nephrin and Nephrotic-Syndrome

Podocyte is one of the main components of glomerular filtration barrier in the kidney; the loss or dysfunction of podocyte could impair the functions of glomerular filtration barrier, leading to development of various renal diseases. Podocyte is a terminally differentiated cell, and thus does not possess any proliferative properties. Accordingly, its number and contribution to renal function are initially determined by its normal development. Information from the literature and results of our research indicate that genetic factors or prenatal adverse environment could cause developmental retardation of podocytes, thereby suggesting the potential fetal developmental origin(s) of kidney diseases, and involvement of epigenetic mechanisms in the regulation of key genes in podocyte development. In this review, we provide a brief overview on the podocyte normal development; discussion on the potential pathogenic mechanisms for developmental disorders of podocytes; as well as renal diseases associated with podocyte developmental retardation. We aim to provide some insights in articulating the strategies for diagnosis and treatments of renal diseases associated with podocyte developmental abnormalities.

Topics: Animals; Glomerulonephritis; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Membrane Proteins; Mutation; Nephrotic Syndrome; Podocytes; Wnt Signaling Pathway

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

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

Mycophenolic acid (MPA) was introduced into clinical practice as immunosuppressive drug therapy to prevent allograft rejection. Since then, its clinical application has widened. Our goal was to review the lessons learned from experimental nontransplant glomerular disease models on the mechanisms of MPA therapy. T and B lymphocytes are preferentially dependent on de novo purine synthesis. By inhibiting the rate-limiting enzyme of de novo purine synthesis, MPA depletes the pool of deoxyguanosine triphosphate (dGTP) and inhibits proliferation of these immune cells. Furthermore, MPA can also induce apoptosis of immune cells and is known to inhibit synthesis of fucose- and mannose-containing membrane glycoproteins altering the surface expression and binding ability of adhesion molecules. However, MPA exerts a direct effect also on nonimmune cells. Mesangial cells are partially dependent on de novo purine biosynthesis and are thus susceptible to MPA treatment. Additionally, MPA can inhibit apoptosis in podocytes and seems to be beneficial in preserving the expression of nephrin and podocin, and by attenuation of urokinase receptor expression leads to decreased foot-process effacement. In summary, our manuscript sheds light on the molecular mechanisms underlying the antiproteinuric effect of MPA. Overall, MPA is an excellent treatment option in many immunologic glomerulopathies because it possesses immunosuppressive properties, has a remarkable effect on nonimmune cells and counteracts the proliferation of mesangial cells, expansion of mesangial matrix, and foot-process effacement of podocytes combined with a low systemic toxicity.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Glomerular Basement Membrane; Glomerulonephritis; Humans; Immunosuppressive Agents; IMP Dehydrogenase; Intracellular Signaling Peptides and Proteins; Lymphocytes; Membrane Proteins; Mesangial Cells; Monocytes; Mycophenolic Acid; Nephrotic Syndrome; Podocytes; Purines; Rats

Steroid-resistant nephrotic syndrome poses a significant clinical challenge. Its pathogenesis has not been fully elucidated. In recent years, numerous studies have shown that podocyte-specific gene mutations may play important roles in the development of steroid-resistant nephrotic syndrome. Among the identified genes mutated in podocytes include NPHS2, NPHS1, WT1, TRPC6, MDR1, PLCE1, LMX1B, and LAMB2. This review aims to summarize the characteristics of these mutated genes in podocytes. The putative role for these podocyte-specific mutated genes in the pathogenesis, diagnosis, treatment and prognosis of steroid-resistant nephrotic syndrome is also discussed.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Genes, Wilms Tumor; Humans; Intracellular Signaling Peptides and Proteins; LIM-Homeodomain Proteins; Membrane Proteins; Mutation; Nephrotic Syndrome; Podocytes; Transcription Factors; TRPC Cation Channels; TRPC6 Cation Channel

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

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

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: Drug Resistance; Hormones; Humans; Membrane Proteins; Nephrotic Syndrome

In recent years there has been an explosion of interest in the glomerular podocyte, which plays a central role in control of glomerular filtration. A host of new molecules have been identified as playing essential roles in the maintenance of podocyte integrity in both humans and mouse models. Of all of these, arguably the most pivotal is nephrin, a transmembrane receptor molecule located at the specialized podocyte cell-cell junction, termed the slit diaphragm. Mutations in this gene cause the most severe form of congenital nephrotic syndrome, and many interacting proteins have now been described to form a large multiprotein complex with complex dynamics. There is little evidence of functional nephrin expression outside the glomerulus, and there are accumulating data that nephrin is essential for the unique properties of podocyte biology. Utilizing a powerful human cell culture model, comparing wild-type with nephrin-null podocytes, we can show that several crucial functional properties of podocytes depend on nephrin, including insulin responsiveness and cytoskeletal reorganization. Thus, it is reasoned that nephrin is a signature molecule required to define distinct podocyte characteristics.

Topics: Animals; Diabetic Nephropathies; Genotype; Humans; Kidney Glomerulus; Membrane Proteins; Mice; Mutation; Nephrotic Syndrome; Phenotype; Podocytes

Topics: Actinin; Adaptor Proteins, Signal Transducing; Animals; Cytoskeletal Proteins; Formins; Genes, Dominant; Genes, Recessive; Homeodomain Proteins; Humans; Intracellular Signaling Peptides and Proteins; Laminin; LIM-Homeodomain Proteins; Membrane Proteins; Microfilament Proteins; Molecular Motor Proteins; Mutation; Myosin Heavy Chains; Nephrotic Syndrome; Phosphoinositide Phospholipase C; Transcription Factors; WT1 Proteins

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

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

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

Congenital nephrotic syndrome (CNS) is a rare kidney disorder characterized by heavy proteinuria, hypoproteinemia, and edema starting soon after birth. The majority of cases are caused by genetic defects in the components of the glomerular filtration barrier, especially nephrin and podocin. CNS may also be a part of a more generalized syndrome or caused by a perinatal infection. Immunosuppressive medication is not helpful in the genetic forms of CNS, and kidney transplantation is the only curative therapy. Before the operation, management of these infants largely depends on the magnitude of proteinuria. In severe cases, daily albumin infusions are required to prevent life-threatening edema. The therapy also includes hypercaloric diet, thyroxin and mineral substitution, prevention of thrombotic episodes, and prompt management of infectious complications. The outcome of CNS patients without major extrarenal manifestations is comparable with other patient groups after kidney transplantation.

Topics: Humans; Intracellular Signaling Peptides and Proteins; Kidney Transplantation; Membrane Proteins; Nephrotic Syndrome; Podocytes

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

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

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

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

Since the discovery of nephrin, the first integral component of the slit diaphragm to be identified, the podocyte slit pore has become a major focus in research concerning the glomerular filtration barrier. Nephrin is a central component of the glomerular ultrafilter, with both structural and signaling functions. The extracellular domain of nephrin and other components of the slit diaphragm seem to form a porous molecular sieve. The intracellular domain of nephrin is associated with linker proteins, such as CD2-associated protein and Nck proteins that can connect nephrin to the actin cytoskeleton. Alterations in nephrin interactions with other proteins during development or injury can lead to complex signaling reactions aimed at establishing or restoring the filter function.

Topics: Animals; Glomerular Filtration Rate; Humans; Kidney; Membrane Proteins; Models, Biological; Nephrotic Syndrome; Podocytes; Signal Transduction

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

Topics: Glomerular Mesangium; Humans; Membrane Proteins; Mutation; Nephrosclerosis; Nephrotic Syndrome; WT1 Proteins

This Review summarizes recent research on the podocyte slit diaphragm. A growing number of molecules that function at the slit diaphragm have been identified in patients with inherited and sporadic nephrotic syndromes. Genetic deletion of nearly all of these molecules results in proteinuria and effacement of foot processes. Nephrin, Neph1 and podocin seem to form a multifunctional receptor complex at the slit diaphragm. Most of the other components of the slit diaphragm interact directly with this complex, in many cases coupling slit diaphragm components to the podocyte's actin cytoskeleton. These molecular findings are being applied to patients with glomerular disease. Over the next decade, these data might help to improve disease classification and prediction of which patients will respond to immunosuppressive treatment.

Topics: Gene Expression; Glomerular Filtration Rate; Humans; Intercellular Junctions; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nephrotic Syndrome; Podocytes; Signal Transduction

For more than three decades, the molecular composition of the interpodocyte slit diaphragm of the glomerular filtration barrier has remained elusive. The first electron microscopic studies described the slit diaphragm as a porous, 'zipper-like' structure, but it was not until 1998 that the first transmembrane molecule of the slit diaphragm was identified: nephrin is a cell surface receptor of the immunoglobulin superfamily participating in cell-cell adhesion and signaling functions. Mutations in nephrin lead to the congenital nephrotic syndrome of the Finnish type, suggesting that nephrin is of pivotal importance for maintaining the filtration barrier. In recent years, the mapping of the genetic background of other inherited and acquired nephropathies and generation of transgenic animal models have led to a beginning of a new era in nephrology, possibly promising new targeted therapies and advanced diagnostics. This review article will briefly summarize the main findings that explain the molecular architecture of the glomerular filter itself and causes of some glomerular diseases that lead to proteinuria and, eventually, to renal failure.

Topics: Adaptor Proteins, Signal Transducing; Cadherins; Carrier Proteins; Finland; Humans; Kidney Glomerulus; Membrane Proteins; Microscopy, Electron; Nephrotic Syndrome; Nuclear Matrix-Associated Proteins; Podocytes; Sialoglycoproteins

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

Idiopathic nephrotic syndrome is the most common glomerular disorder of childhood. Recurrence of nephrotic syndrome immediately following renal transplantation is rapid, results in a high rate of graft loss, and represents the most severe form of nephrotic syndrome. This review discusses the molecular heterogeneity of pediatric nephrotic syndrome across the spectrum of disease activity. A schema is offered for a molecular approach to pediatric nephrotic syndrome, including immune-mediated and structural/genetic factors.

Topics: Actinin; Child; Genetic Heterogeneity; Humans; Interferon-gamma; Interleukin-12; Interleukin-13; Interleukin-15; Interleukin-18; Interleukin-2; Interleukin-4; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Microfilament Proteins; Nephrotic Syndrome; NF-kappa B; Proteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

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

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

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

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

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

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

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

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

Topics: Adult; Female; Glycoproteins; Humans; Membrane Proteins; Mutation; Nephrotic Syndrome; Pregnancy; Prenatal Diagnosis; Proteins; Reptilian Proteins

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

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

Nephrotic syndrome is characterized by protein loss in the urine, hypoalbuminemia, hyperlipidemia and edema. Several diseases cause a nephrotic syndrome, as they damage the glomerular podocytes. These specialized epithelial cells, together with endothelial cells of the glomerular capillaries and the basal membrane, form a filter that retains plasma proteins in the circulation. A disturbance of this filter causes proteinuria. The three most common primary glomerular diseases are minimal change, membranous glomerulonephritis, and the primary focal segmental glomerulosclerosis. The familiar forms are rare; however, the identification of the relevant gene defects has greatly advanced our understanding of podocyte function as well as the pathogenesis of nephrotic syndrome.

Topics: Gene Expression Regulation; Genetic Predisposition to Disease; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Nephrotic Syndrome; Proteins

Topics: Humans; Membrane Proteins; Mutation; Nephrotic Syndrome; Proteins

Nephrin is identified as a product of the gene mutated in a patient with congenital nephrotic syndrome of the Finnish type. To analyse the function of nephrin and the relationship of nephrin and the target antigen of nephritogenic anti-rat slit diaphragm monoclonal antibody (mAb) 5-1-6, the rat homologue of nephrin was cloned. The deduced amino acid sequence of rat nephrin shows 82.2% homology to human nephrin. Signal sequences of rat nephrin are 36 amino acids, whereas those of human nephrin are reported to be 22 amino acids. The localization of rat nephrin always coincided with that of mAb 5-1-6 antigen. The specific anti-nephrin antibody recognized the mAb 5-1-6-antigen complex. mAb 5-1-6 reacted with COS cells transfected with rat nephrin cDNA. These results demonstrate that nephritogenic mAb 5-1-6 identifies the extracellular domain of rat nephrin, thereby documenting that nephrin is a functional protein of the slit diaphragm. The staining pattern of nephrin shifted from a linear-like pattern to a discontinuous coarse granular pattern not only in mAb 5-1-6-induced nephropathy, but also in puromycin aminonucleoside nephropathy, adriamycin nephropathy and passive Heymann nephropathy. Rat nephrin is detected first on the basal and lateral side below the junctional complex at the S-shaped body stage. With the interdigitation of foot processes, nephrin becomes concentrated in the slit pore, and finally restricted in the site of the slit diaphragm bridging two adjacent foot processes.

Topics: Animals; Capillary Permeability; Embryo, Mammalian; Embryonic and Fetal Development; Kidney Glomerulus; Membrane Proteins; Nephrotic Syndrome; Proteins; Rats

Many acquired and familial renal diseases in man lead to kidney dysfunction and nephrotic syndrome. These diseases share a common pathological fate in the form of glomerular dysfunction and proteinuria. Classification of the disease is difficult because the onset of pathological appearance in congenital nephrotic syndrome (CNS) varies considerably. Recently, classification has been aided by applying molecular genetics to identify genes involved in the pathogenesis of proteinuria. Light has also been shed on the biology and mechanisms of glomerular filtration and the molecular pathogenesis of CNS.

Topics: Animals; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nephrotic Syndrome; Proteins

Recent discoveries in podocyte proteins involved in the renal filtration barrier have shed new light on the ultrastructure of the kidney filter and pathogenesis of proteinuria. The identification of nephrin, a component of the slit diaphragm, and the intracellular slit diaphragm associated proteins CD2AP and podocin has demonstrated the existence of proteins that directly contribute to a functional kidney filter. Mutations in the genes for these three proteins result in proteinuria and nephrotic syndrome, and these proteins are also likely to be involved more generally in the pathomechanisms of proteinuria. This new knowledge has been promoted particularly through the powerful methods of molecular genetics and molecular biology. In this minireview, we present the recent progress in research of the podocyte slit diaphragm.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cadherins; Cytoskeletal Proteins; Humans; Kidney Glomerulus; Membrane Proteins; Nephrotic Syndrome; Phosphoproteins; Proteins; Zonula Occludens-1 Protein

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

The molecular nature of the glomerular slit diaphragm, the site of renal ultrafiltration, has until recently remained a mystery. However, the identification of the gene affected in congenital nephrotic syndrome has revealed the presence of a novel protein, possibly specific for the slit diaphragm. This protein, which has been termed nephrin, is a transmembrane protein that probably forms the main building block of an isoporous zipper-like slit diaphragm filter structure. Defects in nephrin lead to abnormal or absent slit diaphragm leading to massive proteinuria and renal failure. The discovery of nephrin sheds new light on the glomerular filtration barrier, provides new insight into the pathomechanisms of proteinuria, and even opens up possibilities for the development of novel therapies for this common and severe kidney complication.

Topics: Animals; Gene Expression; Humans; In Situ Hybridization; Kidney; Kidney Glomerulus; Membrane Proteins; Microscopy, Immunoelectron; Nephrotic Syndrome; Proteins

Topics: Animals; Humans; Kidney Glomerulus; Membrane Proteins; Nephrotic Syndrome; Proteins; Ultrafiltration

Congenital nephrotic syndrome (CNS) is a heterogeneous disorder in which massive proteinuria, hypoproteinemia, and hyperlipidemia and marked edema are the main manifestations before 3 months-of-age. Here, we present a case involving the genetic diagnosis of a child with CNS.. A 31-day-old male infant with diarrhea for 25 days and generalized edema for more than 10 days. There was no family history of kidney disease. On proband whole exome sequencing, a compound heterozygous mutation of the NPHS1 gene was identified, including a novel in-frame mutation in exon 14 (c.1864_1866dupACC p. T622dup) and a missense mutation in exon 8 (c.928G>A p. D310N).. Based on the clinical and genetic findings, this patient was finally diagnosed with CNS.. The main treatment options for the patient were 2-fold: anti-infective treatment and symptomatic treatment.. The patient died in follow-up 2 months later; the specific reason for death was unclear.. Whole exome sequencing and Sanger sequencing confirmed that the infant had CNS. Our study identified a novel mutation in an infant, thus expanding the gene-mutation spectrum of the NPHS1 gene, thus providing an efficient prenatal screening strategy and early genetic counseling.

Topics: East Asian People; Humans; Infant; Male; Membrane Proteins; Mutation; Nephrotic Syndrome

Sphingosine 1-phosphate (S1P) lyase (SPL,

Topics: Animals; Humans; Interleukin-6; Mice; Nephrotic Syndrome; Podocytes; Protein Kinase C-delta; RNA, Messenger

Congenital nephrotic syndrome (CNS) of the Finnish type (CNF) is an autosomal recessively disorder. NPHS1 gene mutation is the main gene responsible for CNF. This study aimed to explore the clinical manifestations and the characteristics of genetic variation in Chinese patients with CNS.. A 15-minute-old boy and a 34-day-old girl with CNS were included. NPHS1 gene was detected by next-generation high-throughput sequencing.. Patient 1 carried two novel heterozygous mutations of NPHS1 gene, one was c.204delG, p. (Leu69fs) in exon 2 of NPHS1 gene, a heterozygote frameshift mutation; the other was c.3558delT, p. (Gly1187fs) in exon 28, a heterozygote frameshift mutation. Patient 2 carried three heterozygous mutations of NPHS1, among them, c.1561-G>A. p.Asp521Asn in exon 12 is a heterozygous missense mutation. It was identified as possible de novo pathogenicity gene.. Three novel heterozygous mutations of NPHS1 gene were responsible for the patients with CNS and can enlarge the spectrum of NPHS1 gene mutation.

Topics: East Asian People; Female; Heterozygote; Humans; Infant; Infant, Newborn; Male; Membrane Proteins; Mutation; Nephrotic Syndrome

NPHS2 is the causative gene of nephrotic syndrome type 2 (MIM 600995) which often clinically manifests as steroid-resistant nephrotic syndrome (SRNS). The NPHS2 gene encodes a slit diaphragm (SD) associated protein podocin.. This study reported a novel disease-causing mutation of NPHS2 in a Chinese family with SRNS. We also investigated the pathogenic mechanism of the variants in this family.. A Chinese family with SRNS was recruited. Whole exome sequencing was performed to screen for disease-causing mutation. Sanger sequencing was used to confirm the results. In vitro functional experiments including immunoblotting, co-immunoprecipitation and double immunofluorescence staining were performed to explore the pathogenic mechanisms of mutations.. In this family, compound heterozygous mutations of NPHS2 (c.467dupT and c.865A > G) were identified and segregated with the disease. The maternal c.865A > G was a novel variant, leading to amino acid substitution (p.K289E). In vitro functional assays indicated that c.467dupT (p.L156FfsX11) mutant lost interaction with nephrin. Both K289E and L156FfsX11 mutants showed sharply diminished plasma membrane localization. Furthermore, abnormal distribution of podocin mutants also altered the cell membrane localization of nephrin.. We reported a family with SRNS caused by compound heterozygous mutations of NPHS2 (c.467dupT and c.865A > G). c.865A > G (p.K289E) in NPHS2 was a novel causative variant associated with SRNS. Both variants in this family not only affected the normal cell membrane localization of podocin, but also altered the cell membrane localization of nephrin which is the major architectural protein of SD.

Topics: China; DNA Mutational Analysis; Female; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Steroids

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

Podocytes are key to the glomerular filtration barrier by forming a slit diaphragm between interdigitating foot processes; however, the molecular details and functional importance of protein folding and degradation in the ER remain unknown. Here, we show that the SEL1L-HRD1 protein complex of ER-associated degradation (ERAD) is required for slit diaphragm formation and glomerular filtration function. SEL1L-HRD1 ERAD is highly expressed in podocytes of both mouse and human kidneys. Mice with podocyte-specific Sel1L deficiency develop podocytopathy and severe congenital nephrotic syndrome with an impaired slit diaphragm shortly after weaning and die prematurely, with a median lifespan of approximately 3 months. We show mechanistically that nephrin, a type 1 membrane protein causally linked to congenital nephrotic syndrome, is an endogenous ERAD substrate. ERAD deficiency attenuated the maturation of nascent nephrin, leading to its retention in the ER. We also show that various autosomal-recessive nephrin disease mutants were highly unstable and broken down by SEL1L-HRD1 ERAD, which attenuated the pathogenicity of the mutants toward the WT allele. This study uncovers a critical role of SEL1L-HRD1 ERAD in glomerular filtration barrier function and provides insights into the pathogenesis associated with autosomal-recessive disease mutants.

Topics: Animals; Endoplasmic Reticulum-Associated Degradation; Glomerular Filtration Rate; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Transgenic; Nephrotic Syndrome; Podocytes; Proteins; Ubiquitin-Protein Ligases

Mutations in the NPHS1 gene, which encodes NEPHRIN, cause congenital nephrotic syndrome, resulting from impaired slit diaphragm (SD) formation in glomerular podocytes. We previously reported NEPHRIN and SD abnormalities in the podocytes of kidney organoids generated from patient-derived induced pluripotent stem cells (iPSCs) with an NPHS1 missense mutation (E725D). However, the mechanisms underlying the disease may vary depending on the mutations involved, and thus generation of iPSCs from multiple patients is warranted. Here we established iPSCs from two additional patients with different NPHS1 mutations and examined the podocyte abnormalities in kidney organoids derived from these cells. One patient had truncating mutations, and NEPHRIN was undetectable in the resulting organoids. The other patient had a missense mutation (R460Q), and the mutant NEPHRIN in the organoids failed to accumulate on the podocyte surface to form SD precursors. However, the same mutant protein behaved normally when overexpressed in heterologous cells, suggesting that NEPHRIN localization is cell context-dependent. The localization of another SD-associated protein, PODOCIN, was impaired in both types of mutant organoids in a cell domain-specific manner. Thus, the new iPSC lines and resultant kidney organoids will be useful resources for dissecting the disease mechanisms, as well as for drug development for therapies.

Topics: Amino Acid Sequence; HEK293 Cells; Humans; Induced Pluripotent Stem Cells; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Mutant Proteins; Mutation, Missense; Nephrotic Syndrome; Organoids

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

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

Salvianolic acid A (SAA) is extracted from traditional Chinese medicine Salvia miltiorrhiza and is the main water-soluble and the biologically active ingredient. SAA possesses a variety of pharmacological activities and has an excellent protective effect on kidney disease, especially steroid resistant nephrotic syndrome (SRNS), and has advantages in improving the efficacy of glucocorticoids, but its mechanism needs to be further explored.. The study was designed to explore the effect of suPAR and uPAR in SRNS patients and evaluate the potential effect of SAA in improving podocyte steroid resistance and explore its mechanism.. The ELISA kits were used to detect the levels of suPAR in the blood and urine of subjects. The levels of uPAR, GRα, and GRβ expression in renal tissues of SRNS patients was detected by immunohistochemistry and analyzed using the Pearson method. In vitro studies, steroid resistance model was induced by the TNF-α and IFN-γ. The protein and mRNA expression of Nephrin, GR, GRα and GRβ were analyzed using western blot and qRT-PCR. The activity of GR-DNA binding was detected by using TransAM™ GR kits. Adriamycin further induced steroid resistance podocyte. Flow cytometry was used to detect the effect of SAA on podocyte apoptosis. ELISA assay was used to detect the suPAR expression in the podocyte supernatant. Western blot and qRT-PCR were used to detect the protein and mRNA expression of uPAR and Nephrin in podocytes.. The serum and urine levels of suPAR were conspicuously higher in SRNS patients than healthy volunteers and SSNS patients, and the expression of uPAR in renal tissue of SRNS patients is negatively correlated with GRα, but positively correlated with GRβ. The combination of TNF-α and IFN-γ could conspicuously increase the GRβ expression and reduce GRα/GRβ, and induce steroid resistance in podocytes. Moreover, we found that SAA could reduce the apoptosis of podocytes and suppress the expression of suPAR/uPAR, and increase the expression of Nephrin.. The level of suPAR and uPAR expression may have important value in predicting glucocorticoids resistance in patients with idiopathic nephrotic syndrome (INS). The combination of TNF-α and IFN-γ induce podocytes can establish steroid resistance model in vitro. SAA could improve glucocorticoids resistance of podocyte which can be attributed in part to regulate the suPAR/uPAR-αvβ3 signaling pathway.

