aged; Nephrosis, Lipoid; Podocytes; RNA, Messenger

The aim of our study was to examine NPHS1, NPHS2, WT1 and LAMB2 mutations, previously reported in two thirds of patients with nephrotic syndrome with onset before the age of one year old. Genomic DNA samples from Polish children (n=33) with Steroid-Resistant Nephrotic Syndrome (SRNS) due to focal segmental glomerulosclerosis (FSGS), manifesting before the age of 13 years old, underwent retrospective analysis of NPHS1, NPHS2, WT1 (exons 8, 9 and adjacent exon/intron boundaries) and LAMB2. No pathogenic NPHS1 or LAMB2 mutations were found in our FSGS cohort. SRNS-causing mutations of NPHS2 and WT1 were detected in 7 of 33 patients (21%), including those with nephrotic syndrome manifesting before one year old: five of seven patients. Four patients had homozygous c.413G>A (p.Arg138Gln) NPHS2 mutations; one subject was homozygous for c.868G>A (p.Val290Met) NPHS2. A phenotypic female had C>T transition at position +4 of the WT1 intron 9 (c.1432+4C>T) splice-donor site, and another phenotypic female was heterozygous for G>A transition at position +5 (c.1432+5G>A). Genotyping revealed a female genotypic gender (46, XX) for the first subject and male (46, XY) for the latter. In addition, one patient was heterozygous for c.104dup (p.Arg36Profs*34) NPHS2; two patients carried a c.686G>A (p.Arg229Gln) NPHS2 non-neutral variant. Results indicate possible clustering of causative NPHS2 mutations in FSGS-proven SRNS with onset before age one year old, and provide additional evidence that patients with childhood steroid-resistant nephrotic syndrome due to focal segmental glomerulosclerosis should first undergo analysis of NPHS2 coding sequence and WT1 exons 8 and 9 and surrounding exon/intron boundary sequences, followed by gender genotyping.

Topics: Adolescent; Age Factors; Child; Child, Preschool; Female; Genes, Wilms Tumor; Glomerulosclerosis, Focal Segmental; Humans; Infant; Intracellular Signaling Peptides and Proteins; Laminin; Male; Membrane Proteins; Mutation; Retrospective Studies; Sex Factors; WT1 Proteins

To observe the effects and mechanisms of Bushenhuoxue on desmin and nephrin expression in mice podocytes, and to investigate its effects on wt1 expression in Wilms' tumor.. Adriamycin (ADR) was used to induce focal segmental glomerulous sclerosis (FSGS) in mice. Bushenhuoxue was used to treat FSGS for 6 weeks. We measured body mass and right renal mass, and determined serum albumin (ALB) levels, protein content in urine, and urinary protein and albumin creatinine ratio (UACR). Changes in renal tissue morphology were evaluated by microscopy. wt1 and nephrin expression in podocytes were detected using immunofluorescence. Expression levels of desmin, wt1 and nephrin mRNAs in renal tissue were determined using reverse transcription polymerase chain reaction assays.. Protein levels in urine and UACR were significantly increased in FSGS model mice compared with Bushenhuoxue-treated and control mice. Body mass and ALB levels were decreased in FSGS mice compared with control and Bushenhuoxue-treated mice. Expression of the wt1 protein was observed in control mice. Compared with controls, wt1 expression levels were reduced in Bushenhuoxue-treated mice, and to a greater extent in FSGS mice. Nephrin protein expression was widespread in FSGS mice, and significantly reduced in control and Bushenhuoxue mice. Expression levels of wt1 and nephrin mRNAs in FSGS mice were lower compared with those in control and Bushenhuoxue-treated mice. Desmin mRNA levels in FSGS mice were reduced compared with those in control and Bushenhuoxue-treated mice.. Bushenhuoxue ameliorated albuminuria in FSGS mice; this was possibly related to the up-regulation of wt1 and nephrin, and down-regulation of desmin.

