Nephrin, Podocin And CD2AP Mutation And Expression Profile In Children With Primary Nephrotic Syndrome

IntroductionNephrotic syndrome is a clinical diagnosis defined by the presence of massive proteinuria, hypoalbuminemia, hyperlipidemia, and edema. According to its etiology, Nephrotic syndrome can be classified as the primary, secondary and congenital nephrotic syndrome (CNS). Often as the target of injury, Glomerular filtration barrier consists of glomerular visceral epithelial cells (Podocytes), fenestrated capillary endothelial cells and glomerular basement membrane.There are many exciting advances in our understanding of genetic causes of nephrotic syndrome since 1998 with Nephrin was found to be encoded by the NPHS1 gene and mutated in severe congenital nephrotic syndrome of Finnish type. The identification of Nephrin and Podocin as genes that underlie two forms of Nephrotic syndrome has provided a critical molecular foothold into podocyte biology. A number of slit diaphragm molecules have now been identified including CD-associated protein, ZO-1, FAT-1, Neph-1,2, and 3, P-cadherin, and so on.Materials and MethodsNephrin, Podocin and CD2AP mutation profile are investigated in 60 children with primary nephrotic syndrome, mRNA expression profile were studied by Quantitative Real-time PCR in aspirated renal biopsy tissues from 30 samples with a clinical and renal immunopathologic diagnosis of minimal change nephrotic syndrome (MCNS), primary IgAN (by the presence of predominant IgA deposits in the mesangium associated with focal or diffuse mesangial cell proliferation) or isolated hematuria. Meanwhile, protein expression profiles of Nephrin, Podocin and CD2AP were analyzed in 23 samples with MCNS, IgAN or isolated hematuria patients by in-direct Immunofluorescence and Confocal Microscope and Immunohistochemistry. ResultsIn NPHS1 gene mutation analysis, 4 of the 60 primary nephrotic syndrome patients had novel heterozygous missense mutation leading to amino acid substitutions (R800C, Q453R), 3 known Single Nucleotide Polymorphism (SNP) were also found (T741T, V763V, S1105S) which resulting no amino acid substitutions. 1 patient with samesense mutation (G223G) was found in CD2AP gene analysis. No mutation was found in NPHS2 gene in this study.Compared with renal samples from isolated hematuria patients, CD2AP mRNA level was significantly down-regulated in renal samples from 11 primary nephrotic syndrome patients (MCNS and IgAN) by quantitative real-time PCR (P=0.044), though no significant down-regulation was found in mRNA level of Nephrin (P=0.196). Meanwhile, the expression of CD2AP and Nephrin proteins was significantly reduced (CD2AP P=0.028, Nephrin P=0.038) in 11 primary nephrotic syndrome patients while compared with the samples from isolated hematuria patients, though no significant reduction of Podocin protein expression was found in these patients (P=0.728).ConclusionThe results suggest that genetic, transcript and translation expression changes of Nephrin and CD2AP may have pathogenetic roles in some patients with primary nephrotic syndrome, though no correlationship was found in Podocin in this study.