Topics: Adult; Caffeic Acids; Case-Control Studies; Female; Glucocorticoids; Humans; Lactates; Male; Membrane Proteins; Middle Aged; Nephrotic Syndrome; Podocytes; Receptors, Glucocorticoid; Receptors, Urokinase Plasminogen Activator; Salvia miltiorrhiza; Signal Transduction; Young Adult

Congenital nephrotic syndrome (CNS) is a rare autosomal recessive disorder that occurs in the first 0 to 3 months of life. The course of CNS is progressive, often leading to end-stage renal disease within 2 to 3 years. Most patients with CNS are resistant to glucocorticoids and immunosuppressive drugs. We report a girl aged 1 month and 20 days who was admitted to hospital with a history of abdominal distension and palpebral edema. She was diagnosed with CNS and administered a glucocorticoid (methylprednisolone) for 2 years. Targeted high-throughput next-generation sequencing showed mutations in the NPHS1 gene. She had a favorable outcome after 2 years of treatment. She has remained in complete remission for the last 6 months. From a clinical point of view, the outcome of CNS may be associated with end-stage renal disease or even death. Appropriate pharmacotherapy is beneficial to maintain a normal function and integrity of the glomerular barrier. An aggressive treatment plan is required to save the life of patients with CNS, even if a heterozygous mutation is detected by genetic analysis.

Topics: Female; Genetic Testing; Heterozygote; Humans; Infant; Membrane Proteins; Mutation; Nephrotic Syndrome

Ephrin-B1 plays a critical role at slit diaphragm. Partitioning-defective (Par)-6 is down-regulated in podocyte of ephrin-B1 knockout mouse, suggesting that Par-6 is associated with ephrin-B1. Par polarity complex, consisting of Par-6, Par-3, and atypical protein kinase C, is essential for tight junction formation. In this study, the expression of Par-6 was analyzed in the normal and nephrotic syndrome model rats, and the molecular association of Par-6, Par-3, ephrin-B1, and nephrin was assessed with the human embryonic kidney 293 cell expression system. Par-6 was concentrated at slit diaphragm. Par 6 interacted with ephrin-B1 but not with nephrin, and Par-3 interacted with nephrin but not with ephrin-B1. The complexes of Par-6-ephrin-B1 and Par-3-nephrin were linked via extracellular sites of ephrin-B1 and nephrin. The Par-6-ephrin-B1 complex was delinked from the Par-3-nephrin complex, and Par-6 and ephrin-B1 were clearly down-regulated already at early phase of nephrotic model. The alteration of Par-6/ephrin-B1 advanced that of Par-3/nephrin. Stimulation to nephrin phosphorylated not only nephrin but also ephrin-B1, and consequently inhibited the interaction between ephrin-B1 and Par-6. Par-6 appeared at presumptive podocyte of early developmental stage and moved to basal area at capillary loop stage to participate in slit diaphragm formation at the final stage. Par-6-ephrin-B1 interaction is crucial for formation and maintenance of slit diaphragm of podocyte.

Topics: Adaptor Proteins, Signal Transducing; Animals; Animals, Newborn; Carrier Proteins; Diaphragm; Ephrin-B1; HEK293 Cells; Humans; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Knockout; Nephrotic Syndrome; Phosphorylation; Podocytes; Rats; Rats, Wistar

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

Connexins (Cxs) are membrane-spanning proteins which enable flow of information important for kidney homeostasis. Changes in their spatiotemporal patterning characterize blood vessel abnormalities and chronic kidney diseases (CKD). We analysed spatiotemporal expression of Cx37, Cx40, Cx43 and Cx45 in nephron and glomerular cells of developing, postnatal kidneys, and nephrotic syndrome of the Finnish type (CNF) by using immunohistochemistry, statistical methods and electron microscopy. During kidney development, strong Cx45 expression in proximal tubules and decreasing expression in glomeruli was observed. In developing distal nephron, Cx37 and Cx40 showed moderate-to-strong expression, while weak Cx43 expression gradually increased. Cx45/Cx40 co-localized in mesangial and granular cells. Cx43 /Cx45 co-localized in podocytes, mesangial and parietal epithelial cells, and with podocyte markers (synaptopodin, nephrin). Different Cxs co-expressed with endothelial (CD31) and VSMC (α -SMA) markers in vascular walls. Peak signalling of Cx37, Cx43 and Cx40 accompanied kidney nephrogenesis, while strongest Cx45 signalling paralleled nephron maturation. Spatiotemporal Cxs patterning indicate participation of Cx45 in differentiation of proximal tubules, and Cx43, Cx37 and Cx40 in distal tubules differentiation. CNF characterized disorganized Cx45 expression in proximal tubules, increased Cx43 expression in distal tubules and overall elevation of Cx40 and Cx37, while Cx40 co-localized with increased number of interstitial myofibroblasts.

Topics: Actins; Connexins; Gap Junctions; Gene Expression Regulation, Developmental; Gestational Age; Humans; Infant; Kidney; Male; Membrane Proteins; Mesenchymal Stem Cells; Microfilament Proteins; Mutation, Missense; Nephrotic Syndrome; Organ Specificity; Platelet Endothelial Cell Adhesion Molecule-1

To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (P

Topics: Alleles; Child; Genome-Wide Association Study; Haplotypes; Humans; Membrane Proteins; Mutation; Nephrotic Syndrome; Steroids; Tumor Necrosis Factor Ligand Superfamily Member 15

Podocyte injury has recently been described as unifying feature in idiopathic nephrotic syndromes (INS). Puumala hantavirus (PUUV) infection represents a unique RNA virus-induced renal disease with significant proteinuria. The underlying pathomechanism is unclear. We hypothesized that PUUV infection results in podocyte injury, similar to findings in INS. We therefore analyzed standard markers of glomerular proteinuria (e.g. immunoglobulin G [IgG]), urinary nephrin excretion (podocyte injury) and serum levels of the soluble urokinase plasminogen activator receptor (suPAR), a proposed pathomechanically involved molecule in INS, in PUUV-infected patients. Hantavirus patients showed significantly increased urinary nephrin, IgG and serum suPAR concentrations compared to healthy controls. Nephrin and IgG levels were significantly higher in patients with severe proteinuria than with mild proteinuria, and nephrin correlated strongly with biomarkers of glomerular proteinuria over time. Congruently, electron microcopy analyses showed a focal podocyte foot process effacement. suPAR correlated significantly with urinary nephrin, IgG and albumin levels, suggesting suPAR as a pathophysiological mediator in podocyte dysfunction. In contrast to INS, proteinuria recovered autonomously in hantavirus patients. This study reveals podocyte injury as main cause of proteinuria in hantavirus patients. A better understanding of the regenerative nature of hantavirus-induced glomerulopathy may generate new therapeutic approaches for INS.

Topics: Adolescent; Adult; Female; Glomerular Filtration Barrier; Hemorrhagic Fever with Renal Syndrome; Humans; Kidney Glomerulus; Male; Membrane Proteins; Middle Aged; Nephrotic Syndrome; Podocytes; Puumala virus; Receptors, Urokinase Plasminogen Activator; Young Adult

To detect genetic variant in a sib-pair with Finnish type congenital nephrotic syndrome (CNF).. Clinical data of the sib-pair was reviewed. Coding regions of the NPHS1 gene was analyzed for the sib-pair and both parents.. The sister and brother respectively developed severe proteinuria 1 month and 28 days after birth, in addition with low serum albumin, hypercholesterolemia and severe edema, which were suggestive of CNF. Genetic testing identified that the sib-pair has both carried two heterozygous variants of NPHS1 gene, namely c.2605G>C (p.P869>A) and c.-61G>A, for which their father and mother were heterozygous carriers.. The c.2605G>C (p.869P>A) and c.-61G>A variants of the NHPS1 gene probably underlay the CNF in both sibs. The c.2605G>C(p.869P>A) was unreported previously.

Topics: Adult; Female; Humans; Infant, Newborn; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Siblings

Steroid-resistant nephrotic syndrome is a frequent cause of chronic kidney disease almost inevitably progressing to end-stage renal disease. More than 58 monogenic causes of SRNS have been discovered and majority of known steroid-resistant nephrotic syndrome causing genes are predominantly expressed in glomerular podocytes, placing them at the center of disease pathogenesis. Herein, we describe two unrelated families with steroid-resistant nephrotic syndrome with homozygous mutations in the KIRREL1 gene. One mutation showed high frequency in the European population (minor allele frequency 0.0011) and this patient achieved complete remission following treatment, but later progressed to chronic kidney disease. We found that mutant KIRREL1 proteins failed to localize to the podocyte cell membrane, indicating defective trafficking and impaired podocytes function. Thus, the KIRREL1 gene product has an important role in modulating the integrity of the slit diaphragm and maintaining glomerular filtration function.

Topics: Adolescent; Age of Onset; Cell Line; Child; Child, Preschool; Consanguinity; Disease Progression; DNA Mutational Analysis; Drug Resistance; Exome Sequencing; Female; Follow-Up Studies; Gene Frequency; Glomerular Basement Membrane; Glucocorticoids; Homozygote; Humans; Male; Membrane Proteins; Microscopy, Electron, Transmission; Mutation; Nephrotic Syndrome; Pedigree; Podocytes; Renal Insufficiency, Chronic

To explore the genetic basis for a fetus suspected for congenital nephrotic syndrome of Finland (CNF).. Genomic DNA was extracted from peripheral and umbilical cord blood samples derived from both parents and the fetus. Potential variants were detected by using next-generation sequencing. Suspected variants were confirmed by Sanger sequencing.. The fetus was found to carry compound heterozygous variants c.1440+1G>A and c.925G>T of the NPHS1 gene, which were respectively inherited from its mother and father.. Identification of the compound heterozygous NPHS1 variants has enabled diagnosis of CNF in the fetus and genetic counseling for the affected family.

Topics: Female; Fetus; Finland; Heterozygote; Humans; Membrane Proteins; Nephrotic Syndrome; Pregnancy; Prenatal Diagnosis

Mutations in about 50 genes have been identified as monogenic causes of nephrotic syndrome, a frequent cause of CKD. These genes delineated the pathogenetic pathways and rendered significant insight into podocyte biology.. We used whole-exome sequencing to identify novel monogenic causes of steroid-resistant nephrotic syndrome (SRNS). We analyzed the functional significance of an SRNS-associated gene. We identified hemizygous missense mutations in the gene. Novel mutations in

Topics: Animals; Autophagy; Calcium-Binding Proteins; Cell Line, Transformed; Dogs; Drosophila melanogaster; Drosophila Proteins; Exocytosis; Exome Sequencing; Gene Silencing; HEK293 Cells; Humans; Immunoglobulins; Madin Darby Canine Kidney Cells; Membrane Proteins; Mutation, Missense; Nephrotic Syndrome; Phenotype; Podocytes; Protein Interaction Mapping; rab GTP-Binding Proteins; RNA Interference; RNA, Small Interfering; Transport Vesicles; Vesicular Transport Proteins

Recommendations for management of Finnish-type congenital nephrotic syndrome (CNS) followed by many teams include daily albumin infusions, early bilateral nephrectomy, dialysis and transplantation. We aimed to assess the treatment and outcome of patients with CNS in France.. We conducted a nationwide retrospective study on 55 consecutive children born between 2000 and 2014 treated for non-infectious CNS.. The estimated cumulative incidence of CNS was 0.5/100 000 live births. The underlying defect was biallelic mutations in NPHS1 (36/55, 65%), NPHS2 (5/55, 7%), PLCE1 (1/55, 2%), heterozygous mutation in WT1 (4/55, 7%) and not identified in nine children (16%). Fifty-three patients (96%) received daily albumin infusions from diagnosis (median age 14 days), which were spaced and withdrawn in 10 patients. Twenty children (35%) were managed as outpatients. Thirty-nine patients reached end-stage kidney disease (ESKD) at a median age of 11 months. The overall renal survival was 64% and 45% at 1 and 2 years of age, respectively. Thirteen children died during the study period including four at diagnosis, two of nosocomial catheter-related septic shock, six on dialysis and one after transplantation. The remaining 13 patients were alive with normal renal function at last follow-up [median 32 months (range 9-52)]. Renal and patient survivals were longer in patients with NPHS1 mutations than in other patients. The invasive infection rate was 2.41/patient/year.. Our study shows: (i) a survival free from ESKD in two-thirds of patients at 1 year and in one-half at 2 years and (ii) a significant reduction or even a discontinuation of albumin infusions allowing ambulatory care in a subset of patients. These results highlight the need for new therapeutic guidelines for CNS patients.

Topics: Disease Progression; Female; France; Humans; Incidence; Infant; Infant, Newborn; Male; Membrane Proteins; Mutation; Nephrectomy; Nephrotic Syndrome; Retrospective Studies; Survival Rate; Treatment Outcome

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

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

Topics: Adolescent; Biopsy; Cardiomyopathy, Dilated; Child; Child, Preschool; Deafness; Genetic Testing; Humans; Infant; Infant, Newborn; Kidney; Kidney Transplantation; Longitudinal Studies; Male; Membrane Proteins; Mitochondrial Diseases; Nephrectomy; Nephrotic Syndrome; Renal Insufficiency, Chronic; Young Adult

Topics: Adolescent; Biopsy; Cardiomyopathy, Dilated; Child; Child, Preschool; Deafness; Genetic Testing; High-Throughput Nucleotide Sequencing; Humans; Infant; Infant, Newborn; Introns; Kidney; Longitudinal Studies; Male; Membrane Proteins; Mitochondrial Diseases; Mutation; Nephrotic Syndrome; Renal Insufficiency, Chronic; Young Adult

Neonatal-onset Denys-Drash syndrome (NODDS) is a distinctive clinical entity and has a poor renal and life outcome. Early diagnosis of NODDS is important for managing disorders of sexual development and determining assigned gender. Although patients with NODDS and congenital nephrotic syndrome of the Finnish type (CNF) present with nephrotic syndrome in neonatal life or infancy, the clinical course of NODDS and factors distinguishing these diseases at onset is unknown.. We performed a retrospective cohort study of patients with NODDS and CNF between 1997 and 2017. Patients with nephrotic syndrome and WT1 or NPHS1 mutations with neonatal onset (within 30 days) were eligible.. We studied eight patients with NODDS and 15 with CNF. The median serum creatinine level at onset in the NODDS group was significantly higher (1.85 mg/dL) than that in the CNF group (0.15 mg/dL; P = 0.002). The median placental/fetal weight ratio in the NODDS and CNF group was 41.8% and 21.0%, respectively (P = 0.001). Kaplan-Meier analysis showed that the median number of days for progression to ESRD from onset in the NODDS and CNF groups was 6 and 910 days, respectively (P < 0.001). All patients in the NODDS group were alive at follow-up. Only one patient in the CNF group died of cardiac complications during follow-up.. CNS, renal dysfunction at onset, and a relatively large placenta are prominent signs of NODDS. Prognosis for patients with NODDS is satisfactory if appropriate and active management is performed.

Topics: Adolescent; Age of Onset; Child; Child, Preschool; Denys-Drash Syndrome; Disease Progression; Genetic Predisposition to Disease; Humans; Infant; Infant, Newborn; Kidney Failure, Chronic; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype; Renal Dialysis; Retrospective Studies; Risk Factors; Tokyo; WT1 Proteins

Topics: Exons; Female; Genotype; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Pedigree; Phenotype; Phosphoinositide Phospholipase C; Sequence Analysis, DNA; Vietnam; WT1 Proteins

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

In the neonatal period, pulmonary thromboembolic episodes are rare and unknown events. Case of a preterm newborn who presented a bilateral thrombosis of the pulmonary arteries associated with a congenital nephrotic syndrome. Pediatricians should consider pulmonary thromboembolic episodes in cases of newborns with unexplained severe and hypoxic respiratory distress syndrome.

Topics: Anticoagulants; Blood Transfusion; Fatal Outcome; Female; Humans; Infant, Newborn; Membrane Proteins; Nephrotic Syndrome; Plasma; Pulmonary Artery; Thrombosis

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

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

Genetic mutations in dozens of monogenic genes can lead to serious podocyte dysfunction, which is a major cause of steroid-resistant nephrotic syndrome (SRNS). The NUP160 gene is expressed in both human kidney and mouse kidney. However, whether knockdown of NUP160 impairs podocytes has not yet been established. Therefore, we knocked down NUP160 by targeted short hairpin RNA (shRNA) in conditionally immortalized mouse podocytes and observed the effect of NUP160 knockdown on the proliferation, apoptosis, autophagy and cell migration of podocytes. We also investigated the effect of NUP160 knockdown on the expression and localization of podocyte associated molecules, such as nephrin, podocin, CD2AP and α-actinin-4. The knockdown of NUP160 significantly inhibited the proliferation of podocytes by decreasing the expression of both cyclin D1 and CDK4, increasing the expression of p27, and inducing S phase arrest. The knockdown of NUP160 promoted the apoptosis and autophagy of podocytes, and enhanced cell migration. The knockdown of NUP160 decreased the expression of nephrin, podocin and CD2AP in podocytes, and increased the expression of α-actinin-4. The knockdown of NUP160 also altered the subcellular localization of nephrin, podocin and CD2AP in podocytes. These results suggest that the knockdown of NUP160 impairs mouse podocytes, i.e. inhibiting cell proliferation, inducing apoptosis, autophagy and cell migration of mouse podocytes, and altering the expression and localization of podocyte associated molecules, including nephrin, podocin, CD2AP and α-actinin-4.

Topics: Actinin; Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Autophagy; Cell Line; Cell Movement; Cell Proliferation; Cytoskeletal Proteins; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Nephrotic Syndrome; Nuclear Pore Complex Proteins; Podocytes; RNA Interference; RNA, Small Interfering; S Phase Cell Cycle Checkpoints

Steroid-resistant nephrotic syndrome (SRNS) has a heterogeneous spectrum of monogenic causes that substantially differ among populations. The aim of this study was to analyse the genetic aetiology of SRNS in Czech and Slovak paediatric patients.. We analysed clinical data from 74 patients (38 boys) with congenital (15%), infant (14%), and childhood-onset (71%) SRNS collected from the Czech Republic and Slovakia from 2000 to 2017 (inclusive). The DNA samples were first analysed by Sanger sequencing (genes NPHS2, NPHS1, and WT1) and then by next generation sequencing (NGS) using a targeted panel of 48 genes previously associated with SRNS. Family segregation of the causative variants was confirmed by Sanger sequencing when possible.. Genetic diagnosis was established in 28/74 patients (38%) based on findings of pathogenic or likely pathogenic causative variants in genotypes conforming to the expected mode of inheritance. Sanger sequencing diagnosed 26% of patients, whereas second-tier testing by a targeted NGS panel diagnosed a further 12%. Frequent causative genes were NPHS2 (15%), WT1 (9.5%), and surprisingly NUP93 with four (5.4%) unrelated cases. Additional causative genes included COQ2 (two patients), NPHS1, INF2, DGKE, and LMX1B (one patient each).. Compared with outright use of NGS, our tiered genetic testing strategy was considerably more rapid and marginally less expensive. Apart from a high aetiological fraction of NPHS2 and WT1 genes, our study has identified an unexpectedly high frequency of a limited set of presumably ancestral causative mutations in NUP93. The results may aid in tailoring testing strategies in Central European populations.

Topics: Adolescent; Child; Child, Preschool; Czech Republic; DNA Mutational Analysis; Female; Genetic Predisposition to Disease; Genetic Testing; Genotype; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Longitudinal Studies; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Nuclear Pore Complex Proteins; Prospective Studies; Slovakia; WT1 Proteins

The slit diaphragm is a specialized adhesion junction between opposing podocytes, establishing the final filtration barrier that prevents passage of proteins from the capillary lumen into the urinary space. Nephrin, the key structural and signaling adhesion molecule expressed in the slit diaphragm, contains an evolutionally conserved, atypical PDZ-binding motif (PBM) reported to bind to a variety of proteins in the slit diaphragm. Several mutations in. Using a combination of biochemic, biophysic, and cell biologic approaches, we systematically investigated the interactions between nephrin-PBM and PDZ domain-containing proteins in the slit diaphragm.. We found that nephrin-PBM specifically binds to one member of the membrane-associated guanylate kinase family of scaffolding proteins, MAGI1, but not to another, MAGI2. The complex structure of MAGI1-PDZ3/nephrin-PBM reveals that the Gly at the -3 position of nephrin-PBM is the determining feature for MAGI1-PDZ3 recognition, which sharply contrasts with the typical PDZ/PBM binding mode. A single gain-of-function mutation within MAGI2 enabled nephrin-PBM binding. In addition, using our structural analysis, we developed a highly efficient inhibitory peptide capable of specifically blocking the nephrin/MAGI1 interaction.. MAGI1 interacts with nephrin-PBM with exquisite specificity. A newly developed, potent inhibitory peptide that blocks this interaction may be useful for future functional investigations

Topics: Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins; Cell Adhesion Molecules; Cell Adhesion Molecules, Neuronal; Cell Communication; Cell Cycle Proteins; Cells, Cultured; Cytoskeletal Proteins; Guanylate Kinases; Humans; Kidney Glomerulus; Membrane Proteins; Nephrotic Syndrome; Podocytes; Qualitative Research; Sensitivity and Specificity; Signal Transduction

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

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

Mutations in the NPHS1 gene, which encodes NEPHRIN, cause congenital nephrotic syndrome, resulting from impaired slit diaphragm (SD) formation in glomerular podocytes. However, methods for SD reconstitution have been unavailable, thereby limiting studies in the field. In the present study, we established human induced pluripotent stem cells (iPSCs) from a patient with an NPHS1 missense mutation, and reproduced the SD formation process using iPSC-derived kidney organoids. The mutant NEPHRIN failed to become localized on the cell surface for pre-SD domain formation in the induced podocytes. Upon transplantation, the mutant podocytes developed foot processes, but exhibited impaired SD formation. Genetic correction of the single amino acid mutation restored NEPHRIN localization and phosphorylation, colocalization of other SD-associated proteins, and SD formation. Thus, these kidney organoids from patient-derived iPSCs identified SD abnormalities in the podocytes at the initial phase of congenital nephrotic disease.

Topics: Animals; Cells, Cultured; HEK293 Cells; Humans; Induced Pluripotent Stem Cells; Kidney; Membrane Proteins; Mice, SCID; Mutation, Missense; Nephrotic Syndrome; Organoids; Podocytes

Podocytes are postmitotic, highly specialized cells which maintain the glomerular filtration barrier (GFB). Their injury is characterized by foot processes effacement and change in protein expression leading to proteinuria and end-stage kidney disease.. Our study focuses on the morphological and immunohistochemical changes of human podocytes during normal development and postnatal period, compared to congenital nephrotic syndrome of the Finnish type (CNF). Kidney tissues taken from 17 human conceptuses 8th-38th weeks old, two healthy and three CNF kidneys were embedded in paraffin for immunohistochemical or double immunofluorescence methods, or were embedded in resin for electron microscopy. Paraffin sections were stained with markers for proliferation (Ki-67), proteins nephrin and nestin, and alpha-tubulin. Quantification of positive cells were performed using Mann Whitney and Kruskal-Wallis test.. Tissue analysis showed that proliferation of podocytes gradually decreased during development and disappeared in postnatal period. Decrease in number of ciliated glomerular cells and visceral podocytes (from 47% to 3%), and parietal epithelial cells (from 32% to 7%) characterized normal development. Nestin and nephrin co-expressed in developing podocytes in different cellular compartments. During development, nephrin expression increased (from 17% to 75%) and postnatally changed its pattern, while nestin positive glomerular cells decreased from 98% to 40%. CNF glomeruli displayed increased number of immature ciliated podocytes (6%) and parietal epithelial cells (9%).. Changes in cytoplasmic alpha-tubulin expression and reduced nephrin expression (20%) indicating association of incomplete podocyte maturation with failure of GFB function and appearance of prenatal proteinuria in CNF patients.