Topics: Animals; Drugs, Chinese Herbal; Glomerulosclerosis, Focal Segmental; Humans; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Podocytes; WT1 Proteins

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

Mutations in inverted formin 2 INF2 are a common cause of familial FSGS. INF2 interacts with diaphanous-related formins (mDia) and antagonizes mDia-mediated actin polymerization in response to active Rho signaling, suggesting that dysregulation of these pathways may mediate the development of INF2-related FSGS. However, the precise mechanisms by which INF2 regulates actin-dependent podocyte behavior remain largely unknown. Here, we investigated the possible role of INF2 in both lamellipodia-associated actin dynamics and actin-dependent slit diaphragm (SD) protein trafficking by manipulating the expression of INF2 and the activity of Rho/mDia signaling in cultured podocytes. Activation of mDia in the absence of INF2 led to defective formation of lamellipodia and abnormal SD trafficking. Effects of mutations disrupting the INF2-mDia interaction suggested the specificity of the mDia-antagonizing effect of INF2 in maintaining the lamellipodium. Furthermore, we found that SD trafficking requires INF2 interaction with lipid raft components. In summary, INF2 regulates lamellipodial actin dynamics and the trafficking of slit diaphragm proteins by opposing Rho/mDia-mediated actin polymerization. Thus, in podocytes, INF2 appears to be an important modulator of actin-dependent behaviors that are under the control of Rho/mDia signaling.

Topics: Actins; Carrier Proteins; Cell Line, Transformed; Cytosol; Dimerization; Formins; Glomerulosclerosis, Focal Segmental; Humans; Membrane Microdomains; Membrane Proteins; Microfilament Proteins; Podocytes; Point Mutation; Protein Structure, Quaternary; Protein Transport; Pseudopodia; rhoA GTP-Binding Protein; RNA, Small Interfering; Signal Transduction

A girl aged 6 presented with haematuria and her sister (aged 5) presented with haematuria and proteinuria. Family history showed multiple individuals suffering from end stage renal failure from the paternal side of the pedigree. Following kidney biopsy in the father and paternal grandmother, the pathological diagnosis was of focal segmental glomerulosclerosis (FSGS). Exome sequencing was undertaken in the proband's sister and grandmother. Genetic variants shared by both affected individuals were interrogated to identify the genetic cause of disease. Candidate variants were then sequenced in all the family members to determine segregation with the disease. A mutation of COL4A5 known to cause Alport syndrome segregated with disease from the paternal side of the pedigree and a variant in NPHS1 was present in both paediatric cases and inherited from their mother. This study highlights the advantages of exome sequencing over single gene testing; disease presentation can be heterogeneous with several genes representing plausible candidates; candidate gene(s) may be unavailable as a diagnostic test; consecutive, single gene testing typically concludes once a single causal mutation is identified. In this family, we were able to confirm a diagnosis of Alport syndrome, which will facilitate testing in other family members.

Topics: Adult; Biopsy; Child; Child, Preschool; Collagen Type IV; Diagnosis, Differential; Exome; Female; Glomerulosclerosis, Focal Segmental; Humans; Kidney; Male; Membrane Proteins; Middle Aged; Mutation; Nephritis, Hereditary; Pedigree; Sequence Analysis, DNA

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

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

Podocyte damage and apoptosis are thought to be important if not essential in the development of glomerulosclerosis. Female estrogen receptor knockout mice develop glomerulosclerosis at 9 months of age due to excessive ovarian testosterone production and secretion. Here, we studied the pathogenesis of glomerulosclerosis in this mouse model to determine whether testosterone and/or 17β-estradiol directly affect the function and survival of podocytes. Glomerulosclerosis in these mice was associated with the expression of desmin and the loss of nephrin, markers of podocyte damage and apoptosis. Ovariectomy preserved the function and survival of podocytes by eliminating the source of endogenous testosterone production. In contrast, testosterone supplementation induced podocyte apoptosis in ovariectomized wild-type mice. Importantly, podocytes express functional androgen and estrogen receptors, which, upon stimulation by their respective ligands, have opposing effects. Testosterone induced podocyte apoptosis in vitro by androgen receptor activation, but independent of the TGF-β1 signaling pathway. Pretreatment with 17β-estradiol prevented testosterone-induced podocyte apoptosis, an estrogen receptor-dependent effect mediated by activation of the ERK signaling pathway, and protected podocytes from TGF-β1- or TNF-α-induced apoptosis. Thus, podocytes are target cells for testosterone and 17β-estradiol. These hormones modulate podocyte damage and apoptosis.

Topics: Animals; Apoptosis; Cell Dedifferentiation; Desmin; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression; Glomerulosclerosis, Focal Segmental; In Vitro Techniques; Membrane Proteins; Mice; Mice, Knockout; Ovariectomy; Podocytes; Receptors, Androgen; Smad7 Protein; Testosterone; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

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

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and α-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling.

