Protein analysis of steroid resistant congenital nephrotic syndrome gene: podocin

类固醇抵抗性先天性肾病综合征（SRN）是一种遗传性肾脏疾病，出生3个月后出现大量蛋白尿。本研究的目的是通过对足docin突变研究的分析来确定足docin的蛋白质功能及其在获得性肾病综合征中蛋白尿状态的可能含义。本项目研制的特异性抗podocin抗体明确地确定了podocin和肾小球中起过选择性屏障作用的主要分子nephrin在足细胞狭隔膜中的共定位。该抗体与离体肾小球蛋白样品共免疫沉淀表明，podocin是人足细胞足突中真正的肾素细胞内配体。为了阐明蛋白尿在SRN中的作用机制，我们通过SRN患者错义突变体的表达实验进行了诱变研究。c端错义足蛋白突变体不能被运输到质膜上，可能是因为它聚集在内质网或溶酶体中。此外，由于它们具有很强的蛋白质亲和力，这些足蛋白突变体也抑制了其配体肾素的正确质膜组装。为了研究足docin在蛋白尿发病机制中的作用，建立了puromycin - aminonucle苷大鼠肾病模型，展示了肾病综合征的微小变化。有趣的是，podocin和nephrin的蛋白表达在蛋白尿发生前明显下降。这一结果提示podocin和nephrin在蛋白尿及其突变肾病以及获得性肾病综合征的发病机制中起着关键作用。

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.