Topics: Cell Differentiation; Cell Proliferation; Cilia; Humans; Immunohistochemistry; Kidney Glomerulus; Membrane Proteins; Mutation; Nephrotic Syndrome; Podocytes; Reference Standards; Tissue Embedding

Mutations in the transmembrane protein nephrin (encoded by NPHS1) underlie nearly half of all cases of congenital nephrotic syndrome (CNS), which is caused by aberrations in the blood filtering function of glomerular podocytes. Nephrin directly contributes to the structure of the filtration barrier, and it also serves as a signaling scaffold in podocytes, undergoing tyrosine phosphorylation on its cytoplasmic tail to recruit intracellular effector proteins. Nephrin phosphorylation is lost in several human and experimental models of glomerular disease, and genetic studies have confirmed its importance in maintenance of the filtration barrier. To date, however, the effect of CNS-associated NPHS1 variants on nephrin phosphorylation remains to be determined, which hampers genotype-phenotype correlations. Here, we have characterized a novel nephrin sequence variant, A419T, which is expressed along with C623F in a patient presenting with CNS. Nephrin localization is altered in kidney biopsies, and we further demonstrate reduced surface expression and ER retention of A419T and C623F in cultured cells. Moreover, we show that both mutations impair nephrin tyrosine phosphorylation, and they exert dominant negative effects on wildtype nephrin signaling. Our findings thus reveal that missense mutations in the nephrin extracellular region can impact nephrin signaling, and they uncover a potential pathomechanism to explain the spectrum of clinical severity seen with mild NPHS1 mutations.

Topics: Adolescent; Amino Acid Substitution; Animals; Cell Membrane; Chlorocebus aethiops; COS Cells; Female; HEK293 Cells; Humans; Kidney; Luminescent Proteins; Male; Membrane Proteins; Microscopy, Confocal; Mutant Proteins; Mutation, Missense; Nephrotic Syndrome; Pedigree; Phosphorylation; Podocytes; Recombinant Proteins; Signal Transduction

Steroid resistant nephrotic syndrome (SRNS) is a genetically heterogeneous disease with significant phenotypic variability. More than 53 podocyte-expressed genes are implicated in SRNS which complicates the routine use of genetic screening in the clinic. Next generation sequencing technology (NGS) allows rapid screening of multiple genes in large number of patients in a cost-effective manner.. We developed a targeted panel of 17 genes to determine relative frequency of mutations in south Indian ethnicity and feasibility of using the assay in a clinical setting. Twenty-five children with SRNS and 3 healthy individuals were screened.. In this study, novel variants including 1 pathogenic variant (2 patients) and 3 likely pathogenic variants (3 patients) were identified. In addition, 2 novel variants of unknown significance (VUS) in 2 patients (8% of total patients) were also identified.. The results show that genetic screening in SRNS using NGS is feasible in a clinical setting. However the panel needs to be screened in a larger cohort of children with SRNS in order to assess the utility of the customised targeted panel in Indian children with SRNS. Determining the prevalence of variants in Indian population and improvising the bioinformatics-based filtering strategy for a more accurate differentiation of pathogenic variants from those that are benign among the VUS will help in improving medical and genetic counselling in SRNS.

Topics: Adolescent; Case-Control Studies; Child; Child, Preschool; Drug Resistance; Female; Gene Expression; Genetic Testing; High-Throughput Nucleotide Sequencing; Humans; India; Infant; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype; Phosphoinositide Phospholipase C; Severity of Illness Index; Steroids; WT1 Proteins

The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.

Topics: Cell Culture Techniques; Cell Line; Cells, Cultured; Collagen; Drug Evaluation, Preclinical; Female; Gene Expression; Gene Expression Profiling; Humans; Immunohistochemistry; Induced Pluripotent Stem Cells; Insulin; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Glomerulus; Laminin; Membrane Proteins; Mutation; Nephrotic Syndrome; Organoids; Podocytes; Sequence Analysis; Sequence Analysis, RNA; Stem Cells

Congenital nephrotic syndrome (CNS) is characterised by increased proteinuria, hypoproteinemia, and edema beginning in the first 3 months of life. Recently, molecular genetic studies have identified several genes involved in the pathogenesis of CNS. A systematic investigation of the genes for CNS in China has never been performed; therefore, we conducted a mutational analysis in 12 children with CNS,with the children coming from 10 provinces and autonomous regions in China.. Twelve children with CNS were enrolled from 2009 to 2016. A mutational analysis was performed in six children by Sanger sequencing in eight genes (NPHS1, NPHS2, PLCE1, WT1, LAMB2, LMXIB, COQ6 and COQ2) before 2014, and whole-exome sequencing was used from 2014 to 2016 in another six children. Significant variants that were detected by next generation sequencing were confirmed by conventional Sanger sequencing in the patients' families.. Of the 12 children, eight patients had a compound heterozygous NPHS1 mutation, one patient had a de novo mutation in the WT1 gene, and another patient with extrarenal symptoms had a homozygous mutation in the COQ6 gene. No mutations were detected in genes NPHS2, PLCE1, LAMB2, LMXIB, and COQ2 in the 12 patients.. This study demonstrates that the majority of CNS cases (67%, 8/12 patients) are caused by genetic defects, and the NPHS1 mutation is the most common cause of CNS in Chinese patients. A mutational analysis of NPHS1 should be recommended in Chinese patients with CNS in all exons of NPHS1 and in the intron-exon boundaries.

Topics: Alkyl and Aryl Transferases; Asian People; China; DNA Mutational Analysis; Exome Sequencing; Female; Heterozygote; Homozygote; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Laminin; LIM-Homeodomain Proteins; Male; Membrane Proteins; Nephrotic Syndrome; Phosphoinositide Phospholipase C; Transcription Factors; Ubiquinone; WT1 Proteins

Approximately 20% of children with idiopathic nephrotic syndrome do not respond to steroid therapy. More than 30 genes have been identified as disease-causing genes for the steroid-resistant nephrotic syndrome (SRNS). Few reports were from the Chinese population. The coding regions of genes commonly associated with SRNS were analyzed to characterize the gene mutation spectrum in children with SRNS in central China. The first phase study involved 38 children with five genes (NPHS1, NPHS2, PLCE1, WT1, and TRPC6) by Sanger sequencing. The second phase study involved 33 children with 17 genes by next generation DNA sequencing (NGS. 22 new patients, and 11 patients from first phase study but without positive findings). Overall deleterious or putatively deleterious gene variants were identified in 19 patients (31.7%), including four NPHS1 variants among five patients and three PLCE1 variants among four other patients. Variants in COL4A3, COL4A4, or COL4A5 were found in six patients. Eight novel variants were identified, including two in NPHS1, two in PLCE1, one in NPHS2, LAMB2, COL4A3, and COL4A4, respectively. 55.6% of the children with variants failed to respond to immunosuppressive agent therapy, while the resistance rate in children without variants was 44.4%. Our results show that screening for deleterious variants in some common genes in children clinically suspected with SRNS might be helpful for disease diagnosis as well as prediction of treatment efficacy and prognosis.

Topics: Adolescent; Child; Child, Preschool; Collagen; Female; Humans; Infant; Laminin; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Phosphoinositide Phospholipase C; Polymorphism, Single Nucleotide

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an inhibitor of megakaryopoiesis and platelet function. Recently, PACAP deficiency was observed in children with nephrotic syndrome (NS), associated with increased platelet count and aggregability and increased risk of thrombosis. To further study PACAP deficiency in NS, we used transgenic Tg(cd41:EGFP) zebrafish with GFP-labeled thrombocytes. We generated two models for congenital NS, a morpholino injected model targeting nphs1 (nephrin), which is mutated in the Finnish-type congenital NS. The second model was induced by exposure to the nephrotoxic compound adriamycin. Nephrin RNA expression was quantified and zebrafish embryos were live-screened for proteinuria and pericardial edema as evidence of renal impairment. Protein levels of PACAP and its binding-protein ceruloplasmin were measured and GFP-labeled thrombocytes were quantified. We also evaluated the effects of PACAP morpholino injection and the rescue effects of PACAP-38 peptide in both congenital NS models. Nephrin downregulation and pericardial edema were observed in both nephrin morpholino injected and adriamycin exposed congenital NS models. However, PACAP deficiency was demonstrated only in the adriamycin exposed condition. Ceruloplasmin levels and the number of GFP-labeled thrombocytes remained unchanged in both models. PACAP morpholino injections worsened survival rates and the edema phenotype in both congenital NS models while injection with human PACAP-38 could only rescue the adriamycin exposed model. We hereby report, for the first time, PACAP deficiency in a NS zebrafish model as a consequence of adriamycin exposure. However, distinct from the human congenital NS, both zebrafish models retained normal levels of ceruloplasmin and thrombocytes. We further extend the renoprotective effects of the PACAP-38 peptide against adriamycin toxicity in zebrafish.

Topics: Animals; Blood Platelets; Ceruloplasmin; Doxorubicin; Membrane Proteins; Nephrotic Syndrome; Peptide Fragments; Pericardium; Pituitary Adenylate Cyclase-Activating Polypeptide; Zebrafish; Zebrafish Proteins

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

Many cellular structures directly imply specific biological functions. For example, normal slit diaphragm structures that extend from podocyte foot processes ensure the filtering function of renal glomeruli. These slits are covered by a number of surface proteins, such as nephrin, podocin, podocalyxin and CD2AP. Here we report a human patient presenting with congenital nephrotic syndrome, omphalocele and microcoria due to two loss-of-function mutations in PODXL, which encodes podocalyxin, inherited from each parent. This set of symptoms strikingly mimics previously reported mouse Podxl

Topics: Adaptor Proteins, Signal Transducing; Animals; Cytoskeletal Proteins; Disease Models, Animal; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Mice; Mutation; Nephrotic Syndrome; Podocytes; Sialoglycoproteins

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

Ten pages, selected from a total of 127, of the last manuscript of Master i Margarita, written by Bulgakov in the last four years of his life, have been analysed in order to harvest and identify any trace proteome left on the margin by the novelist, in the hope of finding biomarkers of his fatal nephrotic syndrome. To that aim, we prepared a special ethyl-vinyl acetate film as binder of ground AG 501 Bio-Rad mix-bed strong cation/strong anion exchange resins for adsorbing any protein left on the margins of the pages via saliva and/or sweat. After eluting, digesting and interrogating the peptides by LC-MS/MS, we could identify three proteins, periostin, N-acetyl-β-glucosaminidase and nephrin, reported as biomarkers of renal pathologies. Additionally a further 29 unique gene products, of saliva and skin origin, have been identified, together with two bacterial proteins. The novel method here reported could be safely applied to any other research on manuscripts stored in public libraries and repositories of the World Cultural Heritage.. The present manuscript aims at finding proteomics traces in a 75-year old manuscript in order to confirm the health state of the author. In the case of Bulgakov it was known that he died of renal disease, possibly leaving traces and/or biomarkers of this pathology on the margins of the pages analysed. Three proteins, stated to be biomarkers of nephrotic syndrome, could be identified. In order not to contaminate the manuscript pages with resin particles, we have devised a novel harvesting film, by which strong cation and anion exchangers are embedded in ethyl-vinyl acetate foils. It is felt that this technology could be safely applied to other specimens belonging to the Word Heritage.

Topics: Biomarkers; Cell Adhesion Molecules; Hexosaminidases; Humans; Manuscripts as Topic; Membrane Proteins; Nephrotic Syndrome; Paper; Proteome; Technology; Vinyl Compounds

Aldosterone (Aldo) has been shown as an important contributor of podocyte injury. However, the underlying molecular mechanisms are still elusive. Recently, the pathogenic role of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in mediating renal tubular damage was identified while its role in podocyte injury still needs evidence. Thus the present study was undertaken to investigate the role of NLRP3 inflammasome in Aldo-induced podocyte damage. In vitro, exposure of podocytes to Aldo enhanced NLRP3, caspase-1, and IL-18 expressions in dose- and time-dependent manners, indicating an activation of NLRP3 inflammasome, which was significantly blocked by the mineralocorticoid receptor antagonist eplerenone or the antioxidant

Topics: Albuminuria; Aldosterone; Animals; Apoptosis; Caspase 1; Cell Line; Dose-Response Relationship, Drug; Genotype; Humans; Inflammasomes; Interleukin-18; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Nephrotic Syndrome; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Phenotype; Podocytes; Receptors, Mineralocorticoid; RNA Interference; Signal Transduction; Time Factors; Transfection

Steroid Resistant Nephrotic Syndrome (SRNS) in children and young adults has differing etiologies with monogenic disease accounting for 2.9-30% in selected series. Using whole exome sequencing we sought to stratify a national population of children with SRNS into monogenic and non-monogenic forms, and further define those groups by detailed phenotypic analysis. Pediatric patients with SRNS were identified via a national United Kingdom Renal Registry. Whole exome sequencing was performed on 187 patients, of which 12% have a positive family history with a focus on the 53 genes currently known to be associated with nephrotic syndrome. Genetic findings were correlated with individual case disease characteristics. Disease causing variants were detected in 26.2% of patients. Most often this occurred in the three most common SRNS-associated genes: NPHS1, NPHS2, and WT1 but also in 14 other genes. The genotype did not always correlate with expected phenotype since mutations in OCRL, COL4A3, and DGKE associated with specific syndromes were detected in patients with isolated renal disease. Analysis by primary/presumed compared with secondary steroid resistance found 30.8% monogenic disease in primary compared with none in secondary SRNS permitting further mechanistic stratification. Genetic SRNS progressed faster to end stage renal failure, with no documented disease recurrence post-transplantation within this cohort. Primary steroid resistance in which no gene mutation was identified had a 47.8% risk of recurrence. In this unbiased pediatric population, whole exome sequencing allowed screening of all current candidate genes. Thus, deep phenotyping combined with whole exome sequencing is an effective tool for early identification of SRNS etiology, yielding an evidence-based algorithm for clinical management.

Topics: Adolescent; Age of Onset; Child; Child, Preschool; Cohort Studies; Disease Progression; Exome; Female; Genetic Association Studies; Genetic Predisposition to Disease; Genomics; Heredity; High-Throughput Nucleotide Sequencing; Humans; Infant; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Kidney; Kidney Failure, Chronic; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Pedigree; Phenotype; Precision Medicine; Predictive Value of Tests; Prognosis; Registries; Risk Factors; United Kingdom; WT1 Proteins; Young Adult

Topics: China; Heterozygote; Humans; Infant; Membrane Proteins; Mutation; Nephrotic Syndrome

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

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

Altered expression of nephrin underlies the pathophysiology of proteinuria in both congenital and acquired nephrotic syndrome. However, the epigenetic mechanisms of nephrin gene regulation remain elusive. Here, we show that Wolf-Hirschhorn syndrome candidate 1-like 1 long form (WHSC1L1-L) is a novel epigenetic modifier of nephrin gene regulation. WHSC1L1-L was associated with histone H3K4 and H3K36 in human embryonic kidney cells. WHSC1L1-L gene was expressed in the podocytes, and functional protein product was detected in these cells. WHSC1L1-L was found to bind nephrin but not other podocyte-specific gene promoters, leading to its inhibition/suppression, abrogating the stimulatory effect of WT1 and NF-κB. Gene knockdown of WHSC1L1-L in primary cultured podocytes accelerated the transcription of nephrin but not CD2AP. An in vivo zebrafish study involving the injection of Whsc1l1 mRNA into embryos demonstrated an apparent reduction of nephrin mRNA but not podocin and CD2AP mRNA. Immunohistochemistry showed that both WHSC1L1-L and nephrin emerged at the S-shaped body stage in glomeruli. Immunofluorescence and confocal microscopy displayed WHSC1L1 to colocalize with trimethylated H3K4 in the glomerular podocytes. Chromatin immunoprecipitation assay revealed the reduction of the association of trimethylated H3K4 at the nephrin promoter regions. Finally, nephrin mRNA was upregulated in the glomerulus at the early proteinuric stage of mouse nephrosis, which was associated with the reduction of WHSC1L1. In conclusion, our results demonstrate that WHSC1L1-L acts as a histone methyltransferase in podocytes and regulates nephrin gene expression, which may in turn contribute to the integrity of the slit diaphragm of the glomerular filtration barrier.

Topics: Adaptor Proteins, Signal Transducing; Animals; Binding Sites; Cytoskeletal Proteins; Disease Models, Animal; Doxorubicin; Epigenesis, Genetic; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; HEK293 Cells; Histone-Lysine N-Methyltransferase; Histones; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Methylation; Mice, Inbred BALB C; Mice, Inbred C57BL; Nephrotic Syndrome; Nuclear Proteins; Podocytes; Promoter Regions, Genetic; RNA Interference; Transfection; Zebrafish; Zebrafish Proteins

Autosomal recessive mutations in NPHS1 gene are a common cause of congenital nephrotic syndrome (CNS). The disorder is characterized by massive proteinuria that manifests in utero or in the neonatal period during the first 3 months of life. NPHS1 encodes nephrin, a member of the immunoglobulin family of cell adhesion molecules and the main protein expressed at the renal slit diaphragm. Currently, there are approximately 250 mutations described in the NPHS1 gene distributed among all nephrin domains. The main objective of this study was to perform the analysis of the NPHS1 gene in patients with congenital nephrotic syndrome in order to determine the molecular cause of the disease.. Direct sequencing of NPHS1 gene in four children was performed.. Each patient was heterozygous for two pathogenic mutations disclosing the molecular cause of the disease in 100% of the cases. We identified six different mutations, consisting of one in-frame deletion, one frameshift, and four missense substitutions. The p.Val736Met mutation that is described here for the first time was considered pathogenic by different mutation predictive algorithms. Regardless of the type of mutation, three patients had a bad outcome and died. Despite the small size of the cohort, this study contributed to the increasing number of deleterious mutations in the NPHS1 gene by describing a new mutation. Also, since we identified NPHS1 pathogenic mutations as the cause of the disease in all cases analyzed, it might be a frequent cause of CNS in the South Eastern region of Brazil, although the analysis of a larger sample is required to obtain more indicative epidemiological data.

Topics: Brazil; Child, Preschool; DNA Mutational Analysis; Fatal Outcome; Female; Genetic Markers; Genetic Predisposition to Disease; Heterozygote; Humans; Infant; Infant, Newborn; Male; Membrane Proteins; Mutation; Nephrectomy; Nephrotic Syndrome; Phenotype; Treatment Outcome

Treatment of congenital nephrotic syndrome (CNS) and steroid-resistant nephrotic syndrome (SRNS) is demanding, and renal prognosis is poor. Numerous causative gene mutations have been identified in SRNS that affect the renal podocyte. In the era of high-throughput sequencing techniques, patients with nongenetic SRNS frequently escape the scientific interest. We here present the long-term data of the German CNS/SRNS Follow-Up Study, focusing on the response to cyclosporin A (CsA) in patients with nongenetic versus genetic disease.. Cross-sectional and longitudinal clinical data were collected from 231 patients with CNS/SRNS treated at eight university pediatric nephrology units with a median observation time of 113 months (interquartile range, 50-178). Genotyping was performed systematically in all patients.. The overall mutation detection rate was high at 57% (97% in CNS and 41% in SRNS); 85% of all mutations were identified by the analysis of three single genes only (NPHS1, NPHS2, and WT1), accounting for 92% of all mutations in patients with CNS and 79% of all mutations in patients with SRNS. Remission of the disease in nongenetic SRNS was observed in 78% of patients after a median treatment period of 2.5 months; 82% of nongenetic patients responded within 6 months of therapy, and 98% of patients with nongenetic SRNS and CsA-induced complete remission (normalbuminemia and no proteinuria) maintained a normal renal function. Genetic SRNS, on the contrary, is associated with a high rate of ESRD in 66% of patients. Only 3% of patients with genetic SRNS experienced a complete remission and 16% of patients with genetic SRNS experienced a partial remission after CsA therapy.. The efficacy of CsA is high in nonhereditary SRNS, with an excellent prognosis of renal function in the large majority of patients. CsA should be given for a minimum period of 6 months in these patients with nongenetic SRNS. In genetic SRNS, response to CsA was low and restricted to exceptional patients.

Topics: Adolescent; Austria; Biopsy; Child; Child, Preschool; Cross-Sectional Studies; Cyclosporine; Disease Progression; DNA Mutational Analysis; Female; Genetic Predisposition to Disease; Germany; Humans; Immunosuppressive Agents; Infant; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Failure, Chronic; Longitudinal Studies; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype; Recovery of Function; Remission Induction; Risk Factors; Time Factors; Treatment Outcome; WT1 Proteins

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

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

Topics: China; Female; Humans; Infant, Newborn; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Pedigree

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

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

Identification of the NPHS1 gene, which encodes nephrin, was followed by many studies demonstrating its mutation as a frequent cause of congenital nephrotic syndrome (CNS). While this gene is found in 98% of Finnish children with this syndrome, non-Finnish cases have lower level of incidence ranging from 39 to 80%.. This report describes the clinical presentation of a two-week-old neonate who presented with periorbital and lower extremities edema, abdominal distention, heavy proteinuria, serum hypoproteinemia and failure to thrive. Genetic analysis revealed NHPS1 gene mutation leading to CNS-Finnish type diagnosis.. Through this case we want to create awareness about diagnosis and treatment challenges in developing countries for rare congenital diseases.

Topics: Albumins; Diuretics; Failure to Thrive; Genetic Testing; Humans; Infant; Infusions, Intravenous; Male; Membrane Proteins; Mutation, Missense; Nephrotic Syndrome; Peritoneal Dialysis; Referral and Consultation

Congenital nephrotic syndrome (CNS) of the Finnish type, NPHS1, is the most severe form of CNS. Outcomes of renal replacement therapy (RRT) in NPHS1 patients in Europe were analysed using data from the ESPN/ERA-EDTA Registry. As NPHS1 is most prevalent in Finland and the therapeutic approach differs from that in many other countries, we compared outcomes in Finnish and other European patients.. NPHS1 mutations were confirmed in 170 children with CNS who initiated RRT (dialysis or renal transplantation) between 1991 and 2012. Finnish (n = 66) and non-Finnish NPHS1 patients (n = 104) were compared with respect to treatment policy, age at first RRT and renal transplantation (RTX), patient and graft survival, estimated glomerular filtration rate (eGFR) and growth. Age-matched patients with congenital anomalies of the kidney and urinary tract (CAKUT) served as controls.. Finnish NPHS1 patients were significantly younger than non-Finnish patients, both at the start of RRT and at the time of RTX. We found similar overall 5-year patient survival on RRT (91 %) and graft survival (89 %) in both NPHS1 groups and CAKUT controls. At the start of RRT, height standard deviation score (SDS) was higher in Finnish patients than in non-Finnish patients (mean [95 % CI]: -1.31 [-2.13 to -0.49] and -3.0 [-4.22 to -1.91], p < 0.01 respectively), but not at 5 years of age. At 5 years of age height and body mass index (BMI) SDS were similar to those of CAKUT controls.. Overall, 5-year patient and graft survival of both Finnish and non-Finnish NPHS1 patients on RRT were excellent and comparable with CAKUT patients with equally early RRT onset and was independent of the timing of RRT initiation and RTX.

Topics: Age Factors; Body Mass Index; Finland; Glomerular Filtration Rate; Graft Survival; Growth; Kidney Transplantation; Membrane Proteins; Mutation; Nephrotic Syndrome; Registries; Renal Replacement Therapy; Survival Analysis; Time-to-Treatment; Treatment Outcome

Congenital nephrotic syndrome of the Finnish type (CNF) is a rare autosomal recessive disorder. The incidence of CNF is relatively high in Finland but considerably lower in other countries. We encountered a male newborn with CNF, associated with compound heterozygous mutations in nephrosis 1, congenital, Finnish type (NPHS1). The patient was admitted to hospital as a preterm infant. Physical and laboratory findings fulfilled the diagnostic criteria of nephrotic syndrome, and were compatible with a diagnosis of CNF, but there was no family history of the disease. On genetic analysis of NPHS1 a paternally derived heterozygous frame-shift mutation caused by an 8 bp deletion, resulting in a stop codon in exon 16 (c.2156-2163 delTGCACTGC causing p.L719DfsX4), and a novel, maternally derived nonsense mutation in exon 15 (c.1978G>T causing p.E660X) were identified. Early genetic diagnosis of CNF is important for proper clinical management and appropriate genetic counseling.