Topics: Actinin; Albuminuria; Animals; Bradykinin; Bradykinin B1 Receptor Antagonists; Disease Models, Animal; Doxorubicin; Gene Expression Regulation; Glomerulosclerosis, Focal Segmental; Heme Oxygenase-1; Interleukin-1beta; Intracellular Signaling Peptides and Proteins; Macrophages; Male; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Inbred BALB C; Podocytes; Receptor, Bradykinin B1; RNA, Messenger; Signal Transduction; Time Factors; Tissue Inhibitor of Metalloproteinase-1

Podocyte proteins are involved in the pathogenesis of glomerular kidney disease (GKD). However, there is little information on messenger RNA (mRNA) expression patterns of B7-1 and NPHS1 in urinary sediment of patients with GKD. The objective of this study was to analyse the gene expression of B7-1 in urinary sediment and correlate it with the expression of podocyte-specific genes in patients with GKD.. Adult patients with proliferative and non-proliferative GKD, proteinuria and stable renal function, were included. A group of healthy subjects was used to determine normal levels of urinary markers and to obtain reference RNA. Biochemical, clinical and experimental procedures included measurement of creatinine level and total urinary protein, renal biopsy, identification of urinary podocytes, gene expression analysis of B7-1, NPHS1, NPHS2 and SyNPO genes and urinary B7-1 protein analysis by enzyme-linked immunosorbent assay.. Between June 2006 and November 2009, 69 patients with GKD (median age: 46 ± 15 years, 64% men) and 14 healthy subjects (median age: 34 ± 12 years, 43% men) were included. In both groups, urinary mRNA levels of B7-1 and NPHS1 were significantly higher in patients with GKD compared to healthy subjects (P = 0.050 and P = 0.008, respectively). Regarding GKD subtypes, patients with focal segmental glomerulosclerosis (FSGS), but not patients with minimal change disease (MCD), had a significantly higher mRNA expression of B7-1 and NPHS1 than healthy subjects (P = 0.012 and P = 0.030, respectively). Patients with MCD had a significantly lower NPHS1 mRNA expression than patients with FSGS (P = 0.012). The B7-1:NPHS1 urinary mRNA ratio was significantly higher in patients with MCD compared with patients with FSGS (P = 0.027).. mRNA expression analysis of B7-1 and NPHS1 in urinary sediment may be useful to differentiate between different histologic subtypes of GKD, particularly between MCD and FSGS.

Topics: Adult; Diagnosis, Differential; Female; Glomerulosclerosis, Focal Segmental; Humans; Inducible T-Cell Co-Stimulator Ligand; Male; Membrane Proteins; Middle Aged; Nephrosis, Lipoid; Podocytes; Prospective Studies; RNA, Messenger

Transient receptor potential canonicals (TRPCs) play important roles in the regulation of intracellular calcium concentration. Mutations in the TRPC6 gene are found in patients with focal segmental glomerulosclerosis (FSGS), a proteinuric disease characterized by dysregulated function of renal glomerular epithelial cells (podocytes). There is as yet no clear picture for the activation mechanism of TRPC6 at the molecular basis, however, and the association between its channel activity and pathogenesis remains unclear. We demonstrate here that tyrosine phosphorylation of TRPC6 induces a complex formation with phospholipase C (PLC)-γ1, which is prerequisite for TRPC6 surface expression. Furthermore, nephrin, an adhesion protein between the foot processes of podocytes, binds to phosphorylated TRPC6 via its cytoplasmic domain, competitively inhibiting TRPC6-PLC-γ1 complex formation, TRPC6 surface localization, and TRPC6 activation. Importantly, FSGS-associated mutations render the mutated TRPC6s insensitive to nephrin suppression, thereby promoting their surface expression and channel activation. These results delineate the mechanism of TRPC6 activation regulated by tyrosine phosphorylation, and imply the cell type-specific regulation, which correlates the FSGS mutations with deregulated TRPC6 channel activity.

Topics: Animals; Calcium Signaling; Cell Membrane; Glomerulosclerosis, Focal Segmental; HEK293 Cells; Humans; Membrane Proteins; Mutation, Missense; Peptide Fragments; Phospholipase C gamma; Phosphorylation; Podocytes; Protein Binding; Protein Transport; Rats; Recombinant Proteins; src-Family Kinases; TRPC Cation Channels; TRPC6 Cation Channel; Tyrosine

