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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.

Background: 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., Methods: 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., Results: 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., Conclusion: 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.

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

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.

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.

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

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.

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.

The mechanism by which glucocorticoids govern antiproteinuric effect in nephrotic syndrome remains unknown. Present study examined the protective role of dexamethasone (DEX) in the intracellular trafficking of nephrin under endoplasmic reticulum (ER) stress. Human embryonic kidney-293 cell line expressing a full-length human nephrin was cultured in mediums containing 5.5 or 25 mM glucose with or without DEX. The result revealed that glucose starvation evoked a rapid ER stress leading to formation of underglycosylated nephrin that was remained in the ER as a complex with calreticulin/calnexin. DEX rescued this interfered trafficking through binding to its receptor and stimulating the mitochondrial transcripts and adenosine 5′ triphosphate (ATP) production, leading to synthesis of fully glycosylated nephrin. These results suggest that ER-stress in podocytes may cause alteration of nephrin N-glycosylation, which may be an underlying factor in the pathomechanism of the proteinuria in nephrotic syndrome. DEX may restore this imbalance by stimulating expression of mitochondrial genes, resulted in the production of ATP that is essential factor for proper folding machinery aided by the ER chaperones.Kidney International (2006) 69, 1350–1359. doi:10.1038/sj.ki.5000317; published online 8 March 2006 [ABSTRACT FROM AUTHOR]