Topics: DNA Mutational Analysis; Genetic Testing; Humans; Infant, Newborn; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Polymerase Chain Reaction

Renin-angiotensin system (RAS) inhibitors reduce glomerular injury and proteinuria, indicating that angiotensin II (Ang II) is involved in glomerular diseases. Although the local RAS is reported to play an essential role in maintaining local tissue functions, the role of the local RAS in regulating glomerular function is not well evaluated. In this study, we analyzed the glomerular expression of RAS components in nephrotic models and the effect of Ang II receptor blockers (ARB) on the expression of angiotensinogen (AGT).. The levels of glomerular expression of RAS components were analyzed in two nephrotic models: anti-nephrin antibody-induced nephropathy and PAN nephropathy, a mimic of human minimal change nephrotic syndrome. The effect of the ARB irbesartan on the expression of AGT in the nephrotic model was analyzed.. Glomerular expression of AGT and the receptors for Ang II was clearly increased in the nephrotic models, while the expression levels of renin, ACE and ACE2 were decreased. ARB treatment suppressed the increase of glomerular expression of AGT in the nephrotic model.. It is conceivable that the promoted local RAS action participated in the glomerular dysfunction, and that ARB treatment ameliorated slit diaphragm injury by inhibiting the positive feedback loop of the activated local Ang II action.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensinogen; Animals; Cells, Cultured; Disease Models, Animal; Female; Kidney Glomerulus; Membrane Proteins; Nephrotic Syndrome; Podocytes; Rats, Wistar; Renin-Angiotensin System; RNA, Messenger

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

The purpose of this study was to detect the urinary podocytes and its related protein, nephrin, in the urine of the children with glomerular disease in order to analyze the relationship of the clinical testing with the significance of the glomerular disease. A total of 65 children with nephrotic syndrome were selected for this study. The podocytes and nephrin were detected in the urinary sediment by indirect immunofluorescence, enzyme-linked immunosorbent assay, and Western blotting. The urinary podocytes and nephrin positive rates were 53.8% and 50.8%, respectively, in the children with glomerular disease. The serum total protein and albumin decreased in the podocyte-positive children, while the urine total protein at 24 h, urinary albumin/creatinine ratio, blood urea nitrogen, and serum creatinine were significantly elevated as compared to those of the podocyte-negative patients. Furthermore, the results were the same in the patients with positive nephrin as compared to that of the patients with negative nephrin. The podocyte number and nephrin level were significantly higher in the lupus nephritis group as compared to those of the other groups. Likewise, the podocyte number and nephrin level dramatically increased in the focal segmental glomerulosclerosis group as compared to those of the mesangial proliferative glomerulonephritis and minimal change disease groups. In addition, the podocyte numbers and nephrin expression were significantly higher in severe proteinuria group as compared to those of the mild proteinuria group. The urinary nephrin expression was positively related to podocyte and urinary albumin/creatinine ratio. We concluded that the detection of the urinary podocytes and nephrin could be taken as markers for children with glomerular disease, reflecting the type of the disease. Therefore, this can be used as a noninvasive method to evaluate the severity of the kidney disease in children.

Topics: Albuminuria; Biomarkers; Child; Creatinine; Female; Humans; Lupus Nephritis; Male; Membrane Proteins; Nephrotic Syndrome; Podocytes

Nephrotic syndrome (NS), the association of gross proteinuria, hypoalbuminaemia, edema, and hyperlipidemia, can be clinically divided into steroid-sensitive (SSNS) and steroid-resistant (SRNS) forms. SRNS regularly progresses to end-stage renal failure. By homozygosity mapping and whole exome sequencing, we here identify recessive mutations in Crumbs homolog 2 (CRB2) in four different families affected by SRNS. Previously, we established a requirement for zebrafish crb2b, a conserved regulator of epithelial polarity, in podocyte morphogenesis. By characterization of a loss-of-function mutation in zebrafish crb2b, we now show that zebrafish crb2b is required for podocyte foot process arborization, slit diaphragm formation, and proper nephrin trafficking. Furthermore, by complementation experiments in zebrafish, we demonstrate that CRB2 mutations result in loss of function and therefore constitute causative mutations leading to NS in humans. These results implicate defects in podocyte apico-basal polarity in the pathogenesis of NS.

Topics: Amino Acid Sequence; Animals; Carrier Proteins; Child; Child, Preschool; Chromosome Mapping; Exome; Genes, Recessive; Homozygote; Humans; Infant; Kidney Failure, Chronic; Membrane Proteins; Molecular Sequence Data; Mutation; Nephrotic Syndrome; Podocytes; Rats; Zebrafish

Nephrin and podocin proteins, encoded by NPHS1 and NPHS2 genes, are essential for the integrity of the glomerular filter. The present study was aimed to investigate whether NPHS1 rs437168 and NPHS2 rs61747728 genetic variants are involved in the susceptibility to nephrotic syndrome (NS).. This case-control study was performed on 108 children with NS and 97 healthy children. Genomic DNA was extracted from whole blood using the salting-out method. Polymorphism of the NPHS1 rs437168 and NPHS2 rs61747728 were detected by amplification refractory mutation system- and tetra primers amplification refractory mutation system-polymerase chain reaction, respectively.. The results showed that the NPHS1 rs437168 GA as well as GA+AA genotypes increased the risk of NS in comparison with GG genotype (odds ratio, 4.76, 95% confidence interval, 2.31 to 9.80; P < .001 and odds ratio, 4.57; 95% confidence interval, 2.31 to 9.04, ; P < .001, respectively). The A allele was associated with increased risk of NS (odds ratio, 3.53; 95% confidence interval, 1.94 to 6.42, ; P < .001) in comparison to the G allele. No association was observed between NPHS2 rs61747728 polymorphism and NS.. Our findings indicate that NPHS1 rs437168, but not NPHS2 rs61747728 variant, is associated with NS.

Topics: Age Factors; Case-Control Studies; Chi-Square Distribution; Child; Child, Preschool; Female; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Genetic Variation; Humans; Infant; Intracellular Signaling Peptides and Proteins; Iran; Male; Membrane Proteins; Nephrotic Syndrome; Odds Ratio; Phenotype; Polymerase Chain Reaction; Risk Assessment; Risk Factors

Congenital nephrotic syndrome (CNS) is a rare disease inherited as an autosomally recessive trait and defined as proteinuria manifesting at birth or in the first 3 months of life. The classical form is the Finnish type of CNS (CNF), which is caused by mutations in the nephrin gene (NPHS1). The classical findings include prematurity, large placenta and massive proteinuria. Minor cardiac findings have been reported as a minor functional disorder but CNS with major cardiac malformation is rare. Here we report the case of a Turkish child with CNS with small indel mutation (c.614_621delCACCCCGGinsTT) in exon 6 of NPHS1 and also major cardiac malformation who did not develop end-stage renal disease until the age of 5 years.

Topics: Abnormalities, Multiple; Biopsy; DNA; DNA Mutational Analysis; Echocardiography; Female; Genotype; Heart Defects, Congenital; Humans; Infant, Newborn; Kidney; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype; Polymerase Chain Reaction

Congenital nephrotic syndrome (CNS) and infantile nephrotic syndrome (INS) are caused primarily by mutations in genes that encode structural and regulatory proteins of the glomerular filtration barrier. The aim of this study was to determine genotype-phenotype correlations and prognosis in patients with CNS and INS.. NPHS1, NPHS2, LAMB2 and the eighth and ninth exons of WT1 were sequenced in 80 and 22 patients with CNS and INS, respectively. Genotype-phenotype correlations and survival were evaluated.. Causative mutations were identified in 64.7 % of patients, of which NPHS1 mutations were the most common (37.4 %). The mutation detection rate was twofold higher in CNS patients than in INS patients (72.5 vs. 36.2 %). The most commonly mutated gene in CNS patients was NPHS1 (46.3 %) versus NPHS2 (13.6 %) and WT1 (13.6 %) in INS patients. NPHS2 mutations, female patients with NPHS1 mutations, and NPHS1 mutations affecting the transmembrane or intracellular domains of nephrin were associated with longer survival.. Based on our present findings, the likelihood of identification of a genetic cause decreases with increasing age at diagnosis. The underlying genetic abnormality should be identified as early as possible, as this knowledge will facilitate clinicians in their prognostic prediction and enable patients to receive appropriate genetic counseling.

Topics: Age of Onset; DNA Mutational Analysis; Female; Genes, Wilms Tumor; Genetic Association Studies; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Laminin; Male; Membrane Proteins; Nephrotic Syndrome; Prognosis

Congenital nephrotic syndrome (CNS) is defined as heavy proteinuria or nephrotic syndrome occurring before 3 months of age. It is characterized by early onset and progresses to end-stage renal disease. Recently, several genes associated with CNS have been identified, including NPHS1 and NPHS2. Mutations in the NPHS1 gene have been identified in patients with CNS in Finland with relatively high frequency. Thus far, only a few case reports about CNS have described an NPHS1 mutation in China. In this study, mutational analyses of NPHS1 and NPHS2 were performed in a Chinese child with CNS. Mutations were analyzed in all exons and exon/intron boundaries of NPHS1 and NPHS2 in the patient and his parents as well as in 50 unrelated controls using polymerase chain reaction and direct sequencing techniques. No mutations were detected in NPHS2. A novel splice site mutation (IVS11+1G>A) within intron 11 and a missense mutation within exon 8 (c.928G>A) in the NPHS1 gene were detected in the child. The child's mother had normal urinalysis and a c.928G>A (D310N) heterozygous mutation, and his father had normal urinalysis and IVS11+1G>A. These were not identified in the 50 unrelated controls. The novel splice site mutation of IVS11+1G>A and a missense mutation at c.928G>A in NPHS1 were found to cause CNS in this Chinese child.

Topics: Asian People; Base Sequence; Child; Humans; Infant, Newborn; Male; Membrane Proteins; Molecular Sequence Data; Mutation; Nephrotic Syndrome; RNA Splice Sites

Topics: Aortic Valve Stenosis; Base Sequence; Codon, Nonsense; DNA Mutational Analysis; Female; Genetic Predisposition to Disease; Humans; Male; Membrane Proteins; Molecular Sequence Data; Nephrotic Syndrome; Pedigree

Nephrotic syndrome is traditionally classified on the basis of the response to standard steroid treatment. Mutations in more than 24 genes have been associated with nephrotic syndrome in children, although the great majority of steroid-resistant cases have been attributed to mutations in three main genes: NPHS1, NPHS2 and WT1. The aims of this study were to identify mutations in these genes more frequently reported as mutated and to characterize each variation using different in silico prediction algorithms in order to understand their biological functions.. We performed direct sequence analysis of exons 8 and 9 of WT1, 8 exons of NPHS2 and 29 exons of NPHS1, including NPHS2 and NPHS1 intron-exon boundary sequences, as well as 700 bp of the 5' UTR from both genes in 27 steroid-resistant patients aged between 3 months and 18 years.. Analysis of the NPHS2 gene revealed four missense mutations, one frameshift mutation and three variations in the 5' UTR. Four patients presented compound heterozygosis, and four other patients presented one heterozygous alteration only. WT1 and NPHS1 gene analysis did not reveal any mutations.. This is the first study focusing on genetics of SRNS in Brazilian children. Identification of mutations is important because it could influence physicians' decision on patient treatment, as patients carrying mutations can be spared the side effects of immunosuppressive therapy and ultimately could be considered for kidney transplantation from a living donor.. After molecular analysis of the genes more frequently reported as mutated in 27 steroid-resistant nephrotic syndrome patients, we identified NPHS2 mutations confirming the hereditary character of the kidney disease in only 14.8% of patients. Therefore, the next step is to perform a next generation sequencing based analysis of glomeluropathy-related panel of genes for the remaining patients in order to search for mutations in other genes related to steroid-resistant nephrotic syndrome.

Topics: 5' Untranslated Regions; Adolescent; Base Sequence; Child; Child, Preschool; Computational Biology; DNA Primers; Frameshift Mutation; Humans; Infant; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Molecular Sequence Data; Mutation, Missense; Nephrotic Syndrome; Sequence Analysis, DNA; WT1 Proteins

Progresses in podocyte biology have been strictly connected with genetic advances; the identification of genes mutated in familial and sporadic forms of nephrotic syndrome has been followed by functional studies of the encoded proteins, revealing numerous properties of the cell. The molecules uncovered so far belong to three main categories: a) proteins located at the slit diaphragm, the intercellular junction which laterally connects podocyte processes and is responsible for selectivity of the glomerular filter, b) molecules involved in regulation of actin dynamics, which are essential for the maintenance of podocyte structure and function, and c) molecules belonging to intracellular organelles, such as mitochondria and lysosomes, which are central players in podocyte metabolism. Considering the key role of the podocyte in health and disease of the glomerular filter, better knowledge of this cell is a pre-requisite for developing targeted therapies of glomerular diseases.

Topics: Animals; Cytoskeleton; Humans; Lysosomes; Membrane Proteins; Mitochondria; Nephrotic Syndrome; Podocytes

Many mutations in the NPHS1 gene were detected among patients with congenital nephrotic syndrome. Functional analysis of those mutations was done with a stable-expression cell line. Nevertheless, establishing such a cell line is time-consuming.. We established an easier method using automatic counting software for functional analysis with transient-transfection cells rather than a stable-expression cell line. We demonstrated maltrafficking to the plasma membrane of abnormal nephrin for immunostaining on transient-expression cells by comparison without Triton X (detecting proteins on the cell membrane only) and with Triton X (detecting proteins both on the cell membrane and inside the cell cytoplasm). We obtained relevant results with data obtained previously using a stable-expression cell line. Furthermore, we conducted functional analysis of NPHS1 mutations in Japanese patients with congenital nephrotic syndrome using this simple method, which revealed that all pathogenic mutations impaired trafficking to the protein plasma membrane.. Functional analysis using transient-expression cells with automatic counting software was useful to demonstrate maltrafficking to the plasma membrane of a protein. All pathogenic mutations detected in Japanese patients impaired trafficking to the protein plasma membrane.

Topics: Asian People; Genotype; HEK293 Cells; Humans; Japan; Membrane Proteins; Mutation; Nephrotic Syndrome

Nephrotic syndrome (NS) is a clinical syndrome with a variety of causes, mainly characterized by heavy proteinuria. Podocyte injury plays a key role in proteinuria, one of the principal means for the control of NS is to prevent podocyte injury. Qi-Dan Fang consists of two of the most extensively applied herbal remedies among Traditional Chinese Medicine (TCM) (Radix Astragali Mongolici and Radix Salviae Miltiorrhizae, with a weight ratio of 5:1) which are specifically used for the treatment of various kidney diseases. In previous studies, we found that Qi-Dan Fang provides improvement to patients with adriamycin-induced nephrotic syndrome by alleviating proteinuria and serum lipid. The aim of this study is to study the efficiency of Qi-Dan Fang on NS model rat with renal dysfunction and podocyte injury, something which has not been carried out yet.. The rats were divided into Normal, Model, Jin Gui Shen Qi Pill (4.12 g/kg), Qi-Dan Fang (3.09, 6.17 and 12.34 g/kg/d) groups, they were each given a single tail intravenous injection of Adriamycin (6.0 mg/kg) except for the Normal group and were orally administered dosages of Qi-Dian Fang and Jin Gui Shen Qi pills once daily for 7 weeks. Following the treatment, the content of cystation C (CysC), blood urea nitrogen (BUN), serum creatinine (Scr) were measured with an autobiochemical analyser. The pathomorphological changes to the glomeruli, the mRNA expressions of nephrin, podocin, CD2AP genes and p53, bax, bcl-2 proteins expressions were also carried out to probe the effects of Qi-Dan Fang.. (1) Qi-Dan Fang treatment raised the level of CysC in blood serum while lowering the content of BUN and Scr in the adriamycin-induced nephrotic syndrome rat model; (2) Long-term administration of Qi-Dan Fang was able to ameliorate pathomorphological change of glomeruli and repair the organization structure of Glomerulus; (3) Qi-Dan Fang could increase the mRNA expression of nephrin, podocin and CD2AP genes, down-regulate the expression of p53, bax proteins, while increased bcl-2 protein to protect the podocyte and restore Glomerular selective filtration function.. Results of our present studies reveal that Qi-Dan Fang is able to enhance renal function, inhibit podocyte injury to provide improvements to the Adriamycin-induced nephrotic syndrome.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibiotics, Antineoplastic; bcl-2-Associated X Protein; Cytoskeletal Proteins; Disease Models, Animal; Doxorubicin; Drugs, Chinese Herbal; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Microscopy, Electron, Transmission; Nephrotic Syndrome; Phytotherapy; Podocytes; Protective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tumor Suppressor Protein p53

Topics: Adult; alpha-Fetoproteins; Female; Genetic Testing; Heterozygote; Humans; Male; Maternal Serum Screening Tests; Membrane Proteins; Nephrotic Syndrome; Pregnancy

To investigate urinary nephrin and podocalyxin standardized by aquaporin (AQP)-2 using the enzyme-linked immunosorbent assay (ELISA) method in adult nephrotic syndrome (NS) patients.. In 107 adult NS patients (27 proliferative nephritis, 77 non-proliferative, and 3 amyloidosis) undergoing renal biopsy, urinary nephrin, podocalyxin and AQP2 were measured by ELISA. Urinary nephrin and podocalyxin were standardized by AQP2 (neph/AQP and PCX/AQP) and values were compared with 11 healthy controls.. Urinary neph/AQP correlated positively to PCX/AQP (r = 0.51, p < 0.001). Urinary neph/AQP and PCX/AQP were lower in controls than NS patients. Both proliferative and non-proliferative NS patients excreted high urinary neph/AQP and PCX/AQP without a significant difference between them (p > 0.05). Patients with focal segmental glomerular sclerosis (FSGS) excreted higher urinary neph/AQP (p = 0.09) and PCX/AQP (p < 0.05) compared to the other patients. Urinary neph/AQP and PCX/AQP were increased in the immunoglobulin M nephropathy patients. Amyloidosis patients excreted lower neph/AQP and PCX/AQP. The sensitivity was 0.87 and specificity 0.37 when the neph/AQP borderline value of 0.16 was adopted [area under the curve (AUC) = 0.61]. The sensitivity was 0.74 and specificity 0.61 when the PCX/AQP borderline value was 3.06 (AUC = 0.69).. Urinary neph/AQP and PCX/AQP are increased in NS patients, with FSGS patients showing the highest levels. To distinguish FSGS from other NS forms, the measurement of urinary PCX/AQP may be a practical method, and superior to neph/AQP.

Topics: Adolescent; Adult; Aged; Amyloidosis; Aquaporin 2; Case-Control Studies; Child; Enzyme-Linked Immunosorbent Assay; Female; Glomerulonephritis; Humans; Male; Membrane Proteins; Middle Aged; Nephrosis, Lipoid; Nephrotic Syndrome; Podocytes; Sensitivity and Specificity; Sialoglycoproteins; Urine; Young Adult

In steroid-resistant nephrotic syndrome (SRNS), >21 single-gene causes are known. However, mutation analysis of all known SRNS genes is time and cost intensive. This report describes a new high-throughput method of mutation analysis using a PCR-based microfluidic technology that allows rapid simultaneous mutation analysis of 21 single-gene causes of SRNS in a large number of individuals.. This study screened individuals with SRNS; samples were submitted for mutation analysis from international sources between 1996 and 2012. For proof of principle, a pilot cohort of 48 individuals who harbored known mutations in known SRNS genes was evaluated. After improvements to the method, 48 individuals with an unknown cause of SRNS were then examined in a subsequent diagnostic study. The analysis included 16 recessive SRNS genes and 5 dominant SRNS genes. A 10-fold primer multiplexing was applied, allowing PCR-based amplification of 474 amplicons in 21 genes for 48 DNA samples simultaneously. Forty-eight individuals were indexed in a barcode PCR, and high-throughput sequencing was performed. All disease-causing variants were confirmed via Sanger sequencing.. The pilot study identified the genetic cause of disease in 42 of 48 (87.5%) of the affected individuals. The diagnostic study detected the genetic cause of disease in 16 of 48 (33%) of the affected individuals with a previously unknown cause of SRNS. Seven novel disease-causing mutations in PLCE1 (n=5), NPHS1 (n=1), and LAMB2 (n=1) were identified in <3 weeks. Use of this method could reduce costs to 1/29th of the cost of Sanger sequencing.. This highly parallel approach allows rapid (<3 weeks) mutation analysis of 21 genes known to cause SRNS at a greatly reduced cost (1/29th) compared with traditional mutation analysis techniques. It detects mutations in about 33% of childhood-onset SRNS cases.

Topics: Adult; Age of Onset; Child, Preschool; DNA Mutational Analysis; Drug Resistance; Female; Genetic Testing; High-Throughput Nucleotide Sequencing; Humans; Infant; Infant, Newborn; Laminin; Male; Membrane Proteins; Nephrotic Syndrome; Phosphoinositide Phospholipase C; Steroids

Nephrotic syndrome (NS) is a renal disease characterized by heavy proteinuria, hypoalbuminemia, edema and hyperlipidemia. Its presentation within the first 3 months of life or in multiple family members suggests an underlying inherited cause. To determine the frequency of inherited NS, 62 cases (representing 49 families with NS) from Saudi Arabia were screened for mutations in NPHS1, NPHS2, LAMB2, PLCE1, CD2AP, MYO1E, WT1, PTPRO and Nei endonuclease VIII-like 1 (NEIL1). We detected likely causative mutations in 25 out of 49 families studied (51%). We found that the most common genetic cause of NS in our cohort was a homozygous mutation in the NPHS2 gene, found in 11 of the 49 families (22%). Mutations in the NPHS1 and PLCE1 genes allowed a molecular genetic diagnosis in 12% and 8% of families, respectively. We detected novel MYO1E mutations in three families (6%). No mutations were found in WT1, PTPRO or NEIL1. The pathogenicity of novel variants was analyzed by in silico tests and by genetic screening of ethnically matched control populations. This is the first report describing the molecular genetics of NS in the Arabian Peninsula.

Topics: Child; Child, Preschool; Female; Genetic Testing; Homozygote; Humans; Infant; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mutation; Myosin Type I; Nephrotic Syndrome; Phosphoinositide Phospholipase C; Saudi Arabia

Topics: Agammaglobulinemia; Humans; Immunoglobulins, Intravenous; Infant, Newborn; Male; Membrane Proteins; Nephrotic Syndrome

To report the first case of congenital nephrotic syndrome of the Finnish type (SNCF) in Togo associated with a new mutation of NPHS1.. Our study focused on a female infant of 10months, born premature at 34weeks 6days, followed from birth to pure SNC discovered the 10th day of life. Monitoring and pregnancy outcome unremarkable. It is the third in a family of three children, the first two are killed in a similar table but not explored before 1year of age. The diagnosis is confirmed by the SNCF genetic study NPHS1 gene encoding nephrin performed in our patient and her parents showed a double mutation of which c.[106delG]+[2728T>C] and p. at the nucleotide level. [Ala36fs*6]+[Ser91OPro] at the protein level inherited from each parent. The change was made to the 10th month of death in life after sepsis in a third of cortico-resistance.. The SNCF, autosomal recessive disease early, which remains a serious diagnosis, is genetic. This new mutation could she explained the severity of the SNCF in this family?

Topics: Humans; Infant; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Togo

Infants with congenital nephrotic syndrome (CNS) develop severe nephrotic syndrome that is resistant to medical therapy, and bilateral nephrectomy is recommended toward the end of the first year of life followed by renal replacement therapy. CNS infants in New Zealand have been observed to exhibit a different course to those with the typical Finnish mutation.. A database of CNS children at our center was retrospectively examined. All cases diagnosed between 1975 and 2011 were reviewed. Demographic data, clinical features, genetic mutations, treatment, and outcome were extracted from clinical records.. Thirty-five patients with CNS, 23 children of Maori descent, and 12 Caucasians . Fourteen had died of either bacterial sepsis or intracranial thrombosis. Maori children had displayed a highly variable and protracted timeline to end-stage renal disease (ESRD) with median renal survival of 30 years versus 0.7 years in Caucasian patients. Mutation analysis of NPHS1 showed a founder mutation in the Maori population.. Congenital nephrotic syndrome in New Zealand Maori children exhibit a different clinical course to Caucasian children and have a mutation that was first described in this ethnic group.