Mutations in the TRPC6 gene have been recently identified as the cause of late-onset autosomal-dominant focal segmental glomerulosclerosis (FSGS). To extend the screening, we analyzed TRPC6 in 33 Italian children with sporadic early-onset SRNS and three Italian families with adult-onset FSGS.. TRPC6 mutation analysis was performed through PCR and sequencing. The effects of the detected amino acid substitutions were analyzed by bioinformatics tools and functional in vitro studies. The expression levels of TRPC6 and nephrin proteins were evaluated by confocal microscopy.. Three heterozygous missense mutations (c.374A>G_p.N125S, c.653A>T_p.H218L, c.2684G>T_p.R895L) were identified. The first new mutation, p.H218L, was found in a 18-year-old boy who presented a severe form of FSGS at the age of 8 years. The second, p.R895L, a new de novo mutation, was identified in a girl with collapsing glomerulosclerosis at the age of 2 years. The former mutation, p.N125S, was found in two siblings with early-onset steroid-resistant nephrotic syndrome (SRNS) at the ages of 4 and 14 years. Renal immunofluorescence revealed upregulated expression of TRPC6 and loss of nephrin in glomeruli. The intracellular calcium concentrations were significantly higher in the cells expressing all mutant TRPC6 channels compared with cells expressing wild-type TRPC6.. Our findings suggest that TRPC6 variants can also be detected in children with early-onset and sporadic SRNS (4 of 33 patients). Moreover, in one patient a new de novo TRPC6 mutation was associated with a rare severe form of childhood collapsing glomerulosclerosis with rapid progression to uremia.

Topics: Adolescent; Age of Onset; Amino Acid Sequence; Base Sequence; Calcium; Case-Control Studies; Child; Child, Preschool; DNA Mutational Analysis; Drug Resistance; Female; Fluorescent Antibody Technique; Genetic Predisposition to Disease; Glomerulosclerosis, Focal Segmental; HEK293 Cells; Humans; Infant; Italy; Kidney; Male; Membrane Proteins; Microscopy, Confocal; Molecular Sequence Data; Mutation, Missense; Nephrotic Syndrome; Pedigree; Phenotype; Polymerase Chain Reaction; Risk Assessment; Risk Factors; Steroids; Transfection; TRPC Cation Channels; TRPC6 Cation Channel

Glomerular visceral epithelial cells (podocytes) contain interdigitated processes that form specialized intercellular junctions, termed slit diaphragms, which provide a selective filtration barrier in the renal glomerulus. Analyses of disease-causing mutations in familial nephrotic syndromes and targeted mutagenesis in mice have revealed critical roles of several proteins in the assembly of slit diaphragms. The nephrin-podocin complex is the main constituent of slit diaphragms. However, the molecular mechanisms regulating these proteins to maintain the slit diaphragms are still largely unknown. Here, we demonstrate that the PAR3-atypical protein kinase C (aPKC)-PAR6beta cell polarity proteins co-localize to the slit diaphragms with nephrin. Furthermore, selective depletion of aPKClambda in mouse podocytes results in the disassembly of slit diaphragms, a disturbance in apico-basal cell polarity, and focal segmental glomerulosclerosis (FSGS). The aPKC-PAR3 complex associates with the nephrin-podocin complex in podocytes through direct interaction between PAR3 and nephrin, and the kinase activity of aPKC is required for the appropriate distribution of nephrin and podocin in podocytes. These observations not only establish a critical function of the polarity proteins in the maintenance of slit diaphragms, but also imply their potential involvement in renal failure in FSGS.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Adhesion Molecules; Cell Cycle Proteins; Cell Polarity; Glomerulosclerosis, Focal Segmental; Intercellular Junctions; Intracellular Signaling Peptides and Proteins; Isoenzymes; Membrane Proteins; Mice; Multiprotein Complexes; Podocytes; Protein Kinase C

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

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

Podocyte dysfunction, one of the major causes of proteinuria, leads to glomerulosclerosis and end stage renal disease, but its underlying mechanism remains poorly understood. Here we show that Wnt/beta-catenin signaling plays a critical role in podocyte injury and proteinuria. Treatment with adriamycin induced Wnt and activated beta-catenin in mouse podocytes. Overexpression of Wnt1 in vivo activated glomerular beta-catenin and aggravated albuminuria and adriamycin-induced suppression of nephrin expression, whereas blockade of Wnt signaling with Dickkopf-1 ameliorated podocyte lesions. Podocyte-specific knockout of beta-catenin protected against development of albuminuria after injury. Moreover, pharmacologic activation of beta-catenin induced albuminuria in wild-type mice but not in beta-catenin-knockout littermates. In human proteinuric kidney diseases such as diabetic nephropathy and focal segmental glomerulosclerosis, we observed upregulation of Wnt1 and active beta-catenin in podocytes. Ectopic expression of either Wnt1 or stabilized beta-catenin in vitro induced the transcription factor Snail and suppressed nephrin expression, leading to podocyte dysfunction. These results suggest that targeting hyperactive Wnt/beta-catenin signaling may represent a novel therapeutic strategy for proteinuric kidney diseases.

