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.

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.

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.