Topics: Adolescent; Adult; Child; Child, Preschool; Disease Progression; Female; Founder Effect; Genetic Predisposition to Disease; Humans; Infant; Infant, Newborn; Kidney Failure, Chronic; Male; Membrane Proteins; Mutation; Native Hawaiian or Other Pacific Islander; Nephrotic Syndrome; New Zealand; Phenotype; Prognosis; Renal Replacement Therapy; Retrospective Studies; Risk Factors; Time Factors; White People; Young Adult

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

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

Topics: Burkitt Lymphoma; Chromosomes, Human, Pair 19; Consanguinity; Exons; Fatal Outcome; Genotype; Humans; Infant; Iran; Kidney Failure, Chronic; Kidney Transplantation; Male; Membrane Proteins; Nephrotic Syndrome; Phenotype; RNA Splice Sites; RNA Splicing; Sequence Analysis, DNA

Nephrotic syndrome (NS) in the first year of life is uncommon and makes up a heterogeneous group of disorders. Subsequent studies have further defined the phenotype associated with mutations in the NPHS2 gene, revealing that patients usually develop NS from birth to 6 years of age. We report a child aged 4 months with steroid-resistant NS who had polymorphism of NPHS1 (E117K) and mutation of NPHS2 (P118L). Our patient was carrying a polymorphic NPHS1 mutation, while phenotypically she had a poor prognostic NPHS2 mutation. However, it must be questioned whether this polymorphic change (E117K) alters the signaling pathways of the podocytes and leads to P118L mutation, thus making it behave differently. Perhaps, this would be called a genetic modifier in future.

Topics: Female; Humans; Infant; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mutation; Nephrotic Syndrome; Polymorphism, Genetic

Topics: DNA Mutational Analysis; Fatal Outcome; Female; Genetic Association Studies; Genotype; Greece; Humans; Infant; Male; Membrane Proteins; Nephrotic Syndrome; Point Mutation

Idiopathic Nephrotic Syndrome (NS) is the prevalent glomerular disease in childhood. It is treated with steroid and according to its response is defined as steroid sensitive NS (SSNS) and steroid resistance NS (SRNS). Mutation in NPHS 1 gene is reported in children with SRNS and few cases of SSNS. The aim of current study is to evaluate NPHS1 gene mutations in idiopathic NS (SSNS and SSRS) in Northwest Iran. In this cross-sectional analytic study 20 children from Azeri population in Iran with idiopathic NS including 10 cases with SRNS (5 male and 5 female) and 10 cases with SSNS (7 male and 3 female) were evaluated for NPHS1 gene mutations. DNA was extracted from peripheral blood and NPHSI gene analysis was performed by PCR and direct sequencing method with the use of standard primers. Mutations in NPHS1 gene occurred in 6 cases of SSNS including 3 heterozygous and 3 homozygous mutations and in 8 cases of SRNS including 5 homozygous, one compound heterozygous and 2 heterozygous mutations. Overall 6 different mutations were detected in NPHS1 gene: one deletion, one insertion, 3 missense and one nonsense mutations. Mutations in exon 4 and 27 were only seen in SRNS patients. Mutations in NPHS1 gene could occur in both SRNS and SSNS patients; however, considering higher incidence of heterozygous mutations in SSNS, the existence of milder phenotype in these cases would be the reason for steroid response.

Topics: Age Factors; Chi-Square Distribution; Child; Child, Preschool; Cross-Sectional Studies; DNA Mutational Analysis; Female; Genetic Predisposition to Disease; Heterozygote; Homozygote; Humans; Infant; Iran; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype; Polymerase Chain Reaction; Risk Factors; Steroids

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

Mutations in the NPHS1 and NPHS2 genes are among the main causes of early-onset and familial steroid resistant nephrotic syndrome respectively. This study was carried out to assess the frequencies of mutations in these two genes in a cohort of Pakistani pediatric NS patients.. Mutation analysis was carried out by direct sequencing of the NPHS1 and NPHS2 genes in 145 nephrotic syndrome (NS) patients. This cohort included 36 samples of congenital or infantile onset NS cases and 39 samples of familial cases obtained from 30 families.. A total of 7 homozygous (6 novel) mutations were found in the NPHS1 gene and 4 homozygous mutations in the NPHS2 gene. All mutations in the NPHS1 gene were found in the early onset cases. Of these, one patient has a family history of NS. Homozygous p.R229Q mutation in the NPHS2 gene was found in two children with childhood-onset NS.. Our results show a low prevalence of disease causing mutations in the NPHS1 (22% early onset, 5.5% overall) and NPHS2 (3.3% early onset and 3.4% overall) genes in the Pakistani NS children as compared to the European populations. In contrast to the high frequency of the NPHS2 gene mutations reported for familial SRNS in Europe, no mutation was found in the familial Pakistani cases. To our knowledge, this is the first comprehensive screening of the NPHS1 and NPHS2 gene mutations in sporadic and familial NS cases from South Asia.

Topics: Adolescent; Asia; Child; Child, Preschool; Cohort Studies; DNA Mutational Analysis; Europe; Female; Homozygote; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Pakistan

Congenital nephrotic syndrome (CNS) is defined as nephrotic syndrome that manifests within the first 3 months of life. Mutations in the NPHS1 gene encoding nephrin, are a major cause for CNS. Currently, more than 173 different mutations of NPHS1 have been published as causing CNS, affecting most exons.. We performed mutation analysis of NPHS1 in a worldwide cohort of 20 families (23 children) with CNS. All 29 exons of the NPHS1 gene were examined using direct sequencing. New mutations were confirmed by demonstrating their absence in 96 healthy control individuals.. We detected disease-causing mutations in 9 of 20 families (45%). Seven of the families showed a homozygous mutation, while two were compound heterozygous. In another 2 families, single heterozygous NPHS1 mutations were detected. Out of 10 different mutations discovered, 3 were novel, consisting of 1 splice site mutation and 2 missense mutations.. Our data demonstrate that the spectrum of NPHS1 mutations is still expanding, involving new exons, in patients from a diverse ethnic background.

Topics: Asian People; Case-Control Studies; Cohort Studies; DNA Mutational Analysis; Exons; Female; Heterozygote; Homozygote; Humans; Infant; Infant, Newborn; Male; Membrane Proteins; Nephrotic Syndrome; White People

Since the identification of the NPHS1 gene, which encodes nephrin, various investigators have demonstrated that the NPHS1 mutation is a frequent cause of congenital nephrotic syndrome (CNS); it is found in 98% of Finnish children with this syndrome and in 39-80% of non-Finnish cases. In China, compound heterozygous mutations in the NPHS1 gene have been identified in two Chinese families with CNS. To our knowledge, however, whether or not NPHS1 is the causative gene in sporadic Chinese CNS cases has not been established. We identified a homozygous mutation of NPHS1, 3250insG (V1084fsX1095), in a Chinese child with sporadic CNS. This finding leads us to suggest that NPHS1 mutations are also present in sporadic Chinese CNS cases. This gives additional support for the necessity for genetic examination of mutations in the NPHS1 gene in Chinese children with sporadic CNS.

Topics: Asian People; Humans; Infant; Male; Membrane Proteins; Mutation; Nephrotic Syndrome

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

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

To explore the correlation between Chinese medicine (CM) syndromes and the NPHS1 gene and NPHS2 gene polymorphism as well as corticosteroid sensitivity in patients with minimal change disease (MCD).. A total of 94 MCD patients were recruited, including 58 steroid-sensitive nephritic syndrome (SSNS) patients and 36 steroid-resistant nephritic syndrome (SRNS) patients. Genomic DNA was obtained from peripheral blood lymphocytes and sequence analysis of single nucleotide polymorphisms (SNPs) in the genes was performed.. (1) The SNPs of G349A-3 in NPHS1 gene was found in MCD, but the SNPs of G686A-5 and C695T-5 in NPHS2 gene were not discovered in MCD. (2) When comparing the frequency of genotype AA and allele A in NPHS1 gene (G349A-3), genotype AA and allele A were higher in the SRNS group than in the SSNS group (P < 0.05). (3) When compared with the SRNS group, qi yang deficiency syndrome had a higher incidence in the SSNS group, and yin deficiency syndrome and qi-yin deficiency syndrome had a less incidence in the SSNS, but with no statistical difference (P > 0.05). The rheumatism syndrome had a higher incidence in the SSNS group (P < 0.05). The blood stasis syndrome had a lower incidence in the SSNS with statistical difference (P < 0.05). (4) There was no statistical difference in the correlation between GG, AA, GA and CM syndromes (P > 0.05).. Homozygous mutations of AA and allele A in NPHS1 gene were correlated to SRNS patients of MCD. Rheumatism syndrome patients were prone to be sensitive to corticosteroids, while patients of blood stasis syndrome were prone to be insensitive to corticosteroids. We didn't discover the correlation between NPHS1 gene polymorphism and CM syndrome distribution.

Topics: Adolescent; Adrenal Cortex Hormones; Adult; Aged; Female; Genotype; Homozygote; Humans; Intracellular Signaling Peptides and Proteins; Male; Medicine, Chinese Traditional; Membrane Proteins; Middle Aged; Nephrosis, Lipoid; Nephrotic Syndrome; Polymorphism, Single Nucleotide; Yang Deficiency; Yin Deficiency; Young Adult

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

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

Steroid-resistant nephrotic syndrome (NS) remains one of the most intractable causes of end-stage renal disease in the first two decades of life. Several genes have been involved including NPHS1, NPHS2, WT1, PLCE1, and LAMB2. Our aim was to identify causative mutations in these genes, in 24 children belonging to 13 families with NS manifesting with various ages of onset. We performed haplotype analysis and direct exon sequencing of NPHS1, NPHS2, PLCE1, LAMB2, and the relevant exons 8 and 9 of WT1. Ten different pathogenic mutations were detected in seven families concerning four genes (NPHS1 (3/7), LAMB2 (2/7), NPHS2 (1/7), and WT1 (1/7)). Five of the detected mutations were novel; IVS9+2 T>C and p.D616G in NPHS1; p.E371fsX16 in NPHS2, and p.E705X and p.D1151fsX23 in LAMB2. Nine of 24 patients failed to be categorized by mutational analysis. Our study extends the spectrum of abnormalities underlying NS, by reporting novel mutations in the NPHS1 and NPHS2 genes and the first cases of LAMB2 mutations in Tunisia. Congenital and infantile NS can be explained by mutations in NPHS1, NPHS2, WT1, or LAMB2 genes. The identification of additional genes mutated in NS can be anticipated.

Topics: Adolescent; Child; Child, Preschool; DNA Mutational Analysis; Exons; Female; Genotype; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Laminin; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Pedigree; Tunisia; WT1 Proteins; Young Adult

The increasing number of podocyte-expressed genes implicated in steroid-resistant nephrotic syndrome (SRNS), the phenotypic variability, and the uncharacterized relative frequency of mutations in these genes in pediatric and adult patients with SRNS complicate their routine genetic analysis. Our aim was to compile the clinical and genetic data of eight podocyte genes analyzed in 110 cases (125 patients) with SRNS (ranging from congenital to adult onset) to provide a genetic testing approach.. Mutation analysis was performed by sequencing the NPHS1, NPHS2, TRPC6, CD2AP, PLCE1, INF2, WT1 (exons 8 and 9), and ACTN4 (exons 1 to 10) genes.. We identified causing mutations in 34% (37/110) of SRNS patients, representing 67% (16/24) familial and 25% (21/86) sporadic cases. Mutations were detected in 100% of congenital-onset, 57% of infantile-onset, 24 and 36% of early and late childhood-onset, 25% of adolescent-onset, and 14% of adult-onset patients. The most frequently mutated gene was NPHS1 in congenital onset and NPHS2 in the other groups. A partial remission was observed in 7 of 26 mutation carriers treated with immunosuppressive agents and/or angiotensin-converting enzyme inhibitors. Patients with NPHS1 mutations showed a faster progression to ESRD than patients with NPHS2 mutations. None of these mutation carriers relapsed after kidney transplantation.. We propose a genetic testing algorithm for SRNS based on the age at onset and the familial/sporadic status. Mutation analysis of specific podocyte-genes has a clinical value in all age groups, especially in children.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Adult; Algorithms; Child; Child, Preschool; Cytoskeletal Proteins; Drug Resistance; Formins; Genetic Testing; Genotype; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Microfilament Proteins; Nephrotic Syndrome; Phenotype; Phosphoinositide Phospholipase C; Steroids; TRPC Cation Channels; TRPC6 Cation Channel; WT1 Proteins; Young Adult

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of end-stage renal failure. Identification of single-gene causes of SRNS has generated some insights into its pathogenesis; however, additional genes and disease mechanisms remain obscure, and SRNS continues to be treatment refractory. Here we have identified 6 different mutations in coenzyme Q10 biosynthesis monooxygenase 6 (COQ6) in 13 individuals from 7 families by homozygosity mapping. Each mutation was linked to early-onset SRNS with sensorineural deafness. The deleterious effects of these human COQ6 mutations were validated by their lack of complementation in coq6-deficient yeast. Furthermore, knockdown of Coq6 in podocyte cell lines and coq6 in zebrafish embryos caused apoptosis that was partially reversed by coenzyme Q10 treatment. In rats, COQ6 was located within cell processes and the Golgi apparatus of renal glomerular podocytes and in stria vascularis cells of the inner ear, consistent with an oto-renal disease phenotype. These data suggest that coenzyme Q10-related forms of SRNS and hearing loss can be molecularly identified and potentially treated.

Topics: Animals; Child; Child, Preschool; Chlorocebus aethiops; COS Cells; Hearing Loss, Sensorineural; HeLa Cells; Homozygote; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Laminin; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype; Podocytes; Rats; Ubiquinone; WT1 Proteins; Zebrafish

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

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

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

Congenital nephrotic syndrome of the Finnish type (CNF) is a lethal, autosomal recessive disorder mainly caused by mutations in the NPHS1 gene; it is found at a relatively high frequency in Finns. We investigated the disease-causing mutations in a Chinese family with CNF and developed a prenatal genetic diagnosis for their latest pregnancy. Mutation analysis was made of all exons and exon/intron boundaries of NPHS1 in the fetus, parents and 50 unrelated controls using PCR and direct sequencing. A heterozygous nonsense mutation within exon 20 (c.2783C>A) and a missense mutation within exon 17 (c.2225T>C) in NPHS1 were detected in the proband's father and mother, respectively, but were not found in the fetus or in 50 unrelated controls. Two novel mutations of c.2783C>A and c.2225T>C in NPHS1 were found to be causative in this Chinese CNF family with no known Finnish ancestry. The most recent sibling did not inherit these two mutations and hence was unaffected with CNF. Determining the cumulative number and ethnic distribution of known mutations can help expedite further study of the pathogenesis of CNF.

Topics: Adult; Asian People; China; Codon, Nonsense; Exons; Family; Female; Humans; Infant, Newborn; Male; Membrane Proteins; Mutation, Missense; Nephrotic Syndrome; Pedigree; Pregnancy

Topics: Adult; alpha-Fetoproteins; Female; Genetic Carrier Screening; Genetic Testing; Gestational Age; Heterozygote; Humans; Membrane Proteins; Nephrotic Syndrome; Pregnancy; Pregnancy Trimester, Second; Prenatal Diagnosis

Topics: Asian People; Genotype; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Laminin; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype; WT1 Proteins

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

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

Congenital nephrotic syndrome of the Finnish type (NPHS1, CNF) is an autosomal recessive disease caused by mutations in a major podocyte protein, nephrin. NPHS1 is associated with heavy proteinuria and the development of glomerular scarring. We studied the cellular and molecular changes affecting the glomerular mesangium in NPHS1 kidneys. Marked hyperplasia of mesangial cells (MC) was mainly responsible for the early mesangial expansion in NPHS1 glomeruli. The levels of the proliferation marker, mindbomb homolog 1 and the major MC mitogen, platelet-derived growth factor, and its receptors, however, were quite normal. Only a small number of cells were positive for CD68 (marker for phagocytic cells) and CD34 (marker for mesenchymal precursor cells) in the NPHS1 mesangium. MCs strongly expressed alpha-smooth muscle actin, indicating myofibloblast transformation. The expression levels of the profibrotic mediators osteopontin and transforming growth factor beta were up-regulated in NPHS1 glomeruli by 3.2 and 1.6-fold, respectively, compared to the controls. The synthesis by MCs of the typical fibroblast products collagen I, fibronectin, and tenascin, however, was low, and the extracellular matrix increase was caused by the accumulation of a normal MC product, collagen IV. The results indicate that severe glomerular sclerosis can develop without major qualitative cellular or molecular changes in the mesangium.

Topics: Actins; Adolescent; Antigens, CD; Antigens, CD34; Antigens, Differentiation, Myelomonocytic; Biopsy; Case-Control Studies; Cell Proliferation; Child; Child, Preschool; Disease Progression; Extracellular Matrix Proteins; Genotype; Glomerular Mesangium; Humans; Hyperplasia; Immunohistochemistry; Infant; Membrane Proteins; Mesangial Cells; Middle Aged; Mutation; Nephrectomy; Nephrotic Syndrome; Osteopontin; Phenotype; Platelet-Derived Growth Factor; Receptors, Platelet-Derived Growth Factor; Sclerosis; Ubiquitin-Protein Ligases

Recessive mutations in the NPHS1 gene encoding nephrin account for approximately 40% of infants with congenital nephrotic syndrome (CNS). CNS is defined as steroid-resistant nephrotic syndrome (SRNS) within the first 90 days of life. Currently, more than 119 different mutations of NPHS1 have been published affecting most exons.. We here performed mutational analysis of NPHS1 in a worldwide cohort of 67 children from 62 different families with CNS.. We found bi-allelic mutations in 36 of the 62 families (58%) confirming in a worldwide cohort that about one-half of CNS is caused by NPHS1 mutations. In 26 families, mutations were homozygous, and in 10, they were compound heterozygous. In an additional nine patients from eight families, only one heterozygous mutation was detected. We detected 37 different mutations. Nineteen of the 37 were novel mutations (approximately 51.4%), including 11 missense mutations, 4 splice-site mutations, 3 nonsense mutations and 1 small deletion. In an additional patient with later manifestation, we discovered two further novel mutations, including the first one affecting a glycosylation site of nephrin.. Our data hereby expand the spectrum of known mutations by 17.6%. Surprisingly, out of the two siblings with the homozygous novel mutation L587R in NPHS1, only one developed nephrotic syndrome before the age of 90 days, while the other one did not manifest until the age of 2 years. Both siblings also unexpectedly experienced an episode of partial remission upon steroid treatment.

Topics: Cohort Studies; Exons; Family; Female; Genotype; Global Health; Heterozygote; Homozygote; Humans; Infant; Infant, Newborn; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype; Prognosis

Mutations in NPHS1, which encodes nephrin, are the main causes of congenital nephrotic syndrome (CNS) in Finnish patients, whereas mutations in NPHS2, which encodes podocin, are typically responsible for childhood-onset steroid-resistant nephrotic syndrome in European populations. Genotype-phenotype correlations are not well understood in non-Finnish patients. We evaluated the clinical presentation, kidney histology, and disease progression in non-Finnish CNS cases by mutational screening in 107 families (117 cases) by sequencing the entire coding regions of NPHS1, NPHS2, PLCE1, WT1, LAMB2, PDSS2, COQ2, and NEPH1. We found that CNS describes a heterogeneous group of disorders in non-Finnish populations. We identified nephrin and podocin mutations in most families and only rarely found mutations in genes implicated in other hereditary forms of NS. In approximately 20% of cases, we could not identify the underlying genetic cause. Consistent with the major role of nephrin at the slit diaphragm, NPHS1 mutations associated with an earlier onset of disease and worse renal outcomes than NPHS2 mutations. Milder cases resulting from mutant NPHS1 had either two mutations in the cytoplasmic tail or two missense mutations in the extracellular domain, including at least one that preserved structure and function. In addition, we extend the spectrum of known NPHS1 mutations by describing long NPHS1 deletions. In summary, these data demonstrate that CNS is not a distinct clinical entity in non-Finnish populations but rather a clinically and genetically heterogeneous group of disorders.

Topics: Africa, Northern; Biopsy; Child, Preschool; Cohort Studies; Disease Progression; Europe; Female; Genetic Testing; Genotype; Humans; Infant; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Kidney; Kidney Failure, Chronic; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype; Retrospective Studies; Turkey

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

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

Topics: Genotype; Humans; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype

Mutations in podocyte genes are associated with steroid-resistant nephrotic syndrome (SRNS), mostly affecting younger age groups. To date, it is unclear whether these patients benefit from intensified immunosuppression with cyclosporine A (CsA). The aim of this study was to evaluate the influence of podocyte gene defects in congenital nephrotic syndrome (CNS) and pediatric SRNS on the efficacy of CsA therapy and preservation of renal function.. Genotyping was performed in 91 CNS/SRNS patients, irrespective of age at manifestation or response to CsA.. Mutations were identified in 52% of families (11 NPHS1, 17 NPHS2, 11 WT1, 1 LAMB2, 3 TRPC6). Sixty-eight percent of patients with nongenetic SRNS responded to CsA, most of them achieved complete remission. In contrast, none of the patients with genetic CNS/SRNS experienced a complete remission and only two (17%) achieved a partial response, both affected by a WT1 mutation. Preservation of renal function was significantly better in children with nongenetic disease after a mean follow-up time of 8.6 years (ESRD in 29% versus 71%).. The mutation detection rate in our population was high (52%). Most patients with genetic CNS/SRNS did not benefit from CsA with significantly lower response rates compared with nongenetic patients and showed rapid progression to end-stage renal failure. These data strongly support the idea not to expose CNS/SRNS patients with inherited defects related to podocyte function to intensified immunosuppression with CsA.

Topics: Adolescent; Child; Child, Preschool; Cyclosporine; Disease Progression; DNA Mutational Analysis; Drug Resistance; Female; Genes, Wilms Tumor; Genetic Predisposition to Disease; Germany; Heredity; Hospitals, Pediatric; Hospitals, University; Humans; Immunosuppressive Agents; Infant; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Kidney; Kidney Failure, Chronic; Laminin; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Patient Selection; Phenotype; Retrospective Studies; Steroids; Time Factors; Treatment Outcome; TRPC Cation Channels; TRPC6 Cation Channel

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

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

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

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

Congenital nephrotic syndrome is defined as nephrotic syndrome which manifests in utero or during the first 3 months of life. The prototype of congenital nephrotic syndrome is congenital nephrotic syndrome of Finnish type (CNF, OMIM #602716), which is caused by loss-of-function mutations of the nephrin gene (NPHS1). There have been few clinical case reports of CNF in Korea, but none of which was confirmed by genetic study. Here, we report two children with congenital nephrotic syndrome. Genetic analysis of the NPHS1 gene revealed compound heterozygous frame-shifting mutations (c.2156_2163 delTGCACTGC causing p.L719DfsX4 and c.3250_3251insG causing p.V1084GfsX12) in one patient and a missense mutation (c.1381G>A causing p.R460Q) and a nonsense mutation (c.2442C>G causing p.Y814X) in the other patient. The nonsense mutation was novel. The clinical courses of the patients were typical of CNF. This is the first report of genetically confirmed CNF in Korea to date. The early genetic diagnosis of CNF is important for proper clinical management of the patients and precise genetic counseling of the families.

Topics: Base Sequence; Biopsy; Codon, Nonsense; Female; Frameshift Mutation; Humans; Infant; Infant, Newborn; Korea; Male; Membrane Proteins; Microscopy, Electron; Molecular Sequence Data; Mutation; Nephrotic Syndrome

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

Topics: Amino Acid Substitution; Codon; Finland; Humans; Membrane Proteins; Molecular Sequence Data; Mutation, Missense; Nephrotic Syndrome

The NPHS1gene was analysed in different five Japanese patients with congenital nephrotic syndrome (CNS) from the patients in a previous report (Sako M, Nakanishi K, Obana M et al. Analysis of NPHS1, NPHS2, ACTN4, and WT1 in Japanese patients with congenital nephrotic syndrome. Kidney Int 2005; 67: 1248-1255) that suggested that the mutation of NPHS1 was not a major cause of CNS in Japanese patients. Genomic DNA was extracted from leukocytes, and all exons and exon-intron boundaries were analysed for NPHS1 using polymerase chain reaction and direct sequencing.. Compound heterozygous mutations of NPHS1 were found in four patients and homozygous mutations in one patient. Interestingly, three patients out of five had the same mutation in NPHS1: nt2515(delC). Parents who had this mutation heterozygously were from neighbouring prefectures. Two among five patients in this research and one in the previous report (Kidney Int 2005; 67:1248-1255) had the same mutation: 736G > T in exon 7. All mutations including these two mutations except for one have never been reported outside of Japan yet.