Topics: Albuminuria; Animals; beta Catenin; Biopsy; Cell Line, Transformed; Diabetic Nephropathies; Glomerulosclerosis, Focal Segmental; Humans; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Up-Regulation; Wnt1 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

Mutations in the NPHS1 gene cause congenital nephrotic syndrome of the Finnish type presenting before the first 3 months of life. Recently, NPHS1 mutations have also been identified in childhood-onset steroid-resistant nephrotic syndrome and milder courses of disease, but their role in adults with focal segmental glomerulosclerosis remains unknown. Here we developed an in silico scoring matrix to evaluate the pathogenicity of amino-acid substitutions using the biophysical and biochemical difference between wild-type and mutant amino acid, the evolutionary conservation of the amino-acid residue in orthologs, and defined domains, with the addition of contextual information. Mutation analysis was performed in 97 patients from 89 unrelated families, of which 52 presented with steroid-resistant nephrotic syndrome after 18 years of age. Compound heterozygous or homozygous NPHS1 mutations were identified in five familial and seven sporadic cases, including one patient 27 years old at onset of the disease. Substitutions were classified as 'severe' or 'mild' using this in silico approach. Our results suggest an earlier onset of the disease in patients with two 'severe' mutations compared to patients with at least one 'mild' mutation. The finding of mutations in a patient with adult-onset focal segmental glomerulosclerosis indicates that NPHS1 analysis could be considered in patients with later onset of the disease.

Topics: Adult; Age of Onset; Amino Acid Substitution; Child; Child, Preschool; Cohort Studies; Female; Genetic Association Studies; Glomerulosclerosis, Focal Segmental; Heterozygote; Homozygote; Humans; Infant; Infant, Newborn; Male; Membrane Proteins; Mutation; Mutation, Missense; Nephrotic Syndrome; Spain

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

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

Focal segmental glomerulosclerosis (FSGS) is a major cause of steroid-resistant nephrotic syndrome in childhood with a central role for the podocytes in the pathogenesis. Mutated proteins expressed in podocytes cause proteinuria. The role of combined gene defects in the development of FSGS is less clear.. We analysed seven podocyte genes known to cause proteinuria and FSGS in a group of 19 non-familial childhood-onset steroid-resistant FSGS patients. These genes include NPHS1, NPHS2, ACTN4, CD2AP, WT-1, TRPC6 and PLCE1. We also screened for the mitochondrial A3243G DNA transition associated with the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), and occasionally FSGS.. No mutations were found in the ACTN4 and TRPC6 genes, and no mitochondrial A3243G DNA transition was found in our group of patients. Two patients showed mutations in the CD2AP gene, one combined with an NPHS2 mutation. A tri-allelic hit was found in a patient carrying compound heterozygous NPHS2 mutations and a heterozygous NPHS1 mutation. In another patient a de novo WT-1 mutation was found combined with a heterozygous NPHS1 mutation, and finally two patients showed three heterozygous PLCE1 mutations.. In our rather small group of 19 steroid-resistant FSGS patients, we found 11 mutations in podocyte genes in 6 patients. In four of them the found mutations could explain the pathology. Our data suggest that combined gene defects in podocyte genes may play a role in the development of FSGS.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Base Sequence; Child; Child, Preschool; Cytoskeletal Proteins; DNA Mutational Analysis; DNA Primers; Drug Resistance; Female; Genes, Wilms Tumor; Glomerulosclerosis, Focal Segmental; Heterozygote; Humans; Infant; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mutation; Pedigree; Phosphoinositide Phospholipase C; Podocytes; Steroids

