Difference between total and intact assays for N-terminal propeptide of type I procollagen reflects degradation of pN-collagen rather than denaturation of intact propeptide.

BACKGROUND: The concentration of N-terminal propeptide of type I procollagen (PINP) in the serum reflects the rate of type I collagen formation. Intact PINP assay measures the trimeric propeptide while total P1NP assay measures both trimeric and monomeric forms. In this study we compared these two assays emphasizing the possible differences. METHODS: Intact and total PINP were measured from serum in healthy Finnish blood donors (n = 34) and in the patients with chronic renal failure before and after haemodialysis (n = 39). In addition, the serum of a normal man, pooled hospital serum samples and the serum of a patient with haemodialysis treatment were fractioned by gel filtration and trimeric and monomeric forms were located. Fractions were lyophilized and intact and total PINP were measured in each fraction. Samples from bedridden geriatric patients (n = 173) were also measured using intact and total PINP assays and a degradation marker of type I collagen (ICTP). RESULTS: The correlation between intact and total PINP in controls was 0.89 and their PINP concentrations were similar. In haemodialysis or bedridden geriatric patients, the PINP methods gave significantly different results. In gel filtration studies, intact PINP hardly measured monomeric form even if its concentration was disproportionately increased in haemodialysis patients. In bedridden geriatric patients, the difference of total and intact PINP correlated significantly to degradation marker ICTP. CONCLUSIONS: Difference between total and intact assays for PINP seem to reflect degradation of pN-collagen rather than denaturation of intact propeptide.

Recurrence of proteinuria following renal transplantation in congenital nephrotic syndrome of the Finnish type.

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.

Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography.

Nephrin is a key functional component of the slit diaphragm, the structurally unresolved molecular filter in renal glomerular capillaries. Abnormal nephrin or its absence results in severe proteinuria and loss of the slit diaphragm. The diaphragm is a thin extracellular membrane spanning the approximately 40-nm-wide filtration slit between podocyte foot processes covering the capillary surface. Using electron tomography, we show that the slit diaphragm comprises a network of winding molecular strands with pores the same size as or smaller than albumin molecules, as demonstrated in humans, rats, and mice. In the network, which is occasionally stratified, immunogold-nephrin antibodies labeled individually detectable globular cross strands, about 35 nm in length, lining the lateral elongated pores. The cross strands, emanating from both sides of the slit, contacted at the slit center but had free distal endings. Shorter strands associated with the cross strands were observed at their base. Immunolabeling of recombinant nephrin molecules on transfected cells and in vitrified solution corroborated the findings in kidney. Nephrin-deficient proteinuric patients with Finnish-type congenital nephrosis and nephrin-knockout mice had only narrow filtration slits that lacked the slit diaphragm network and the 35-nm-long strands but contained shorter molecular structures. The results suggest the direct involvement of nephrin molecules in constituting the macromolecule-retaining slit diaphragm and its pores.

Disease-causing missense mutations in NPHS2 gene alter normal nephrin trafficking to the plasma membrane.

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.

Monoclonal antibodies to human nephrin.

Nephrin is a 180-200-kDa transmembrane protein of the immunoglobulin superfamily. In the kidney, nephrin localizes to the slit diaphragm (SD) between interdigitating podocyte foot processes and mutations in the nephrin gene cause congenital nephrotic syndrome. In addition to this rare genetic disorder, recent reports indicate that nephrin is more generally involved in the pathogenesis of glomerular disease. In this report, we describe production and characterization of mouse monoclonal antibodies to human nephrin, and discuss their applications. Recombinant human nephrin variants were produced in both prokaryotic and eukaryotic expression systems and purified proteins were used in an immunization protocol. A total of 16 antibodies were characterized for their reactivity with the nephrin by using ELISA, Western blots, immunoprecipitation and immunostaining of frozen and formaldehyde-fixed paraffin embedded tissue sections. The antibody epitopes were mapped using a variety of recombinant human nephrin variants. The detailed screening and characterization proved to be essential in order to find the most suitable antibody for each application. These antibodies will find wide use in studies of human nephrin and its involvement in kidney disease.