Topics: Asian People; Child; Child, Preschool; DNA Mutational Analysis; Female; Genotype; Haplotypes; Humans; Infant; Infant, Newborn; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Phenotype

Mutations in nephrin (NPHS1) and podocin (NPHS2) genes represent a major cause of idiopathic nephrotic syndrome (NS) in children. It is not yet clear whether the presence of a single mutation acts as a modifier of the clinical course of NS.. We reviewed the clinical features of 40 patients with NS associated with heterozygous mutations or variants in NPHS1 (n = 7) or NPHS2 (n = 33). Long-term renal survival probabilities were compared with those of a concurrent cohort with idiopathic NS.. Patients with a single mutation in NPHS1 received a diagnosis before those with potentially nongenetic NS and had a good response to therapies. Renal function was normal in all cases. For NPHS2, six patients had single heterozygous mutations, six had a p.P20L variant, and 21 had a p.R229Q variant. Age at diagnosis and the response to drugs were comparable in all NS subgroups. Overall, they had similar renal survival probabilities as non-NPHS1/NPHS2 cases (log-rank chi(2) 0.84, P = 0.656) that decreased in presence of resistance to therapy (P < 0.001) and in cases with renal lesions of glomerulosclerosis and IgM deposition (P < 0.001). Cox regression confirmed that the only significant predictor of dialysis was resistance to therapy.. Our data indicate that single mutation or variant in NPHS1 and NPHS2 does not modify the outcome of primary NS. These patients should be treated following consolidated schemes and have good chances for a good long-term outcome.

Topics: Adolescent; Child; Child, Preschool; Female; Follow-Up Studies; Genetic Variation; Heterozygote; Humans; Infant; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Male; Membrane Proteins; Nephrotic Syndrome; Point Mutation

Minimal-change disease (MCD) is the most common cause of nephrotic syndrome (NS) and is characterized only by minor morphological alterations in podocytes. A subtype of MCD arises from mutations in nephrin, a major component of the slit diaphragm (SD). Idiopathic MCD is a complex trait where interactions of genetic and immunological factors are implicated. However, the pathogenic mechanisms remain unclear. Here we studied the molecular basis for familial NS characterized by frequent relapses and minimal-change histology. Our previous mutational analysis revealed that the two affected children were compound heterozygotes for nephrin variants C265R and V822M (Kidney Int., 2008). When heterologously expressed, these variants exhibited normal metabolic half-life and raft binding. C265R exhibited substantial ER retention, reflecting an intracellular trafficking defect. In contrast, V822M was able to reach the plasma membrane, but was restricted in lateral diffusion as well as trafficking at the cell surface. Clustering of V822M failed to evoke a maximum tyrosine-phosphorylation and actin reorganization, suggesting the inability to assemble into functioning membrane microdomains. Our results suggest that C265R and V822M compose a dysfunctional SD complex due to their mixed defects comprising reduced cell surface targeting and ineffective assembly of signaling microdomains. The defective SD likely confers a susceptibility to immunogenic stimuli and predisposes to a relapsing phenotype.

Topics: Cell Line; Genetic Predisposition to Disease; Humans; Membrane Proteins; Mutation, Missense; Nephrotic Syndrome; Pedigree; Phosphorylation; Protein Structure, Tertiary; Protein Transport; Recurrence

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

Classically, infants with mutations in NPHS1, which encodes nephrin, present with nephrotic syndrome within the first 3 mo of life (congenital nephrotic syndrome of the Finnish-type), and children with mutations in NPHS2, which encodes podocin, present later with steroid-resistant nephrotic syndrome. Recently, however, NPHS2 mutations have been identified in children with congenital nephrotic syndrome. Whether NPHS1 mutations similarly account for some cases of childhood steroid-resistant nephrotic syndrome is unknown. In this study, 160 patients who belonged to 142 unrelated families and presented with nephrotic syndrome at least 3 mo after birth were screened for NPHS1 variants once mutations in NPHS2 had been excluded. Compound heterozygous NPHS1 mutations were identified in one familial case and nine sporadic cases. Mutations included protein-truncating nonsense and frameshift mutations, as well as splice-site and missense variants. Mutations were classified as "severe" or "mild" using prediction algorithms and functional assays. Most missense variants trafficked normally to the plasma membrane and maintained the ability to form nephrin homodimers and to heterodimerize with NEPH1, suggesting retained function. The presence of at least one "mild" mutation in these patients likely explains the later onset and milder course of disease. These results broaden the spectrum of renal disease related to nephrin mutations.

Topics: Age of Onset; Algorithms; Child; Child, Preschool; Chromosome Mapping; Cohort Studies; Female; Humans; Infant; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Severity of Illness Index

Topics: Age Factors; Humans; Infant, Newborn; Inheritance Patterns; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mutation; Nephrotic Syndrome

Topics: Codon; Gene Deletion; Humans; Membrane Proteins; Nephrotic Syndrome

Congenital nephrotic syndrome is usually presented with heavy proteinuria, hypoproteinaemia, oedema and hyperlipidaemia in a child from its birth until the age of 3 months. Aetiology of the disease is mutation in the relevant gene or it develops secondary to various infections. The most common form of congenital nephrotic syndrome is caused by mutation in gene for nephrin, the most important protein of the slit diaphragm.. We present the patient with the clinical and laboratory signs of nephrotic syndrome expressed in the first day of life. Despite the adequate and regular substitution, antiproteinuric and antithrombotic therapy, complications occurred and the patient deceased. Genetic analysis revealed homozygous mutation in gene for nephrin (614del8ins2TT). Three years later, in the patient's mother who was in the 12th week of pregnancy at that time, biopsy of chorionic villi was performed and the foetal genetic material showed heterozygosity for the same recessive mutation which meant that the foetus had the status of a carrier. To the best of our knowledge, this is the first family in Serbia in which prenatal molecular--genetic testing for the congenital nephrotic syndrome was accomplished.. We wish to stress the importance of molecular diagnosis in patients with congenital nephrotic syndrome in order to perform early prenatal diagnosis in future pregnancies.

Topics: Humans; Infant, Newborn; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Prenatal Diagnosis

Anti-cancer agent adriamycin (ADR) has demonstrated high anti-tumor efficacy. However, its use in chemotherapy has been limited largely due to its diverse toxicities, including renal toxicity, such as nephrotic syndrome with proteinuria. Podocyte injury leads to glomeruli proteinuria. Wulingsan (WLS) is a blended traditional Chinese herbal medicine specifically used for various kidney diseases. In the present study, we found that a water extract of WLS (480 mg/kg, p.o., x 28 days) reduced ADR-induced increase in urine protein excretion, plasma total cholesterol and triglyceride, and decrease in plasma total protein and albumin in rats. Furthermore, the results of electron microscopy demonstrated suppression by WLS of ADR-induced increase in width of foot process, increase in surface density and decrease in volume density. These results suggest that WLS ameliorates ADR-induced proteinuria and podocyte injury. Gene analysis results demonstrated a suppression of renal overexpression of nephrin mRNA and protein by WLS. Radioimmunoassay showed that WLS suppressed ADR-induced increased renal angiotensin II content in rats. Thus our results demonstrate that WLS ameliorates ADR-induced nephrotic syndrome in rats possibly by suppressing ADR-induced hyperactivity of renal renin-angiotensin system to modulate renal nephrin gene expression, thereby protecting podocyte from injury.

Topics: Angiotensin II; Animals; Antibiotics, Antineoplastic; Blotting, Western; Doxorubicin; Glyceraldehyde-3-Phosphate Dehydrogenases; Kidney; Male; Membrane Proteins; Microscopy, Electron, Transmission; Nephrotic Syndrome; Plant Extracts; Podocytes; Rats; Rats, Sprague-Dawley; RNA, Messenger; Uric Acid

Mutations in the NPHS1 gene cause congenital nephrotic syndrome of the Finnish type. The gene product nephrin is a structural component of the glomerular slit diaphragm formed by neighboring podocytes. Nephrin has also been suggested to be involved in signaling processes that are important for podocyte survival and differentiation. A new study by Doné et al. reports that the absence of nephrin leads to the lack of slit diaphragms but does not affect podocyte apoptosis and gene expression patterns.

Topics: Animals; Apoptosis; Disease Models, Animal; Gene Expression; Humans; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Knockout; Nephrotic Syndrome; Podocytes

Congenital nephrotic syndrome (CNS) is de- fined as nephrotic syndrome that manifests at birth or within the first 3 months of life. Most patients develop end-stage renal disease (ESRD) within 2 to 3 years of life. CNS of the Finnish-type (CNF) features a rather specific renal histology and is caused by recessive mutations in the NPHS1 gene encoding nephrin, a major structural protein of the glomerular slit-diaphragm. So far, more than 80 different mutations of NPHS1 causing CNF have been published.. Here, we performed mutation analysis of NPHS1 by exon sequencing in a worldwide cohort of 32 children with CNS from 29 different families.. Sixteen of the 29 families (55%) were found to have two disease-causing alleles in NPHS1. Two additional patients had a single heterozygous mutation in NPHS1. Thirteen of a total of 20 different mutations detected were novel (65%). These were five missense mutations, one nonsense mutation, three deletions, one insertion and three splice-site mutations.. Our data expand the spectrum of known NPHS1 mutations by >15% in a worldwide cohort. Surprisingly, two patients with disease-causing mutations showed a relatively mild phenotype, as one patient had a partial remission with steroid treatment and one patient had normal renal function 1 year after the onset of disease. The increased number of known mutations will facilitate future studies into genotype/phenotype correlations.

Topics: Codon, Nonsense; Cohort Studies; Female; Gene Deletion; Genotype; Humans; Infant; Infant, Newborn; Male; Membrane Proteins; Mutagenesis, Insertional; Mutation; Mutation, Missense; Nephrotic Syndrome; Phenotype

Recent discoveries indicate that the molecules in glomerular podocytes and slit diaphragms may play an important role in the development of proteinuria and nephrotic syndrome. Mutational analyses of NPHS1 and NPHS2 were performed to verify this hypothesis in sporadic nephrotic syndrome (NS) patients. Clinical characteristics and DNA samples were collected from 38 Chinese children with sporadic steroid-sensitive NS, 22 with steroid-resistant NS and 30 controls. Direct sequencing was performed after PCR amplification of all 29 and 8 exons of the NPHS1 and NPHS2 genes, respectively. In NPHS1, 4 patients had heterozygous missense mutations leading to amino acid substitutions (R800C, Q453R). Furthermore, 3 known single nucleotide polymorphism (SNP) were found (T741T, V763V, S1105S). In NPHS2, 3 patients had novel heterozygous allelic variants leading to amino acid substitutions (S206I, E188D), while 1 patient was found to carry a novel nonsense mutation leading to a truncated protein product (Glu237STOP). Two known polymorphisms were also found (A318A, L346L). The results demonstrate that NPHS1 and NPHS2 mutations are also present in Chinese sporadic NS patients, suggesting that genetic changes of nephrin and podocin may play pathogenetic roles in some patients with sporadic steroid resistant NS.

Topics: Base Sequence; Child; China; Drug Resistance; Female; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Molecular Sequence Data; Nephrotic Syndrome; Polymorphism, Single Nucleotide

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

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

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

The leading causes of albuminuria and end-stage renal failure are secondary to abnormalities in the production or cellular action of insulin, including diabetes and hyperinsulinemic metabolic syndrome. The human glomerular podocyte is a critical cell for maintaining the filtration barrier of the kidney and preventing albuminuria. We have recently shown this cell to be insulin sensitive with respect to glucose uptake, with kinetics similar to muscle cells. We now show that the podocyte protein nephrin is essential for this process. Conditionally immortalized podocytes from two different patients with nephrin mutations (natural human nephrin mutant models) were unresponsive to insulin. Knocking nephrin down with siRNA in wild-type podocytes abrogated the insulin response, and stable nephrin transfection of nephrin-deficient podocytes rescued their insulin response. Mechanistically, we show that nephrin allows the GLUT1- and GLUT4-rich vesicles to fuse with the membrane of this cell. Furthermore, we show that the COOH of nephrin interacts with the vesicular SNARE protein VAMP2 in vitro and ex vivo (using yeast-2 hybrid and coimmunoprecipitation studies). This work demonstrates a previously unsuspected role of nephrin in vesicular docking and insulin responsiveness of podocytes.

Topics: Biological Transport; Child; Deoxyglucose; Humans; Kidney Failure, Chronic; Kidney Glomerulus; Membrane Proteins; Nephrotic Syndrome; Podocytes; RNA, Small Interfering; Vesicle-Associated Membrane Protein 2

Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) is a rare autosomal recessive disease caused by mutations in the NPHS1 gene encoding nephrin. We diagnosed congenital nephrotic syndrome in 12 children living in a village near Jerusalem. Most of the inhabitants are descendants of one Muslim family and have maintained their isolation by preference of consanguineous marriages. The aim of this study was to confirm that the NPHS1 gene is responsible for congenital nephrotic syndrome in our population, applying homozygosity mapping.. DNA samples were genotyped by four microsatellite markers that were in linkage disequilibrium with the NPHS1 gene on chromosome 19q13.1. Immunoperoxidase staining was used to study the expression of nephrin, and mutations were subsequently identified by direct sequencing of the entire coding region of the NPHS1 gene.. Haplotype analysis revealed several different haplotypes, leading us to assume erroneously that there was genetic heterogeneity of congenital nephrotic syndrome. Because nephrin was completely absent in kidney tissue of one patient, direct sequencing of all DNA samples was performed, yielding three novel mutations: c.1138C>T (p.Gln380X), c.2160_ 2161insC (p.Cys721fs), and c.1707C>G (p.Ser569Arg). Patients were either homozygous for one of these mutations or compound heterozygotes, and they differed in their phenotype.. We report the potential pitfalls of performing homozygosity mapping in a highly consanguineous population and discuss the phenomenon of multiple mutations in a given gene within an isolate.

Topics: Chromosomes, Human, Pair 19; Consanguinity; Female; Genetic Diseases, Inborn; Humans; Linkage Disequilibrium; Male; Membrane Proteins; Nephrotic Syndrome; Pedigree; Point Mutation

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

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

Recurrent nephrotic syndrome (NS) is a severe problem after renal transplantation in patients with congenital nephrotic syndrome of the Finnish type (NPHS1). The NPHS1 kidneys do not express nephrin, and antibodies against this major glomerular filter protein have been observed in NPHS1 children with recurrent NS. We evaluated here the use of plasma exchange (PE) therapy and kidney retransplantation in NPHS1 patients with recurrent NS and extended our studies on the pathogenesis of the recurrence.. Clinical data on 65 NPHS1 patients who received 77 kidney transplants between the years 1986 and 2006 was collected. Serum anti-nephrin antibodies were assayed with an enzyme-linked immunosorbent assay method, and the kidney biopsy samples were evaluated by light microscopy and immunohistochemistry.. Twenty-three episodes of recurrent NS occurred in 19 grafts of 13 NPSH1 patients homozygous for Fin-major mutation. Six retransplantations were performed to four NPHS1 patients, who lost their graft because of recurrent NS, and heavy proteinuria developed immediately in all cases. Although 73% of the patients had detectable serum anti-nephrin antibodies, the kidney biopsy findings were minimal. Introduction of PE alongside cyclophosphamide proved effective in the treatment of the proteinuric episodes (one graft loss out of nine). If remission was achieved, recurrent NS did not significantly deteriorate the long term graft function.. The clinical and pathological data suggest that anti-nephrin antibodies effectively impair the glomerular function in kidney grafts of NPHS1 patients homozygous for Fin-major mutation. Plasma exchange is a useful adjunct to the treatment of the recurrent NS.

Topics: Autoantibodies; Child; Child, Preschool; Finland; Follow-Up Studies; Glomerular Filtration Rate; Humans; Kidney Transplantation; Membrane Proteins; Mutation; Nephrotic Syndrome; Plasma Exchange; Recurrence; Reoperation; Retrospective Studies; Treatment Outcome

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

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

IFN-inducible protein-10 (IP-10/CXCL10) is a potent chemoattractant for activated T lymphocytes and was reported recently to have several additional biologic activities. In this study, the pathophysiologic role of IP-10 in the glomerular visceral epithelial cell (podocyte) was investigated. In cultured podocytes subjected to recombinant IP-10 treatment, the expression of slit-diaphragm (SD) components nephrin and podocin clearly was heightened. Rats that had puromycin aminonucleoside nephropathy and anti-nephrin antibody-induced nephropathy and were subjected to anti-IP-10 function-blocking antibody (anti-IP-10 mAb) treatment displayed a decrease in the protein level of SD components, as well as exacerbated proteinuria. For exploration of the mechanisms of this process, the interaction between IP-10 and the cell-cycle regulatory proteins was investigated. Cultured podocytes subjected to recombinant IP-10 treatment displayed an increase in the protein level of p27(Kip1), whereas the levels of cyclins E and A decreased. The expression of IP-10 and SD components was heightened by the treatment of siRNA of cyclin A, whereas these expressions were lowered by the treatment of siRNA of p27(Kip1). Proteinuric rats subjected to anti-IP-10 mAb treatment displayed a heightened expression of cyclin A from the early phase of the disease, which indicates that the anti-IP-10 mAb treatment exacerbates podocyte injury by disturbing the cell-cycle balance. These results raise the possibility that IP-10 could become a novel therapeutic target in nephrotic syndrome and several diseases with altered cell-cycle balance.

Topics: Animals; Cell Culture Techniques; Cell Cycle; Chemokine CXCL10; Chemokines, CXC; Disease Models, Animal; Female; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nephrotic Syndrome; Podocytes; Rats; Rats, Wistar; Receptors, Chemokine; Receptors, CXCR3

The abnormality of a single podocyte molecule, caused by a single gene mutation, such as NPHS1, NPHS2, CD2AP, and ACTN4, can lead to the hereditary/congenital nephrotic syndromes (NS). Further studies suggested that more than one podocyte molecule were together involved in acquired or experimental NS. However, we do not know much on the relationship among these podocyte molecules, and the molecular response induced by the change of each podocyte protein to the remaining ones. We respectively knockdown the nephrin, podocin, CD2AP, or alpha-actinin-4 mRNA by using reconstructed RNA interference vector--psiRNA-hH1GFPzeo in mouse podocyte clone. The molecular behavior or response was revealed by the quantitative expression both at mRNA and protein levels with RT-PCR and Western blot, and by the molecular distribution detected with confocal microscopy. With nephrin knockdown, only CD2AP increased, whereas podocin showed no change. Contrarily, with podocin or CD2AP knockdown, nephrin decreased, while CD2AP or podocin increased. Nephrin, podocin, or CD2AP knockdown did not change the expression of alpha-actinin-4, whereas alpha-actinin-4 knockdown begetted the reduction of nephrin, and the increment of podocin and CD2AP. The redistributions of nephrin, podocin, and CD2AP were revealed around a predominant nuclear staining compared with the membrane surface staining in the control podocytes. Our data imply that the response between the four podocyte molecules is very complicated and evidently different. There is not always an interaction between podocyte molecules. The normal localization of podocyte molecules would depend on their normal expression quantity and the molecular reactions between them.

Topics: Actinin; Adaptor Proteins, Signal Transducing; Animals; Base Sequence; Cloning, Molecular; Cytoskeletal Proteins; Genetic Vectors; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Molecular Sequence Data; Mutation; Nephrotic Syndrome; Podocytes; Proteins; RNA, Messenger

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

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

The glomerular filtration barrier in the kidney is formed in part by a specialized intercellular junction known as the slit diaphragm, which connects adjacent actin-based foot processes of kidney epithelial cells (podocytes). Mutations affecting a number of slit diaphragm proteins, including nephrin (encoded by NPHS1), lead to renal disease owing to disruption of the filtration barrier and rearrangement of the actin cytoskeleton, although the molecular basis for this is unclear. Here we show that nephrin selectively binds the Src homology 2 (SH2)/SH3 domain-containing Nck adaptor proteins, which in turn control the podocyte cytoskeleton in vivo. The cytoplasmic tail of nephrin has multiple YDxV sites that form preferred binding motifs for the Nck SH2 domain once phosphorylated by Src-family kinases. We show that this Nck-nephrin interaction is required for nephrin-dependent actin reorganization. Selective deletion of Nck from podocytes of transgenic mice results in defects in the formation of foot processes and in congenital nephrotic syndrome. Together, these findings identify a physiological signalling pathway in which nephrin is linked through phosphotyrosine-based interactions to Nck adaptors, and thus to the underlying actin cytoskeleton in podocytes. Simple and widely expressed SH2/SH3 adaptor proteins can therefore direct the formation of a specialized cellular morphology in vivo.

Topics: Actins; Adaptor Proteins, Signal Transducing; Amino Acid Motifs; Animals; Cell Line; Cytoskeleton; Humans; Kidney; Membrane Proteins; Mice; Mutation; Nephrotic Syndrome; Oncogene Proteins; Phosphorylation; Phosphotyrosine; src Homology Domains

Congenital nephrotic syndrome of the Finnish type, due to homozygous mutation of NPHS1, is the most common form of congenital nephrotic syndrome. Angiotensin converting enzyme (ACE) and prostaglandin synthesis inhibition along with supportive albumin infusion therapy, with or without unilateral nephrectomy, has allowed management of the disease without dialysis until transplantation in some cases of congenital nephrotic syndrome. Reported here is a case of heterozygous NPHS1 mutation, with normal NPHS2 gene structure, presenting during prenatal screening and developing nephrotic syndrome within days of birth. The patient has responded well to very low doses of ACE inhibitors and indomethacin alone. This case illustrates the importance of an initial trial of conservative medical therapy in milder presentations of the congenital nephrotic syndrome, especially given the current limitations of diagnostic testing and our inadequate knowledge of the complete spectrum of disorders of podocyte proteins.

Topics: Adult; Angiotensin-Converting Enzyme Inhibitors; Female; Heterozygote; Humans; Infant, Newborn; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Prenatal Diagnosis

Congenital nephrotic syndrome of the Finnish type (NPHS1) is a rare genetic disease caused by mutations in the NPHS1 gene encoding a major podocyte slit-diaphragm protein, nephrin. Patients with NPHS1 have severe nephrotic syndrome from birth and develop renal fibrosis in early childhood. In this work, we studied the development of glomerular sclerosis in kidneys removed from 4- to 44-month-old NPHS1 patients. The pathological lesions and expression of glomerular cell markers were studied in nephrectomized NPHS1 and control kidneys using light and electron microscopy and immunohistochemistry. An analysis of 1528 glomeruli from 20 patients revealed progressive mesangial sclerosis and capillary obliteration. Although few inflammatory cells were detected in the mesangial area, paraglomerular inflammation and fibrosis was common. The podocytes showed severe ultrastructural changes and hypertrophy with the upregulation of cyclins A and D1. Podocyte proliferation, however, was rare. Apoptosis was hardly detected and the expression of antiapoptotic B-cell lymphoma-2 and proapoptotic p53 were comparable to controls. Moderate amounts of podocytes were secreted into the urine of NPHS1 patients. Shrinkage of the glomerular tuft was common, whereas occlusion of tubular opening or protrusion of the glomerular tuft into subepithelial space or through the Bowman's capsule were not detected. The results indicate that, in NPHS1 kidneys, the damaged podocytes induce progressive mesangial expansion and capillary obliteration. Podocyte depletion, glomerular tuft adhesion, and misdirected filtration, however, seem to play a minor role in the nephron destruction.

Topics: Apoptosis; Cell Proliferation; Child, Preschool; Disease Progression; Epithelium; Glomerular Mesangium; Humans; Hypertrophy; Infant; Kidney Glomerulus; Membrane Proteins; Mutation; Nephrotic Syndrome; Podocytes; Sclerosis

Topics: Adolescent; Asia; Child; Child, Preschool; Drug Resistance; Female; Humans; Infant; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Nephrotic Syndrome; Prednisolone

Nephrotic syndrome (NS) in infancy includes NS of Finnish type (mutation of the nephrin gene), diffuse mesangial sclerosis (idiopathic or linked to WT1 mutation), idiopathic NS, most often steroid resistant, and NS related to infections during pregnancy (virus, syphilis, toxoplasmosis). Later in life, NS has a large variety of etiologies. It has been described in association with neuromuscular symptoms, deafness, and diabetes in a few children and adults with respiratory chain (RC) disorders. To date, however, NS has never been observed in neonates with RC disorders. Here, we report RC deficiency in one infant with certain congenital NS and two siblings with acute neonatal cardiac and renal disease with probable NS. Although clinical and histopathological presentations were initially close to congenital NS of Finnish type, clinical outcome was atypical and nephrin mutation was excluded. Mitochondrial RC complex II+V deficiency was identified in the three patients. Based on these observations, we suggest that RC disorders should be considered in patients with congenital NS.

