Protein analysis of steroid resistant congenital nephrotic syndrome gene: podocin

激素抵抗型先天性肾病综合征（SRN）是一种遗传性肾病，本研究的目的是通过podocin突变研究来确定podocin的蛋白功能，以及podocin在获得性肾病综合征蛋白尿状态中的可能意义。我们特异性抗podocin抗体，由本项目开发的podocin抗体清楚地确定了podocin和nephrin在足细胞狭缝隔膜中的共定位，nephrin是肾小球中起选择性渗透屏障作用的主要分子。用该抗体和分离肾小球的蛋白质样品进行免疫共沉淀，结果表明podocin是人足细胞足突中nephrin的真实的胞内配体。为了阐明SRN蛋白尿的发生机制，本研究利用SRN患者错义突变体的表达实验进行突变研究。C-末端错义podocin突变体被证明不被运输到质膜，可能是因为其聚集在内质网或溶酶体中。此外，这些podocin突变体也抑制其配体nephrin的正确质膜组装，因为它们的强蛋白亲和力。本研究建立了一种以嘌呤霉素-氨基胍为主要药物的大鼠肾病模型，以探讨podocin在蛋白尿发病机制中的作用。有趣的是，podocin和nephrin的蛋白表达在蛋白尿发作前明显降低。这一结果提示podocin和nephrin在蛋白尿的病理机制中起关键作用，不仅在其突变型肾病中，而且在获得性肾病综合征中也起关键作用。

Steroid resistant congenital nephrotic syndrome (SRN) is one of the hereditary kidney diseases, which presents heavy proteinuria 3 months after its birth.The aim of the present study was to determine the protein function of podocin by the analysis with podocin mutagenesis study and the possible implication in the proteinuric state in the acquired nephrotic syndrome.Our specific anti-podocin antibody developed by the present project clearly determined the co-localization of podocin and nephrin, which is the major molecule serving permselectivity barrier in the glomerulus, in the podocyte slit diaphragm. Co-immunoprecipitation using this antibody and the protein sample from isolated glomeruli showed that podocin is real intracellular ligand of nephrin in human podocyte foot processes. In order to clarify the mechanism of the proteinuria in SRN, the mutagenesis study was done by using the expression experiment with missense mutants demonstrating SRN patient. The C-terminal missense podocin mutants were demonstrated not to be transported to the plasma membrane possibly because of its aggregation in the endoplasmic reticulum or lysozome. Moreover these podocin mutants also inhibited the correct plasma membrane assembly of its ligand, nephrin, because of their strong protein affinity. Puromycin-Aminonucleoside rat nephrosis, which is a modlel demonstrating minimal change nephrotic sydrome, was developed to determine the implication of podocin in the pathomechanism of the proteinuria. Interestingly, the protein expression of podocin as well as nephrin was clearly decreased before the onset of the proteinuria. This result suggested the pivotal role of podocin together with nephrin in the pathomechanism in the proteinuria not only its mutated nephrosis also in the acquired nephrotic syndrome.