Tissue expression of nephrin in human and pig.

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.

Clustering-induced tyrosine phosphorylation of nephrin by Src family kinases.

BACKGROUND: Nephrin is a recently discovered protein of the immunoglobulin (Ig) superfamily. In the kidney, it is located at the slit diaphragm, which forms the decisive size-selective filter of glomerular ultrafiltration barrier and locates between the interdigitating foot processes of podocytes. Nephrin is mutated in congenital nephrosis of the Finnish type (NPHS1) and has been demonstrated to be an essential component of the slit diaphragm. Based on its domain structure, nephrin is likely to be a cell-cell or cell-matrix adhesion protein that may have a signaling function. In this study, we hypothesized that the clustering of nephrin with antibodies on cell surface mimics the situation where the interaction between nephrin and its extracellular ligand(s) is altered. METHODS: Nephrin was clustered on the surface of stably transfected HEK293 cells by a monoclonal antinephrin antibody and polyclonal secondary antibody. Clusters were visualized by immunofluorescence microscopy. Changes in protein phosphorylation were studied employing immunoprecipitations and Western blot analysis. A specific inhibitor and cotransfection experiments were used to investigate role of Src family kinases in nephrin phosphorylation. RESULTS: Clustering of nephrin induced its own tyrosine phosphorylation. This phosphorylation was inhibited by PP2, an inhibitor of Src family kinases. Several members of Src family kinases were able to induce nephrin phosphorylation when cotransfected to HEK293 cells with nephrin. Moreover, the Src family kinase Fyn was consistently found to be coimmunoprecipitated with nephrin. Interestingly, clustering of nephrin induced also tyrosine phosphorylation of a 46 kD protein that was as well found to be coimmunoprecipitated with nephrin. CONCLUSION: Nephrin is a signaling protein phosphorylated by Src family kinases.

Nephrin expression is reduced in human diabetic nephropathy: evidence for a distinct role for glycated albumin and angiotensin II.

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.

Altered ultrastructural distribution of nephrin in minimal change nephrotic syndrome.

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.

Co-localization of nephrin, podocin, and the actin cytoskeleton: evidence for a role in podocyte foot process formation.

The discovery of the genes for nephrin and podocin, which are mutated in two types of congenital nephrotic syndrome, was pivotal in establishing the podocyte as the central component of the glomerular filtration barrier. In vivo the proteins have been localized to the podocyte slit diaphragm, and there is recent evidence for interaction between the two via the adapter molecule CD2AP. We describe in a human podocyte cell line, the subcellular distribution of nephrin, podocins, and CD2AP and their functional interaction with the cytoskeleton. In addition to membrane expression, nephrin and podocin were detected intracellularly in a filamentous pattern. Double immunolabeling and depolymerization studies showed that nephrin and podocin partially co-localize with actin, most strikingly seen protruding from the tips of actin filaments, and are dependent on intact actin polymers for their intracellular distribution. Treatment of differentiated podocytes with puromycin aminonucleoside, an agent that causes foot process effacement in vivo, disrupted actin and nephrin simultaneously, with loss of cell surface localization. We demonstrate an intimate relationship between nephrin podocin and filamentous actin, and reason that disruption of nephrin/podocin could be a final common pathway leading to foot process effacement in proteinuric diseases.

Proteinuria and prenatal diagnosis of congenital nephrosis in fetal carriers of nephrin gene mutations.

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

N-linked glycosylation is critical for the plasma membrane localization of nephrin.

The expression pattern, subcellular localization, and the role of glycosylation of the human nephrin was examined in transfected cells. Stable cell lines, constitutively expressing a full-length human nephrin cDNA construct, were generated from transfected immortalized mouse podocytes (IMP) and a human embryonic kidney cell line (HEK-293). Immunofluorescence confocal microscopy of transfected cells showed plasma membrane localization of the recombinant nephrin. Immunoblotting showed that the recombinant nephrin expressed in transfected cell lines migrated as a double band with a molecular weight of 185 kD. When cells were treated with the N-glycosylation inhibitor, tunicamycin, the molecular weight of nephrin was decreased to a single immunoband of 150 kD, indicating that the shift in the electrophoretic migration of nephrin is due to N-linked carbohydrate moieties. It was further shown that this glycosylation process is highly sensitive to inhibition by tunicamycin, which is a naturally occurring antibiotic, leading to retention of nonglycosylated nephrin molecules in the endoplasmic reticulum. It was concluded that N-glycosylation of nephrin is crucial for its proper folding and thereby plasma membrane localization; therefore, inhibition of this process might be an important factor in the onset of pathogenesis of some acquired glomerular diseases.