Our aim was to correlate an immunohistochemical pattern of selected podocyte cytoskeleton-associated proteins in children diagnosed with focal segmental glomerulosclerosis (FSGS) and diffuse mesangial proliferation accompanied by glomerular immaturity (Im-DMP) with the clinical courses of both diseases. The material included 33 renal biopsies obtained from children diagnosed with DMP with or without signs of glomerular immaturity (ten and 15 participants, respectively) or FSGS (eight patients). Ezrin, podocalyxin, synaptopodin and nephrin expression was evaluated by immunohistochemical assay. A positive reaction for ezrin, podocalyxin, synaptopodin and nephrin was observed in the most superficial, continuous 'layer' of podocytes in Im-DMP patients. This distribution closely mimicked the immunohistochemical pattern observed in FSGS. The severe initial course of Im-DMP was transient. Resistance to steroids (six children) and renal insufficiency (two patients) in these subjects subsided, whilst, in the FSGS patients, the resistance to steroids recognized in all the children and the renal insufficiency diagnosed in three of them were still present. Mimicry between the immunohistochemical pattern of glomerular immaturity in DMP and focal segmental glomerulosclerosis might explain the severe initial course of this nephrotic syndrome in children. The transient clinical character of the former may also indicate that it is not a variant of FSGS.

Topics: Adrenal Cortex Hormones; Biopsy; Child; Cytoskeletal Proteins; Drug Resistance; Glomerular Mesangium; Glomerulosclerosis, Focal Segmental; Humans; Membrane Proteins; Nephrotic Syndrome; Renal Insufficiency; Retrospective Studies; Sialoglycoproteins

The recent advances in understanding the pathophysiology of focal segmental glomerulosclerosis (FSGS) and molecular function of glomerular filtration barrier come directly from genetic linkage and positional cloning studies. The exact role and function of the newly discovered genes and proteins are being investigated by in vitro and in vivo mechanistic studies. Those genes and proteins interactions seem to change susceptibility to kidney disease progression. Better understanding of their exact role in the development of FSGS may influence future therapies and outcomes in this complex disease.

Topics: Actinin; Chromosome Mapping; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Microfilament Proteins; Phosphoinositide Phospholipase C; Type C Phospholipases

We previously reported that increase in plasma homocysteine (Hcys) levels by a 6-week methionine treatment produced remarkable glomerular injury. However, the mechanism by which hyperhomocysteinemia (hHcys) produces glomerular injury remains unknown. The present study was to observe when glomerular injury happens during hHcys and to explore the possible role of podocyte injury in the progression of glomerulosclerosis associated with hHcys.. Uninephrectomized Sprague-Dawley rats treated with methionine were used to examine the time course of glomerular injury induced by hHcys.. Creatinine clearance was not different until rats were treated with methionine for 6 weeks, although plasma Hcys levels significantly increased at the 1st week of methionine treatment. However, urinary albumin excretion increased at the 2nd week of methionine treatment. Morphological examinations showed that mesangial expansion occurred at the 2nd week and podocyte effacement was also observed as processed glomerular damage during hHcys. Immunofluorescence analyses demonstrated that podocin and nephrin expressions were reduced, while alpha-actinin-4 increased during hHcys.. Increased plasma Hcys level is an important pathogenic factor resulting in glomerular injury even in the very early time of hHcys. These pathogenic effects of Hcys are associated with podocyte injury and changed expression and distribution of podocyte-associated proteins.

Topics: Actinin; Albuminuria; Animals; Creatinine; Glomerulosclerosis, Focal Segmental; Homocysteine; Hyperhomocysteinemia; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Methionine; Microfilament Proteins; Podocytes; Rats; Rats, Sprague-Dawley; Time Factors

Topics: Actinin; Adult; Female; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Microfilament Proteins; Mutation; TRPC Cation Channels; TRPC6 Cation Channel

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

The presence of circulating plasma factors (PF) altering renal permeability to proteins has been previously described in patients with focal segmental glomerulosclerosis (FSGS). Since these patients show reduced nephrin and podocin expression at renal biopsy, we evaluated the effect of serum and PF from patients with FSGS on nephrin and podocin expression in human podocytes. We studied 7 sera from patients with steroid-resistant FSGS, 3 from patients with nephrotic syndrome caused by non-immune disease, and 6 from healthy subjects. PF was prepared from plasmapheresis eluates of 2 patients with post-transplant recurrence of FSGS. Purification procedure was based on protein A Sepharose chromatography and differential precipitation in ammonium sulphate. Nephrin and podocin expression was semi-quantitatively evaluated by immunofluorescence. We found that serum and PF from FSGS patients rapidly induced redistribution and loss of nephrin in podocytes. This effect was associated with cytoskeleton redistribution and inhibited by cytochalasin B and sodium azide. On the contrary, podocin expression was unchanged after incubation with serum and PF from FSGS patients for short periods, but markedly reduced at 24 h. Our results demonstrate that serum and PF from FSGS patients may directly affect nephrin and podocin in human podocytes, thus providing new insights into the mechanisms causing proteinuria in FSGS.