Topics: Biopsy; Diagnosis, Differential; Female; Humans; Infant, Newborn; Kidney; Male; Membrane Proteins; Mitochondrial Diseases; Nephrotic Syndrome; Proteins

Congenital nephrotic syndrome (CNS) causes significant renal failure, and is classified into two types: (1) Finnish type; and (2) other, including diffuse mesangial sclerosis. Mutations of NPHS1 and NPHS2, which encode the slit diaphragm components nephrin and podocin, cause CNS and autosomal-recessive familial steroid-resistant nephrotic syndrome, respectively. Most patients with Finnish-type CNS in Europe and the United States have NPHS1 mutations. However, NPHS2 mutations have been detected in some cases. Mutations in ACTN4, encoding alpha-actinin-4, cause an autosomal-dominant focal segmental glomerulosclerosis. alpha-actinin-4 stabilizes the podocyte cytoskeleton structure, connecting with actin filaments. WT1 mutations, causing Wilm's tumor, have been demonstrated in some CNS patients with diffuse mesangial sclerosis. Systematic investigation of genes for CNS in Japan has never been performed.. To clarify the role of mutations in these four genes, we used polymerase chain reaction (PCR) and direct sequencing to investigate all exons and exon-intron boundaries for these genes in 13 unrelated CNS patients from regional pediatric kidney disease centers in Japan.. A novel homozygous nonsense mutation of NPHS1, E246X in exon 7, and a novel homozygous deletion mutation of NPHS1, 2156_2163del in exon 16 were detected in one patient each. A novel homozygous nonsense mutation of NPHS2, R196X in exon 5, was found in one patient, and the same heterozygous nonsense mutation was detected in another. No ACTN4 or WT1 mutations were detected.. These studies demonstrate that mutation of NPHS1 is not a major cause of CNS in Japanese patients, and that mutation of NPHS2 can be responsible for CNS in this population.

Topics: Actinin; Base Sequence; Codon, Nonsense; DNA Primers; Finland; Humans; Intracellular Signaling Peptides and Proteins; Japan; Membrane Proteins; Microfilament Proteins; Nephrotic Syndrome; Racial Groups; United States; WT1 Proteins

Congenital nephrotic syndrome of the Finnish type (CNF) is a rare autosomal recessive disorder, caused by mutations in the NPHS1 gene, coding for nephrin. The aim of this work was to investigate the disease mutations in a CNF Italian family and to perform genetic prenatal diagnosis in the second pregnancy.. Polymerase chain reaction (PCR) and automatic sequence analysis were used to screen the CNF Italian family for NPHS1 mutations.. Two novel heterozygous mutations, including a single nucleotide insertion (c.248insA) and a missense mutation (p.S572N), were detected in the proband. Molecular prenatal diagnosis was performed on fetal DNA sample: the fetus resulted compound heterozygous for the same proband mutations.. To the best of our knowledge, this is the first report of a molecular prenatal diagnosis performed in an Italian family with congenital nephrotic syndrome of Finnish type (CNF). Our findings indicate that, even though CNF is not very common outside Finland, availability and reliability of DNA diagnostics are important issues to confirm the AFP results in prenatal diagnosis.

Topics: Adult; Child, Preschool; Female; Finland; Humans; Italy; Male; Membrane Proteins; Mutation, Missense; Nephrotic Syndrome; Pedigree; Pregnancy; Prenatal Diagnosis; White People

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

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

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

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

The identification of the underlying gene defect in some cases of steroid resistant nephrotic syndrome (SRNS) has recently led to a critical breakthrough in the understanding of the pathogenesis of nephrotic syndromes. The more severe form of hereditary nephrotic syndromes is the congenital nephrotic syndrome of the Finnish type (CNF). The causative gene, NPHS1, encodes a novel protein, nephrin which is a transmembrane protein belonging to the immunoglobulin superfamily specifically expressed in the podocyte at the slit diaphragm. Using a positional cloning approach, our group identified a gene, NPHS2, involved in a specific entity of familial SRNS characterized by early onset, complete steroid-resistance, rapid progression to ESRD and no recurrence after renal transplantation. NPHS2 encodes a novel membrane protein named podocin localized at the cytoplasmic part of the slit diaphragm. Familial autosomal dominant cases of primary FSGS have been described in adulthood. Two corresponding genes have been mapped to date, one to 19q13 and the second to 11q21-22. The former has been identified as ACTN4, the gene encoding the actin-binding protein, a-actinin 4. Other genes involved in the slit-diaphragm or the nephrotic syndrome are CD2-associated protein (CD2AP), FAT1, WT1, LMX1B, SMARCAL1. Altogether, these data demonstrate the pivotal role of the podocyte in the development and the maintenance of the glomerular filtration barrier and the crucial role of the genetic factors in the development of SRNS.

Topics: Actinin; Adaptor Proteins, Signal Transducing; Adult; Child; Cytoskeletal Proteins; Disease Progression; Drug Resistance; Humans; Intracellular Signaling Peptides and Proteins; Kidney Failure, Chronic; Kidney Transplantation; Membrane Proteins; Microfilament Proteins; Mutation; Nephrotic Syndrome; Proteins; Steroids

Human immunodeficiency virus (HIV)-1-associated nephropathy (HIVAN) is characterized by collapsing focal and segmental glomerulosclerosis (FSGS) and microcystic tubular dilatation. HIV-1 infection is also associated with other forms of nephropathy, including mesangial hyperplasia. Since HIV-1 gene products are detected in podocytes and other renal cells, it remains uncertain whether podocyte-restricted HIV-1 gene expression can account for the full spectrum of renal lesions involving nonpodocytes.. To define the role of podocyte-restricted HIV-1 gene expression in the progression of HIVAN, we generated transgenic mice that express nonstructural HIV-1 genes selectively in podocytes.. Four of the seven founder mice developed proteinuria and nephropathy. In a subsequently established transgenic line, reverse transcription-polymerase chain reaction (RT-PCR) analysis detected mRNAs for vif, vpr, nef, and spliced forms of tat and rev, but not vpu, in the kidney. In situ hybridization localized HIV-1 RNA to the podocyte. Transgenic mice on FVB/N genetic background exhibited cuboidal morphology of podocytes with reduced extension of primary and foot processes at 2 weeks of age. After 3 weeks of age, these mice developed massive and nonselective proteinuria with damage of podocytes and other glomerular cells and, after 4 weeks of age, collapsing FSGS and microcystic tubular dilatation. In marked contrast, transgenic mice with C57BL/6 genetic background showed either normal renal histology or only mild mesangial expansion without overt podocyte damage.. The present study demonstrates that podocyte-restricted expression of HIV-1 gene products is sufficient for the development of collapsing glomerulosclerosis in the setting of susceptible genetic background.

Topics: AIDS-Associated Nephropathy; Animals; Female; Gene Expression Regulation, Viral; Genetic Predisposition to Disease; HIV-1; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nephrotic Syndrome; Podocytes; Promoter Regions, Genetic; Transgenes

Congenital nephrotic syndrome (CNS) is defined as heavy proteinuria or nephrotic syndrome occurring before 3 months of age. It is characterized by early onset, resistance to steroid therapy and progressing to end-stage renal disease (ESRD). In recent years, several genes associated with CNS have been identified, such as NPHS1, NPHS2 and WT1. The mutations of these genes have been identified in the patients with CNS in Finland, other European countries, North Africa, North America, and Asia, respectively. However, the investigation of the above genes has not been performed in Chinese CNS patients. In this study, NPHS1 mutations in a Chinese family with CNS were detected and analyzed.. There were two CNS patients in the investigated family. The proband, a 45-day-old boy, was born at fullterm and weighed 2700 g at birth. The placenta weighed 450 g. At the age of 10 days, generalized edema, proteinuria, hypoproteinemia, and hypoalbuminemia were found without renal insufficiency. The proband's sister, with the same phenotype and normal renal function, underwent renal biopsy at 5 years of age. Their parents and elder half-sister all had normal phenotypes. Genomic DNA samples were extracted from peripheral bloods of the proband, his family members and 50 unrelated, normal individuals. All 29 exons and exon-intron boundaries of NPHS1 were detected in the proband by polymerase chain reaction (PCR), direct DNA sequencing, and restriction enzyme analysis.. Three heterozygous mutations of NPHS1, namely, G928A (D310N), 1893-1900del 8 (CGAAACCG), and G2869C (V957L) were identified in the proband. These mutations involved exons 8, 14, and 21. The same genotype was found in the proband's sister who had the same phenotype, but was not detected in proband's elder half-sister who had normal phenotype. Fifty normal individuals had no these mutations. The proband's mother with normal urinalysis had G928A (D310N) heterozygous mutation, and the father with normal urinalysis had two heterozygous mutations of 1893-1900del 8 (CGAAACCG) and G2869C (V957L). At the same time, three types of single nucleotide polymorphisms (SNPs), E117K (rs3814995), S1105S (rs2071327), and IVS27+45c > t, were confirmed in the proband. Another variant, IVS8+68 a > g had also been found.. This is the first report about NPHS1 mutations in Chinese CNS kindred. These three heterozygous mutations of NPHS1 are novel genetic defects of CNS, which have not been described before.

Topics: Asian People; Exons; Genotype; Humans; Infant; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Pedigree; Phenotype; Polymerase Chain Reaction; Sequence Analysis, DNA

Nephrin is a major component of the glomerular filtration barrier. Mutations in the nephrin gene (NPHS1) are responsible for congenital nephrotic syndrome of the Finnish type (NPHS1). Nephrin was at first thought to be podocyte specific, but recent studies have suggested that nephrin is also expressed in nonrenal tissues such as pancreas and CNS. We studied the expression of nephrin in human and porcine tissues at different stages of development and correlated these findings to clinical characteristics of NPHS1 children. Immunofluorescence staining and Western blotting were used to detect nephrin protein in frozen tissue samples. Polyclonal antibodies against the intracellular part of nephrin were used in these analyses. In situ hybridization was used to detect nephrin mRNA in specimens from normal human subjects and patients with NPHS1. Nephrin protein was not detected in nonrenal tissues obtained from human and porcine fetuses, newborns, and infants. Likewise, nephrin mRNA expression was not observed outside kidney glomerulus in normal or NPHS1 children. The phenotype analysis of NPHS1 children with severe nephrin gene mutations supported the findings in the tissue expression studies and revealed no impairment of the neurologic, testicular, or pancreatic function in a great majority of the patients. The studies suggest that nephrin has no major clinical significance outside the kidney.

Topics: Animals; Blotting, Western; Down Syndrome; Female; Glomerular Filtration Rate; Glucose; Humans; In Situ Hybridization; In Situ Hybridization, Fluorescence; Infant; Infant, Newborn; Kidney; Male; Membrane Proteins; Microscopy, Fluorescence; Middle Aged; Mutation; Nephrotic Syndrome; Protein Biosynthesis; Proteins; Species Specificity; Swine; Testis; Time Factors; Tissue Distribution

Autosomal recessive steroid-resistant nephrotic syndrome (SRNS) is a subgroup of familial nephrotic syndrome. A causative gene has been identified, that is NPHS2, in chromosome 1q25-31, which encodes podocin. This study aimed to detect NPHS2 mutation in a Chinese family with SRNS.. Renal biopsy was performed on the proband and her sibling for routine histologic and immunohistochemical investigation and electron microscopic examination. The expressions of podocin, nephrin, alpha-actinin and WT1 in glomeruli of the proband were detected by indirect immunofluorescence. Peripheral blood samples were collected for genetic analysis from the proband and her parents, and 53 adults with normal urinalysis. Genomic DNA was isolated from peripheral blood leucocytes. Eight exons of NPHS2 were amplified by polymerase chain reaction. Mutational analysis was performed using denaturing high-performance liquid chromatography (DHPLC) and DNA fragments with aberrant elution profiles of both strands revealed by DHPLC were re-amplified and sequenced directly.. The histologic findings on kidney biopsies were focal segmental glomerulosclerosis. In controls, the distribution of staining with P35, rabbit against a human podocin recombinant protein (amino acids 135 - 383 = all the C-terminal part of the protein downstream the transmembrane domain), and P21, rabbit against a human podocin recombinant protein (amino acids 15 - 89 = all the N-terminal part of the protein upstream the transmembrane domain) showed a linear pattern along glomerular capillary walls on glomeruli, and the fluorescent intensity of the staining with P35 was intensely positive. The fluorescent intensity of the staining with P21 was positive. In the proband, the distribution of the staining with P35 showed uneven and nonlinear, and the fluorescent intensity of the staining with P35 was weakly positive. The staining with P21 was negative. The area, location, distribution and fluorescent intensity of the staining with nephrin, alpha-actinin and WT1 on glomeruli of the proband were the same as those in the controls. The DHPLC elution profiles of exon 4 of NPHS2 from the proband and her parent were aberrant. The chromatograms by sequencing detected in the exon 4 of NPHS2 showed a composite heterozygous mutation of both 467_468insT and 503G > A in the proband, a heterozygous mutation of 503G > A in her father, and a heterozygous mutation of 467_468insT in her mother, respectively.. The study demonstrated for the first time a novel mutation, 503G > A, of NPHS2 in Chinese kindred with autosomal recessive SRNS. A significantly decreased or negative expression was also revealed in glomeruli of the proband stained with two kinds of anti-podocin antibodies.

Topics: Actinin; Adult; Aged; Base Sequence; Child; DNA Mutational Analysis; Drug Resistance; Female; Fluorescent Antibody Technique, Direct; Humans; Infant; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Pedigree; Polymerase Chain Reaction; Proteins; WT1 Proteins

Kidney podocytes and their slit diaphragms form the final barrier to urinary protein loss. This explains why podocyte injury is typically associated with nephrotic syndrome. The present study uncovered an unanticipated novel role for costimulatory molecule B7-1 in podocytes as an inducible modifier of glomerular permselectivity. B7-1 in podocytes was found in genetic, drug-induced, immune-mediated, and bacterial toxin-induced experimental kidney diseases with nephrotic syndrome. The clinical significance of our results is underscored by the observation that podocyte expression of B7-1 correlated with the severity of human lupus nephritis. In vivo, exposure to low-dose LPS rapidly upregulates B7-1 in podocytes of WT and SCID mice, leading to nephrotic-range proteinuria. Mice lacking B7-1 are protected from LPS-induced nephrotic syndrome, suggesting a link between podocyte B7-1 expression and proteinuria. LPS signaling through toll-like receptor-4 reorganized the podocyte actin cytoskeleton in vitro, and activation of B7-1 in cultured podocytes led to reorganization of vital slit diaphragm proteins. In summary, upregulation of B7-1 in podocytes may contribute to the pathogenesis of proteinuria by disrupting the glomerular filter and provides a novel molecular target to tackle proteinuric kidney diseases. Our findings suggest a novel function for B7-1 in danger signaling by nonimmune cells.

Topics: Actins; Animals; B7-1 Antigen; Base Sequence; DNA; Humans; In Vitro Techniques; Integrin alpha3; Kidney; Lipopolysaccharides; Lupus Nephritis; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Knockout; Mice, SCID; Nephrotic Syndrome; Proteins; Receptors, Cell Surface; Signal Transduction; Toll-Like Receptor 4; Toll-Like Receptors

Mutations in NPHS2, encoding podocin, are a prevalent cause of autosomal-recessive steroid-resistant nephrotic syndrome (SRNS). Podocin is a protein associated with the slit diaphragm that interacts with nephrin and CD2-associated protein (CD2AP) within lipid rafts.. Using renal biopsies of six patients, we analyzed the in vivo consequences of different types of NPHS2 mutations on (1) the podocyte expression and distribution of podocin using in situ hybridization and immunohistology and (2) the distribution of related podocyte proteins and glomerular extracellular matrix components.. In two patients with homozygous 855_856delAA or 419delG mutation, absence of podocyte labeling with the antibodies against the C-terminal domain contrasted with the normal expression of the N-terminal domain of the protein along the glomerular basement membrane (GBM). In patients carrying compound heterozygous mutations or variants (R168S/467_468insT, R138Q/V180M, and R291W/R229Q), or single heterozygous 976_977insA, podocin transcription appeared unchanged but the distribution of the protein was modified. Podocin was restricted to the podocyte body in the patient carrying the R168S/467_468insT mutation whereas strong immunolabeling of the podocyte body was associated with discrete labeling along the GBM in the three others. In all cases, podocin defect was associated with changes in the distribution of nephrin, CD2AP, and alpha-actinin: the proteins were mainly detected in the podocyte body, with mild expression along the GBM. There were no detectable changes in the distribution of other podocyte proteins or glomerular extracellular matrix components.. NPHS2 mutations result in profound alteration of podocin expression and/or distribution. Secondary changes in the distribution of nephrin, CD2AP, and alpha-actinin are additional evidences for the scaffolding role of podocin in the organization of the slit diaphragm.

Topics: Actinin; Adaptor Proteins, Signal Transducing; Adolescent; Child; Child, Preschool; Cytoskeletal Proteins; Extracellular Matrix Proteins; Female; Fluorescent Antibody Technique; Gene Expression; Homozygote; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Nephrotic Syndrome; Point Mutation; Proteins

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

Podocin is a membrane-integrated protein that is located at the glomerular slit diaphragm and directly interacts with nephrin. The gene encoding podocin, NPHS2, is mutated in patients with autosomal-recessive steroid-resistant nephrotic syndrome (SRN). In order to study a potential pathomechanism of massive proteinuria in patients with SRN, we have investigated the trafficking and subcellular localization of five common disease-causing missense mutants of human podocin.. Site-directed mutagenesis was applied to generate cDNA constructs encoding five different missense mutations of human podocin (P20L, G92C, R138Q, V180M, and R291W). To identify the subcellular localization of each mutant in transfected human embryonic kidney (HEK)293 cells, we have generated and characterized a rabbit polyclonal antibody against the human podocin. Specificity of the antibody was determined by light and immunoelectron microscopy, as well as immunoblot analysis using human glomeruli. Confocal microscopy was applied to determine subcellular localization of the wild-type and the mutated podocin molecules, as well as wild-type nephrin in transfected cells. Immunoprecipitation and pull-down studies were carried out to investigate the molecular interaction of podocin mutants and wild-type nephrin.. Immunofluorescence and confocal microscopy showed that wild-type podocin located to the plasma membrane when expressed in HEK293 cells. Two missense mutations, P20L and G92C, located at the N-terminus part of the molecule, were also present at the plasma membrane, indicating that these mutations did not affect the subcellular localization of the mutated podocin molecules. In contrast, subcellular localization of three other missense mutants located in the proximal C-terminus part of the protein was drastically altered, in which R138Q was retained in the endoplasmic reticulum (ER), V180M formed inclusion bodies in the cytoplasm, and the R291W mutant was trapped both in the ER and in small intracellular vesicles. Interestingly, this abnormal subcellular localization of podocin missense mutants also resulted in alteration in protein trafficking of wild-type nephrin in cotransfected cells through the strong protein binding between both molecules.. In patients with SRN, some missense mutations in the NPHS2 gene not only lead to misfolding and mislocalization of the mutated podocin, but they can also interfere with slit diaphragm structure and function by altering the proper trafficking of nephrin to the plasma membrane.

Topics: Cell Line; Cell Membrane; Drug Resistance; Genes, Recessive; Humans; Immunologic Techniques; Intracellular Signaling Peptides and Proteins; Kidney; Membrane Proteins; Microscopy, Immunoelectron; Mutation, Missense; Nephrotic Syndrome; Protein Transport; Proteins; Steroids; Subcellular Fractions; Tissue Distribution; Transfection

Nephrin is a cell-adhesion protein that is defective in congenital nephrotic syndrome of the Finnish type (CNF). Nephrin is synthesized by the podocytes and is localized to the slit membrane between individual foot processes of the podocytes. Although nephrin is apparently pivotal in the development of CNF, the role of nephrin in other causes of nephrotic syndrome is not fully understood.. Renal biopsy specimens from patients with minimal change nephrotic syndrome (MCNS) were investigated. Nephrin distribution was studied with immunohistochemical and semiquantitative immunoelectron microscopic techniques and the results were related to the degree of foot process effacement found under the electron microscope.. In normal kidney, immunofluorescence revealed a linear staining along the capillary basement membranes, corresponding to the localization of nephrin in the slit membranes. In the biopsies from patients with MCNS, the nephrin pattern had become granular. The degree of granularization corresponded to the degree of foot process effacement. Ultrastructural semiquantification showed the amount of nephrin to be reduced both in areas with and without foot process effacement compared with the control specimens. The concentration of nephrin was lowest in the areas with foot process effacement and there was redistribution from the slits into the cytoplasm.. These findings demonstrate that nephrin expression is altered in MCNS. Whether this reflects a pathogenetic role for nephrin in MCNS or a phenomenon secondary to other causes of foot process effacement remains to be elucidated.

Topics: Adult; Aged; Child; Child, Preschool; Humans; Kidney Glomerulus; Membrane Proteins; Microscopy, Immunoelectron; Middle Aged; Nephrotic Syndrome; Proteins

We studied the distribution of nephrin in renal biopsies from 17 patients with diabetes and nephrotic syndrome (7 type 1 and 10 type 2 diabetes), 6 patients with diabetes and microalbuminuria (1 type 1 and 5 type 2 diabetes), and 10 normal subjects. Nephrin expression was semiquantitatively evaluated by measuring immunofluorescence intensity by digital image analysis. We found an extensive reduction of nephrin staining in both type 1 (67 +/- 9%; P < 0.001) and type 2 (65 +/- 10%; P < 0.001) diabetic patients with diabetes and nephrotic syndrome when compared with control subjects. The pattern of staining shifted from punctate/linear distribution to granular. In patients with microalbuminuria, the staining pattern of nephrin also showed granular distribution and reduction intensity of 69% in the patient with type 1 diabetes and of 62 +/- 4% (P < 0.001) in the patients with type 2 diabetes. In vitro studies on human cultured podocytes demonstrated that glycated albumin and angiotensin II reduced nephrin expression. Glycated albumin inhibited nephrin synthesis through the engagement of receptor for advanced glycation end products, whereas angiotensin II acted on cytoskeleton redistribution, inducing the shedding of nephrin. This study indicates that the alteration in nephrin expression is an early event in proteinuric patients with diabetes and suggests that glycated albumin and angiotensin II contribute to nephrin downregulation.

Topics: Adult; Aged; Albuminuria; Angiotensin II; Biopsy; Blotting, Western; Cells, Cultured; Cytochalasin B; Cytoskeleton; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Fluorescent Antibody Technique, Indirect; Gene Expression; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Kidney; Male; Membrane Proteins; Microscopy, Fluorescence; Middle Aged; Nephrotic Syndrome; Proteins; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serum Albumin; Tissue Distribution

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

With the recent molecular findings, the podocyte is emerging as a key cell type involved in glomerular damage, but protein complexes involved remain poorly understood. To systematically search for additional podocyte molecules interacting with nephrin, a key structural molecule of the interpodocyte filtration slit, precipitation of glomerular lysates was set out with anti-nephrin antibodies to identify members of the nephrin-associated protein complex. Proteins of the precipitate were subsequently identified with MALDI-TOF mass analysis. One of the proteins thus obtained showed identity with densin, a protein originally purified from rat forebrain postsynaptic density fraction and so far shown to be highly brain-specific. The expression of densin appeared distinctly in the glomerulus and cultured podocytes by RT-PCR. Immunoblotting studies revealed a specific band of 185 kD in brain and cultured podocytes; in human glomerulus, densin appeared as a 210-kD band. By immunocytochemistry, densin localizes in glomeruli in a podocyte-like pattern. Electron microscopic studies revealed densin localization in the slit diaphragm area. Due to its known involvement in the synaptic organization, maintenance of cell shape and polarity in nerve cells, together with its demonstrated interactions with alpha-actinin-4, densin may share the same functions in podocytes by associating with the nephrin interacting protein complex at the slit diaphragm.

