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Li, Cong, Vesa Ruotsalainen, Karl Tryggvason, Andrey S. Shaw, and Jeffrey H. Miner. CD2AP is expressed with nephrin in developing podocytes and is found widely in mature kidney and elsewhere. Am J Physiol Renal Physiol 279: F785-F792, 2000.--CD2-associated protein (CD2AP) is an adapter molecule that can bind to the cytoplasmic domain of nephrin, a component of the glomerular slit diaphragm. Mice lacking CD2AP exhibit a congenital nephrotic syndrome characterized by extensive foot process effacement, suggesting that CD2AP-nephrin interactions are critical to maintaining slit diaphragm function. We have examined the patterns of expression of both CD2AP and nephrin in developing mouse and human kidney. Both proteins were first detected in developing podocytes at the capillary loop stage of glomerulogenesis and eventually became concentrated near the glomerular basement membrane. CD2AP was also observed diffusely in collecting duct and apically in many cells of proximal and distal tubule. Kidneys from Cd2ap -/- mice initially exhibited normal nephrin localization, but as the mice aged and foot processes became effaced, nephrin disappeared. In laminin-[[Beta].sub.2] mutant mice exhibiting nephrotic syndrome, CD2AP in glomeruli was aberrantly localized in a primarily punctate pattern. Extensive extrarenal expression of CD2AP was observed in endothelial and epithelial cells, in many cases with a specific subcellular localization. Together, these results suggest that CD2AP is not only involved in maintaining the slit diaphragm but may also have a general role in maintaining specialized subcellular architecture. The severity of kidney disease in Cd2ap mutant mice may have eclipsed manifestation of defects in other tissues. CD2-associated protein; renal development; nephrotic syndrome; glomerulus; nephron; epithelia

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. [ABSTRACT FROM AUTHOR]

Disease-causing missense mutations inNPHS2gene 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 theNPHS2gene 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]