Topics: Adolescent; Adult; Blood Proteins; Cells, Cultured; Child; Cytoskeleton; Electrophoresis, Gel, Two-Dimensional; Female; Gene Expression; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Middle Aged; Permeability; Podocytes

Nephrin is a podocyte adhesion molecule located at the slit diaphragm between adjacent glomerular epithelial cells. Mutations in the gene encoding nephrin result in the absence of nephrin or alterations in nephrin causing massive proteinuria in patients with congenital nephrotic syndrome. Given the importance of nephrin to the structural integrity of the glomerular filtration barrier, we postulated that it might also be altered in acquired forms of nephrotic syndrome (NS). To test this hypothesis, frozen kidney biopsy sections from 29 pediatric patients with acquired NS and 5 controls were examined for expression of nephrin. The pathological diagnoses were minimal change disease (MCNS) (19) and focal segmental glomerulosclerosis (FSGS) (10). To determine if nephrin expression differed between children and adults with NS, 10 adult patients and 3 controls were also examined. Nephrin expression was evaluated by immunoperoxidase staining with a monoclonal antibody against the extracellular FnIII portion of human nephrin. In all cases, nephrin expression was seen along the glomerular basement membrane in a finely granular/linear pattern. Expression of nephrin was similar to controls in all 19 patients with MCNS and all 10 patients with FSGS. Areas of sclerosis in patients with FSGS did not demonstrate nephrin expression. A distinctly granular pattern to nephrin expression was seen in adult patients with NS as well as controls. These findings suggest that an alteration in nephrin expression is not a feature of acquired forms of NS in childhood.

Topics: Adolescent; Biopsy; Child; Child, Preschool; Female; Glomerulosclerosis, Focal Segmental; Humans; Immunohistochemistry; Infant; Kidney Glomerulus; Male; Membrane Proteins; Nephrosis, Lipoid; Proteins

Renal failure is a frequent and costly complication of many chronic diseases, including diabetes and hypertension. One common feature of renal failure is glomerulosclerosis, the pathobiology of which is unclear. To help elucidate this, we generated a mouse strain carrying the missense mutation Wt1 R394W, which predisposes humans to glomerulosclerosis and early-onset renal failure (Denys-Drash syndrome [DDS]). Kidney development was normal in Wt1(+/R394W) heterozygotes. However, by 4 months of age 100% of male heterozygotes displayed proteinuria and glomerulosclerosis characteristic of DDS patients. This phenotype was observed in an MF1 background but not in a mixed B6/129 background, suggestive of the action of a strain-specific modifying gene(s). WT1 encodes a nuclear transcription factor, and the R394W mutation is known to impair this function. Therefore, to investigate the mechanism of Wt1 R394W-induced renal failure, the expression of genes whose deletion leads to glomerulosclerosis (NPHS1, NPHS2, and CD2AP) was quantitated. In mutant kidneys, NPHS1 and NPHS2 were only moderately downregulated (25 to 30%) at birth but not at 2 or 4 months. Expression of CD2AP was not changed at birth but was significantly upregulated at 2 and 4 months. Podocalyxin was downregulated by 20% in newborn kidneys but not in kidneys at later ages. Two other genes implicated in glomerulosclerosis, TGFB1 and IGF1, were upregulated at 2 months and at 2 and 4 months, respectively. It is not clear whether the significant alterations in gene expression are a cause or a consequence of the disease process. However, the data do suggest that Wt1 R394W-induced glomerulosclerosis may be independent of downregulation of the genes for NPHS1, NPHS2, CD2AP, and podocalyxin and may involve other genes yet to be implicated in renal failure. The Wt1(R394W) mouse recapitulates the pathology and disease progression observed in patients carrying the same mutation, and the mutation is completely penetrant in male animals. Thus, it will be a powerful and biologically relevant model for investigating the pathobiology of the earliest events in glomerulosclerosis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Base Sequence; Cell Division; Cytoskeletal Proteins; Denys-Drash Syndrome; Disease Models, Animal; DNA; Female; Gene Expression; Genes, Wilms Tumor; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Electron; Phenotype; Point Mutation; Proteins; Renal Insufficiency; Species Specificity

