Functional Molecules on Glomerular Epithelial Cells

用1日龄大鼠肾小球制备cDNA文库，因为用~（13 S）-Methylamine代谢标记<35>显示，mAb 5-1-6识别的抗原（p51）在1日龄大鼠肾脏中的合成比在5日龄或成熟大鼠肾脏中更活跃。使用该文库和COS细胞进行表达，通过mAb 5-1-6的cDNA克隆仍在继续。另一方面，我们利用免疫电镜技术，通过对金颗粒的计数，进一步研究了p51的精确定位。足细胞足突基部的金颗粒数为0.77个/mm肾小球基底膜。结果表明，p51主要定位于裂膜及其胞质面，p51是裂膜蛋白的一个组成部分。为了阐明mAb 5-1-6引起的异常蛋白尿的机制，我们对裂膜的分子本质进行了研究。在mAb 5-1-6诱导的蛋白尿大鼠中，用电子显微镜未检测到裂膈结构的任何形态学改变。注射mAb后第5天，肾小球内p51和紧密连接蛋白ZO-1的表达明显减少，此时尿蛋白量达到峰值。免疫印迹分析也显示，注射mAb后5天肾小球ZO-1条带强度降低。结果表明，p51是ZO-1的一种结合蛋白，这些蛋白不是维持裂膜结构所必需的，而是维持裂膜屏障功能所必需的，并进一步研究了酪氨酸磷酸化是否在p51和ZO-1的分子重排中起作用。在mAb 5-1-6注射后的任何时间点均未检测到酪氨酸磷酸化。

cDNA library was prepar from one day old rat glomeruli, because the metabolic labeling by ^<35>S-methyonine revealed that mAb 5-1-6 recognized antigen (p51) was more actively synthesized in one day old rat kidney than in 5 days old or matured rat kidneys. cDNA cloning by mAb 5-1-6 is being still continued using this library and COS cells for expression. However we have not yet succeeded in identifying p51.On the other hand the precise localization of p51 was further investigated with immunoelectron microscopy by counting the gold particles. The number of gold particles at the base of foot processes of podocytes is 0.77 particles/mm length of glomerular basement membrane. of particles at the lateral surface and at intermittent space of foot processes are 1.61 and 2.14, respectively.From these results we concluded that p51 is mainly localized at the slit diaphragm and its cytoplasmic face and that p51 is a component of slit diaphragin.To clarify the mechanisms of abnormal proteinuria induced by mAb 5-1-6, we have investigated the molecular nature of slit diaphragm. Any morphological alterations of the structure of slit diaphragm was not detected with electron microscopy in proteinuric rats induced by mAb 5-1-6. However, p51 and ZO-1, tight junction protein, staining were definitely decreased at glomeruii of rats 5 days after mAb injection when abnomal proteinuria reached a peak value. lmmunoblot analysis also showed that intensity of ZO-1 band was decreased at glomeruii of 5 days after mAb injection. From these results we conchuded that p51 is an associate protein of ZO-1 and that these proteins are not necessary for maintaining the structure of slit diaphragm but necessary for maintaining the barrier function of slit diaphragm.We have further investigated whether tyrosine phosphorylation plays a role for this molecular rearrangement of p51 and ZO-1. Tyrosine phosphorylation was not detected at any time points after mAb 5-1-6 injection.