Recurrence of nephrotic syndrome in kidney grafts of patients with congenital nephrotic syndrome of the Finnish type: role of nephrin.

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.

Expression of nephrin in acquired human glomerular disease.

BACKGROUND: Nephrin is a recently identified protein, which is synthesized in the podocytes and localized in the slit diaphragm area. Nephrin is a cell adhesion molecule of the immunoglobulin superfamily, and presumably is a part of the zipper-like structure of the slit membrane. As the mutation of the gene coding nephrin induces congenital nephrotic syndrome of Finnish type, which is a prototype of nephrotic syndrome, it has been suggested that nephrin also plays a role in acquired proteinuric kidney disease. METHODS: To address the above issue, the expression of nephrin in acquired human glomerular disease was studied by immunoelectron microscopy employing a polyclonal antibody against nephrin. Four normal human kidneys from nephrectomy specimens and eight kidney biopsy specimens from glomerular disease patients (one minimal change disease, one membranous glomerulonephritis (GN), one membranoproliferative GN, four IgA nephropathy, and one lupus nephritis) were studied. Proteinuria of the patients ranged from 448 to 11725 mg/day. Effacement of the foot processes was observed in all patients. RESULTS: The study demonstrated that the number and distribution of gold particles in the glomerular region, where the podocyte foot process was well preserved, were similar to that found in normal kidneys; however, gold particles were almost always absent in regions where the foot processes were effaced. The number of gold particles per foot process interspace was not different between normal controls and GN patients; however, the number of gold particles per defined length (1000 nm) of the glomerular basement membrane underlying the foot processes was significantly reduced in GN patients. CONCLUSION: Using immunoelectron microscopy, we observed that the expression of nephrin in GN was lower in regions where the foot processes were effaced, and comparable with that of normal controls where the foot process interspaces were preserved. The significance of our observation in the context of proteinuria in acquired GN needs further clarification.

Expression of nephrin in pediatric kidney diseases.

Nephrin is a podocyte cell adhesion protein located at the slit diaphragm area of the kidney glomerulus. Mutations in the nephrin gene (NPHS1) lead to congenital nephrosis, suggesting that nephrin is essential for the glomerular filtration barrier. This prompted this study of the expression of nephrin in acquired pediatric kidney diseases using in situ hybridization and immunohistochemistry. In situ hybridization for nephrin mRNA was performed in biopsy samples from patients with proteinuria caused by minimal change nephrosis, focal segmental glomerulosclerosis, and membranous nephropathy. The expression of nephrin mRNA was evaluated by grading the signal intensity visually and by counting the number of grains in separate glomeruli. No significant difference was observed in these samples as compared with controls. Immunostaining for nephrin was performed using antibodies directed against extra- and intracellular parts of the molecule. Nephrin staining gave a linear pattern along the glomerular capillary loops. In minimal change nephrosis, focal segmental glomerulosclerosis, and membranous nephropathy, the distribution of nephrin was similar to that in controls. In proliferative forms of glomerulonephritides (Henoch-Schönlein nephritis, IgA nephropathy, postinfectious and membranoproliferative glomerulonephritis), crescents and sclerotic lesions were negative for nephrin, and mesangial proliferation led to a scattered and sparse staining pattern. The staining pattern of nephrin was compared to that of ZO-1, a component of the cytoplasmic face of the slit diaphragm. The distributions of these two proteins in capillary tufts were similar in all disease entities studied. In conclusion, immunohistochemistry and in situ hybridization did not reveal major alterations in the expression of nephrin in proteinuric kidney diseases in children. Further studies are needed for more precise evaluation of the role of nephrin in these diseases.