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

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

Mutations of the novel renal glomerular genes NPHS1 and NPHS2 encoding nephrin and podocin cause two types of severe nephrotic syndrome presenting in early life, Finnish type congenital nephrotic syndrome (CNF) and a form of autosomal recessive familial focal segmental glomerulosclerosis (SRN1), respectively. To investigate the mechanisms by which mutations might cause glomerular protein leak, we analysed NPHS1/NPHS2 genotype/phenotype relationships in 41 non-Finnish CNF patients, four patients with congenital (onset 0 to 3 months) focal segmental glomerulosclerosis and five patients with possible SRN1 (onset 6 months to 2 years). We clarify the range of NPHS1 mutations in CNF, detecting mutation 'hot-spots' within the NPHS1 coding sequence. In addition, we describe a novel discordant CNF phenotype characterized by variable clinical severity, apparently influenced by gender. Moreover, we provide evidence that CNF may be genetically heterogeneous by detection of NPHS2 mutations in some CNF patients in whom NPHS1 mutations were not found. We confirm an overlap in the NPHS1/NPHS2 mutation spectrum with the characterization of a unique di-genic inheritance of NPHS1 and NPHS2 mutations, which results in a 'tri-allelic' hit and appears to modify the phenotype from CNF to one of congenital focal segmental glomerulosclerosis (FSGS). This may result from an epistatic gene interaction, and provides a rare example of multiple allelic hits being able to modify an autosomal recessive disease phenotype in humans. Our findings provide the first evidence for a functional inter-relationship between NPHS1 and NPHS2 in human nephrotic disease, thus underscoring their critical role in the regulation of glomerular filtration.

Topics: Adult; Child; Child, Preschool; DNA Mutational Analysis; Female; Genotype; Glomerulosclerosis, Focal Segmental; Humans; Infant; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Nephrotic Syndrome; Phenotype; Proteins

Glomerular disease is one of the most common causes of end-stage renal failure. Increasing evidence suggests that these glomerulopathies are frequently caused by primary lesions in the renal podocytes. One of the major consequences of podocyte lesions is the accumulation of mesangial matrix in the glomerular basement membrane, a process called glomerulosclerosis. Mesangial sclerosis is one of the most consistent findings in Denys-Drash patients and can be caused by dominant mutations in the Wilms' tumor 1 gene (WT1). The underlying mechanism, however, is poorly understood. WT1 is expressed in the podocytes throughout life, but its function in this cell type is unknown. Combining Wt1-knockout and inducible yeast artificial chromosome transgenic mouse models, we demonstrate that reduced expression levels of WT1 result in either crescentic glomerulonephritis or mesangial sclerosis depending on the gene dosage. Strikingly, the two podocyte-specific genes nphs1 and podocalyxin are dramatically downregulated in mice with decreased levels of Wt1, suggesting that these two genes act downstream of Wt1. Taken together, our data provide genetic evidence that reduced levels of Wt1 are responsible for the pathogenesis of two distinct renal diseases and offer a molecular explanation for the increased occurrence of glomerulosclerosis in patients with WAGR syndrome.

Topics: Animals; Down-Regulation; Glomerular Mesangium; Glomerulonephritis; Glomerulosclerosis, Focal Segmental; Humans; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Knockout; Proteins; Sclerosis; Sialoglycoproteins; WT1 Proteins

Topics: Actins; Adaptor Proteins, Signal Transducing; Cytoskeletal Proteins; Epithelial Cells; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Proteins

Podocytes are highly specialized cells that make up a major portion of the glomerular filtration barrier in the kidney. They are also believed to play a pivotal role in the progression of chronic renal disease due to diverse causes that include diabetes (3, 20, 24) and aging (1, 7). Despite the importance of podocytes for kidney function and disease, studies of this cell type have been hindered due to a lack of model systems. Recently, the gene responsible for congenital Finnish nephropathy was identified and named nephrin (13). Nephrin expression is restricted to slit diaphragms of podocytes (11, 30). Infants with congenital Finnish nephropathy develop massive proteinuria and subsequent kidney failure due to podocyte injury. We have identified a 1.25-kb DNA fragment from the human nephrin promoter and 5'-flanking region that is capable of directing podocyte-specific expression in transgenic mice; this represents the first glomerular-specific promoter to be identified. Use of this transgene will facilitate studies of the podocyte in vivo and allow the identification of transacting factors that are required for podocyte-specific expression.

Topics: Base Sequence; beta-Galactosidase; Cloning, Molecular; Glomerulosclerosis, Focal Segmental; Humans; Kidney; Kidney Glomerulus; Lac Operon; Membrane Proteins; Molecular Sequence Data; Promoter Regions, Genetic; Protein Biosynthesis; Proteins; RNA, Messenger