Topics: Actinin; Animals; Brain; Cells, Cultured; Epithelial Cells; Humans; Immunoblotting; Immunohistochemistry; Kidney Glomerulus; Membrane Proteins; Microfilament Proteins; Microscopy, Electron; Microscopy, Immunoelectron; Nephrotic Syndrome; Precipitin Tests; Prosencephalon; Proteins; Rats; Reverse Transcriptase Polymerase Chain Reaction; Sialoglycoproteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

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

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

Recently, nephrin, podocin, alpha-actinin, and WT1, which are located at the slit diaphragm and expressed by the podocyte, were found to be causative in congenital/familial nephrotic syndrome (NS), but their role in acquired NS remains unclear. We studied their expression in NS with the aim of disclosing their possible role in the development of proteinuria. Immunofluorescence, confocal microscopy, and image analysis were used to study the expression and the distribution in 19 children with primary NS, 9 with isolated hematuria, and 9 controls. All the children with NS presented with heavy proteinuria and foot process effacement was identified by electron microscopy. No proteinuria and foot process effacement was seen in the group with hematuria. A dramatic decrease of podocin expression was found in NS (86.66+/-22.74) compared with control groups ( P=0.014). Furthermore, we also found the pattern of distribution of nephrin, podocin, and alpha-actinin changed in children with NS. In conclusion, a dramatic decrease of podocin expression and abnormal distribution of nephrin, podocin, and alpha-actinin were found in children with NS. No differences were found in children with isolated hematuria, suggesting involvement of these molecules in the development of proteinuria in primary NS.

Topics: Actinin; Adolescent; Case-Control Studies; Child; Female; Glomerulonephritis, IGA; Hematuria; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Nephrosis, Lipoid; Nephrotic Syndrome; Proteins; Tissue Distribution; WT1 Proteins

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

Hereditary nephrotic syndrome is a heterogeneous disease, characterized by heavy proteinuria and renal failure. Mutations of NPHS1 or NPHS2, the genes encoding for nephrin and podocin, lead to early onset of heavy proteinuria, and rapid progression to end-stage renal disease, suggesting that both proteins are essential for the integrity of the glomerular filter. Podocin is a stomatin protein family member with a predicted hairpin-like structure localizing to the insertion site of the slit diaphragm of podocytes, the visceral glomerular epithelial cells of the kidney. Here we investigate the pathomechanisms of different disease-causing podocin mutations. We show that wild-type podocin is targeted to the plasma membrane, and forms homo-oligomers involving the carboxy and amino terminal cytoplasmic domains. The association of podocin with specialized lipid raft microdomains of the plasma membrane was a prerequisite for recruitment of nephrin into rafts. In contrast, disease-causing mutations of podocin (R138Q and R138X) failed to recruit nephrin into rafts either because these mutants were retained in the endoplasmic reticulum (R138Q), or because they failed to associate with rafts (R138X) despite their presence in the plasma membrane. None of the mutants did augment nephrin signaling, suggesting that lipid raft targeting facilitates nephrin signaling. Our findings demonstrate that the failure of mutant podocin to recruit nephrin into lipid rafts may be essential for the pathogenesis of NPHS2.

Topics: Cell Membrane; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mutation; Nephrotic Syndrome; Plasmids; Proteins; Signal Transduction; Transfection

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

NPHS1, which encodes nephrin, recently has been identified as the gene in which mutations cause congenital nephrotic syndrome of the Finnish type (CNF). We previously reported novel missense mutations of NPHS1 in a Japanese patient with CNF. However, the mechanism by which these missense mutations cause the disorder remains to be clarified.. Wild-type nephrin and mutated nephrin complementary DNA were each tagged by the green fluorescence protein (GFP) gene; the expressing vectors of the fusion protein were each transfected to human embryonic kidney 293 cells. We compared intracellular localization of mutated nephrin with that of wild-type nephrin by using GFP and immunostaining examination.. In both wild-type and mutated nephrin (Glu(447)Lys), GFP and immunostaining resulted in a colocalized microgranular pattern along the cell membrane that indicated these recombinant proteins were located at the cell surface. Conversely, in mutated nephrin (Asp(819)Val), GFP aggregation was observed in the cytoplasm, and no fluorescence was observed at the cell membrane, indicating that recombinant mutated nephrin (Asp(819)Val) could not be distributed at the cell membrane and instead was retained in cytoplasm.. We confirmed that the missense mutation GAC-to-GTC transversion leading to an Asp(819)Val caused the disorder. The present study analyzes in vitro distribution of nephrin with a missense point mutation. The analysis uses a new convenient method, construction of a nephrin-GFP fusion protein.

Topics: Blotting, Western; Cell Line; Cell Membrane; Cytoplasm; Genetic Vectors; Green Fluorescent Proteins; Humans; Kidney; Luminescent Proteins; Membrane Proteins; Mutation, Missense; Nephrotic Syndrome; Protein Biosynthesis; Protein Sorting Signals; Proteins; Recombinant Fusion Proteins; Transfection

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

Topics: Humans; Kidney Transplantation; Membrane Proteins; Nephrotic Syndrome; Proteins; Recurrence

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

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

Molecules of central functional significance for the glomerular podocytes are rapidly emerging and have been shown to be distinctly involved in diseases with altered glomerular filtration barrier. Here we used the puromycin aminonucleoside (PA) nephrosis model in the rat to study some key proteins associated with the maintenance of the functional glomerular filtration barrier in vivo. The molecules studied included the filtration slit component nephrin, the hairpin-like membrane protein podocin, the basolateral adhesion molecules beta1 integrin and alpha-dystroglycan, and the cytoskeleton-linking intermediary beta-catenin and the actin-associated alpha-actinin-4. The results showed diminished protein levels of podocin and nephrin in the PA-treated group. beta-catenin showed distinct down-regulation at 3 days of induction, and the control level was reached at 10 days. beta1 integrin was markedly up-regulated during induction. alpha-actinin-4 was not changed at the studied time points. The results show distinct differences in the different domains of podocytes during PA-induced proteinuria.

Topics: Actinin; Albuminuria; Animals; beta Catenin; Cytoskeletal Proteins; Disease Models, Animal; DNA Primers; Dystroglycans; Female; Immunohistochemistry; Integrin beta1; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Glycoproteins; Membrane Proteins; Microfilament Proteins; Nephrotic Syndrome; Proteins; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators

This study was undertaken to study the applicability of genetic antenatal screening for the Finnish type of congenital nephrosis (CNF), which is a recessive disorder leading to nephrotic syndrome from birth. At Kuopio University Hospital, a total of 1303 pregnant women were offered carrier screening for CNF at the time of first trimester nuchal fold translucency measurement when fetally derived alpha-fetoprotein is still produced by the yolk sac. Two mutations of the nephrin (NPHS 1) gene, accounting for approximately 95% of affected alleles, were tested by two PCR tests. Uptake of the gene test was 91.0% (n=1183). Altogether 38 female carriers were found; a population carrier frequency of 1 in 31. Their partners were tested and two of them were also found to be carriers. In these two pregnancies invasive prenatal diagnosis was offered and accepted, and the results indicated one carrier and one affected fetus. Carrier screening is an effective and well-accepted method for antenatal screening for fetal CNF. Direct mutation analysis involves markedly less invasive procedures compared with serum alpha-fetoprotein (AFP) screening, and the diagnosis was clear-cut. The results indicate that in single-gene disorders genetic testing is suitable for antenatal screening.

Topics: Alleles; alpha-Fetoproteins; Costs and Cost Analysis; Female; Finland; Humans; Membrane Proteins; Mutation; Nephrotic Syndrome; Polymerase Chain Reaction; Pregnancy; Prenatal Diagnosis; Proteins

Congenital nephrotic syndrome, Finnish type (CNF or NPHS1), is an autosomal recessive disease characterized by massive proteinuria and development of nephrotic syndrome shortly after birth. The disease is most common in Finland, but many patients have been identified in other populations. The disease is caused by mutations in the gene for nephrin which is a key component of the glomerual ultrafilter, the podocyte slit diaphragm. A total of 30 mutations have been reported in the nephrin gene in patients with congenital nephrotic syndrome worldwide. In the Finnish population, two main mutations have been found. These two nonsense mutations account for over 94% of all mutations in Finland. Most mutations found in non-Finnish patients are missense mutations, but they include also nonsense and splice site mutations, as well as deletions and insertions. This mutation update summarizes the nature of all previously reported nephrin mutations and, additionally, describes 20 novel mutations recently identified in our laboratory.

Topics: Codon, Nonsense; DNA Mutational Analysis; Exons; Finland; Genes, Recessive; Genetic Testing; Humans; Membrane Proteins; Molecular Sequence Data; Mutation; Mutation, Missense; Nephrotic Syndrome; Phenotype; Polymorphism, Genetic; Proteins; RNA Splice Sites

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

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

Topics: Child; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nephrotic Syndrome; Proteins

NPHS1 has recently been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type. The respective gene product nephrin is a transmembrane protein expressed in glomerular podocytes and primarily localized to the glomerular slit diaphragm. This interpodocyte junction functions in the glomerular filtration by restricting the passage of plasma proteins into the urinary space in a size-selective manner. The functional role of nephrin in this filtration process is so far not very well understood. In this study, we show that nephrin associates in an oligomerized form with signaling microdomains, also known as lipid rafts, and that these localize to the slit diaphragm. We also show that the nephrin-containing rafts can be immunoisolated with the 27A antibody recognizing a podocyte-specific 9-O-acetylated GD3 ganglioside. In a previous study it has been shown that the in vivo injection of this antibody leads to morphological changes of the filtration slits resembling foot process effacement. Here, we report that, in this model of foot process effacement, nephrin dislocates to the apical pole of the narrowed filtration slits and also that it is tyrosine phosphorylated. We suggest that lipid rafts are important in the spatial organization of the glomerular slit diaphragm under physiological and pathological conditions.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Cell Membrane; Cholesterol; Detergents; Drug Resistance; Endoplasmic Reticulum; Female; Gangliosides; Immunoglobulin G; Kidney Glomerulus; Lipids; Membrane Proteins; Nephrotic Syndrome; Phosphorylation; Precipitin Tests; Proteins; Rats; Rats, Sprague-Dawley; Tyrosine

The novel gene NPHS1 is defective in the patients with congenital nephrotic syndrome of the Finnish type (CNF) leading to abnormal expression of the respective protein product nephrin in glomerular cells. CNF patients are treated with early nephrectomy and renal transplantation, but about 20% show recurrence of nephrotic syndrome (NS). We used indirect immunofluorescence microscopy and immunoblotting and an ELISA assay to search for circulating autoantibodies to nephrin, the protein defect in CNF patient kidneys. In serial serum samples gathered before and after recurrence of NS, we show an increased antibody titer to nephrin prior to the NS episode and a subsequent drop in antibody level after its successful treatment and reactivity of the high titer sera with glomeruli in indirect immunofluorescence microscopy as well. The results show that the transplantation treatment introduces a neoantigen inducing production of autoantibodies, which may be pathogenic for perturbation of the function of the glomerular filtration barrier.

Topics: Amino Acid Sequence; Autoantibodies; Child, Preschool; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Infant; Kidney; Kidney Transplantation; Male; Membrane Proteins; Molecular Sequence Data; Nephrotic Syndrome; Proteins; Recurrence

Congenital nephrotic syndrome of the Finnish type (CNF or NPHS1) is an autosomal recessive kidney disorder resulting in severe proteinurea and renal dysfunction. Although the disease occurs predominantly in the Finnish population, many cases in other populations have also been reported. The disease gene (NPHS1) encodes nephrin, a podocyte transmembrane protein that is an essential component of the podocyte slit diaphragm, the renal ultrafilter. Since the discovery of the gene, many mutations have been reported in the NPHS1 gene in patients with diverse ethnic background. A surprisingly large number of these mutations are missense mutations resulting in single amino acid substitutions. In order to study the pathomechanism of these missense mutations, we have investigated the fate of 21 such mutations hitherto identified in NPHS1 patients. Immunostaining of stable transfected cells expressing the nephrin mutants demonstrated that most of the mutants showed only endoplasmic reticulum (ER) staining and no detectable cell surface localization. Immunoelectron microscopy of cells expressing the wild-type and a mutant nephrin further confirmed that the mutant nephrin could be abundantly found in the ER but not on the plasma membrane. Subcellular fractionation of wild-type and a mutant cell line clearly showed an altered subcellular distribution and molecular mobility of the mutant nephrin. In summary, our data indicate that a defective intracellular nephrin transport, most likely due to misfolding, is the most common consequence of missense mutations in NPHS1.

Topics: Biological Transport; Calcium-Binding Proteins; Calreticulin; Cell Line; Endoplasmic Reticulum; Gene Expression; Humans; Membrane Proteins; Microscopy, Fluorescence; Microscopy, Immunoelectron; Mutation, Missense; Nephrotic Syndrome; Plasmids; Proteins; Ribonucleoproteins; Subcellular Fractions

Galloway-Mowat syndrome is an autosomal recessive disorder characterized by early onset nephrotic syndrome and central nervous system anomalies. Mutations in podocyte proteins, such as nephrin, alpha-actinin 4, and podocin, are associated with proteinuria and nephrotic syndrome. The genetic defect in Galloway-Mowat syndrome is as yet unknown. We postulated that in Galloway-Mowat syndrome the mutation would be in a protein that is expressed both in podocytes and neurons, such as synaptopodin, GLEPP1, or nephrin. We therefore analyzed kidney tissue from normal children (n=3), children with congenital nephrotic syndrome of the Finnish type (CNF, n=3), minimal change disease (MCD, n=3), focal segmental glomerulosclerosis (FSGS, n=3), and Galloway-Mowat syndrome (n=4) by immunohistochemistry for expression of synaptopodin, GLEPP1, intracellular domain of nephrin (nephrin-I), and extracellular domain of nephrin (nephrin-E). Synaptopodin, GLEPP1, and nephrin were strongly expressed in normal kidney tissue. Nephrin was absent, and synaptopodin and GLEPP1 expression were decreased in CNF. The expression of all three proteins was reduced in MCD and FSGS; the decrease in expression being more marked in FSGS. Synaptopodin, GLEPP1, and nephrin expression was present, although reduced in Galloway-Mowat syndrome. We conclude that the reduced expression of synaptopodin, GLEPP1, and nephrin in Galloway- Mowat syndrome is a secondary phenomenon related to the proteinuria, and hence synaptopodin, GLEPP1, and nephrin are probably not the proteins mutated in Galloway-Mowat syndrome.

Topics: Central Nervous System; Female; Humans; Immunohistochemistry; Infant; Infant, Newborn; Kidney Glomerulus; Male; Membrane Proteins; Microfilament Proteins; Nephrotic Syndrome; Protein Tyrosine Phosphatases; Proteins; Receptor-Like Protein Tyrosine Phosphatases, Class 3; Syndrome

Congenital nephrotic syndrome (CNS) of the Finnish type is a rare autosomal-recessive disorder. Kestila et al reported that a positionally cloned gene for a novel glomerular protein nephrin is mutated in CNS. Most Finnish patients have one of two mutations. In this study, we described a Japanese CNS family associated with a novel missense point mutation in the nephrin gene.. Reverse transcription-polymerase chain reaction, polymerase chain reaction, and sequence analysis were used.. The patient had the three missense mutations homozygously. One mutation was already reported as sequence variant. The two other novel mutations were the GAG to AAG transition, leading to a Glu447Lys and the GAC to GTC transition, predicting an Asp819Val substitution in the nephrin protein.. Our findings indicate that an abnormality of nephrin may cause CNS of the Finnish type in Japanese subjects.

Topics: Asian People; DNA Mutational Analysis; Family Health; Finland; Humans; Infant; Japan; Kidney Glomerulus; Male; Membrane Proteins; Nephrotic Syndrome; Nuclear Family; Point Mutation; Polymerase Chain Reaction; Proteins

Topics: Actinin; Adaptor Proteins, Signal Transducing; Animals; Cytoskeletal Proteins; Humans; Kidney Glomerulus; Membrane Proteins; Mice; Mutation; Nephrotic Syndrome; Protein Isoforms; Proteins; Signal Transduction

Congenital nephrotic syndrome of Finnish type (NPHS1) is an autosomal recessive disorder characterized by severe proteinuria of intrauterine onset. Ninety-four percent of the Finnish NPHS1 chromosomes have been reported to carry either a 2-bp deletion in exon 2 (Fin(Major)) or a nonsense mutation in exon 26 (Fin(Minor)) of the NPHS1 gene. The high prevalence of only two mutations in the Finnish population enables the use of molecular techniques in the diagnosis of NPHS1 and for carrier screening.. We describe two different molecular methods for the detection of the NPHS1 mutations: a PCR-restriction fragment length polymorphism (PCR-RFLP) and a dual-color oligonucleotide ligation assay (OLA). The dual-color OLA, which enables simultaneous detection of the NPHS1 Fin(Major) and Fin(Minor) mutations, can be used for rapid analysis of large sets of samples. The analysis of 2004 Finnish blood samples revealed 34 carriers of the Fin(Major) mutation and 1 carrier of the Fin(Minor) mutation, indicating a carrier frequency of 1:59 (95% confidence interval, 1:89-1:44) for the NPHS1 Fin(Major) mutation and 1:2004 (95% confidence interval, 0 to 1:677) for the NPHS1 Fin(Minor) mutation, respectively.. PCR-RFLP and dual-color OLA are suitable for molecular diagnosis and carrier screening of the major mutations that cause NPHS1.

Topics: Color; Enzyme-Linked Immunosorbent Assay; Finland; Humans; Membrane Proteins; Mutation; Nephrotic Syndrome; Oligonucleotides; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Proteins

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

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

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

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

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

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disorder that is caused by mutations in the recently discovered nephrin gene, NPHS1 (AF035835). The disease, which belongs to the Finnish disease heritage, exists predominantly in Finland, but many cases have been observed elsewhere in Europe and North America. The nephrin gene consists of 29 exons spanning 26 kb in the chromosomal region 19q13.1. In the present study, the genomic structure of the nephrin gene was analyzed, and 35 NPHS1 patients were screened for the presence of mutations in the gene. A total of 32 novel mutations, including deletions; insertions; nonsense, missense, and splicing mutations; and two common polymorphisms were found. Only two Swedish and four Finnish patients had the typical Finnish mutations: a 2-bp deletion in exon 2 (Finmajor) or a nonsense mutation in exon 26 (Finminor). In seven cases, no mutations were found in the coding region of the NPHS1 gene or in the immediate 5'-flanking region. These patients may have mutations elsewhere in the promoter, in intron areas, or in a gene encoding another protein that interacts with nephrin.

Topics: Amino Acid Sequence; Base Sequence; Chromosomes, Human, Pair 19; Cosmids; DNA; DNA Mutational Analysis; Finland; Humans; Incidence; Infant, Newborn; Membrane Proteins; Molecular Sequence Data; Mutation, Missense; Nephrotic Syndrome; Proteins

Nephrin recently has been identified as a putative adhesion molecule, expressed in the glomerulus, in which mutations cause congenital nephrotic syndrome of Finnish type. We sought to determine whether expression of nephrin is altered in human glomeruli in patients with acquired nephrotic syndrome.. We performed PCR amplification of nephrin cDNA, using cDNA previously prepared from single human glomeruli plucked fresh from the surface of human renal biopsies. We had available four cases of nephrotic syndrome (one membranous, three minimal change) and six normal controls. PCR product quantitation was by gel densitometry, confirmed by enzyme-linked immunosorbent assay using a specific oligonucleotide probe. Results were corrected for reaction efficiency and glomerular cellularity by expression as a ratio to levels of the 'housekeeping gene' glyceraldehyde phosphate dehydrogenase.. Glomerular levels ofnephrin mRNA are significantly decreased in cases of minimal change nephrotic syndrome. An apparent reduction was also seen in the single case of membranous nephropathy which was available for study.. Abnormalities of nephrin expression appear to be associated with acquired as well as congenital causes of human nephrotic syndrome.

Topics: Adult; Aged; Base Sequence; Case-Control Studies; DNA Primers; Gene Expression; Humans; Kidney Glomerulus; Membrane Proteins; Middle Aged; Nephrotic Syndrome; Polymerase Chain Reaction; Proteins; RNA, Messenger

We describe here the size and location of nephrin, the first protein to be identified at the glomerular podocyte slit diaphragm. In Western blots, nephrin antibodies generated against the two terminal extracellular Ig domains of recombinant human nephrin recognized a 180-kDa protein in lysates of human glomeruli and a 150-kDa protein in transfected COS-7 cell lysates. In immunofluorescence, antibodies to this transmembrane protein revealed reactivity in the glomerular basement membrane region, whereas the podocyte cell bodies remained negative. In immunogold-stained thin sections, nephrin label was found at the slit between podocyte foot processes. The congenital nephrotic syndrome of the Finnish type (NPHS1), a disease in which the nephrin gene is mutated, is characterized by massive proteinuria already in utero and lack of slit diaphragm and foot processes. These features, together with the now demonstrated localization of nephrin to the slit diaphragm area, suggests an essential role for this protein in the normal glomerular filtration barrier. A zipper-like model for nephrin assembly in the slit diaphragm is discussed, based on the present and previous data.

Topics: Cell Membrane; Cloning, Molecular; Epithelial Cells; Finland; Glomerular Filtration Rate; Humans; Kidney Glomerulus; Membrane Proteins; Microscopy, Immunoelectron; Molecular Sequence Data; Mutation; Nephrotic Syndrome; Polymerase Chain Reaction; Proteins; Recombinant Proteins

CD2-associated protein (CD2AP) is an 80-kilodalton protein that is critical for stabilizing contacts between T cells and antigen-presenting cells. In CD2AP-deficient mice, immune function was compromised, but the mice died at 6 to 7 weeks of age from renal failure. In the kidney, CD2AP was expressed primarily in glomerular epithelial cells. Knockout mice exhibited defects in epithelial cell foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition. Supporting a role for CD2AP in the specialized cell junction known as the slit diaphragm, CD2AP associated with nephrin, the primary component of the slit diaphragm.

Topics: Adaptor Proteins, Signal Transducing; Animals; Basement Membrane; Cytoskeletal Proteins; Epithelial Cells; Extracellular Matrix Proteins; Glomerular Mesangium; Intercellular Junctions; Kidney Glomerulus; Lymphocyte Activation; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Electron; Nephrotic Syndrome; Proteins; Recombinant Fusion Proteins; T-Lymphocytes

Defects in the newly reported gene NPHS1 in chromosome 19 cause the massive proteinuria of Finnish type congenital nephrotic syndrome (CNF). Together with its gene product, nephrin, NPHS1 is providing new understanding of the pathophysiological mechanisms of glomerular filtration. Here we show the characteristic splicing of NPHS1 mRNA in the normal and CNF kidneys and localize nephrin exclusively in the glomerulus and to the filtration slit area by light and immunoelectron microscopy. These results indicate that nephrin is a new protein of the interpodocyte filtration slit area with a profound role in the pathophysiology of the filtration barrier.

Topics: Amino Acid Sequence; Base Sequence; Humans; Immunohistochemistry; Kidney Glomerulus; Membrane Proteins; Microscopy, Immunoelectron; Molecular Sequence Data; Nephrotic Syndrome; Protein Isoforms; Proteins; RNA Splicing; RNA, Messenger

Topics: Adaptor Proteins, Signal Transducing; Animals; Cells, Cultured; Chromosomes, Human, Pair 19; Cytoskeletal Proteins; Humans; Intercellular Junctions; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Electron; Mutation; Nephrotic Syndrome; Proteins

Topics: Alleles; Child, Preschool; Christianity; Female; Finland; Gene Frequency; Genetic Heterogeneity; Haplotypes; Humans; Incidence; Infant; Linkage Disequilibrium; Male; Membrane Proteins; Mutation; Nephrotic Syndrome; Pedigree; Pennsylvania; Proteins

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

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

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal-recessive disorder, characterized by massive proteinuria in utero and nephrosis at birth. In this study, the 150 kb critical region of NPHS1 was sequenced, revealing the presence of at least 11 genes, the structures of 5 of which were determined. Four different mutations segregating with the disease were found in one of the genes in NPHS1 patients. The NPHS1 gene product, termed nephrin, is a 1241-residue putative transmembrane protein of the immunoglobulin family of cell adhesion molecules, which by Northern and in situ hybridization was shown to be specifically expressed in renal glomeruli. The results demonstrate a crucial role for this protein in the development or function of the kidney filtration barrier.

Topics: Amino Acid Sequence; Cloning, Molecular; Cosmids; DNA Mutational Analysis; DNA, Complementary; Exons; Family Health; Gene Deletion; Gene Expression; Haplotypes; Humans; Immunoglobulins; Kidney Glomerulus; Membrane Proteins; Molecular Sequence Data; Nephrotic Syndrome; Protein Structure, Tertiary; Proteins; RNA, Messenger