PATHOGENESIS OF MEMBRANOUS GLOMERULONEPHROPATHY

本项目的长期目标是了解 膜性肾小球肾病（MGN）的发病机制， 人类的肾脏疾病 大鼠Heymann肾炎（HN）， MGN的公认模型，将用于深入了解 这种疾病的机制。 具体目的是：1）检测自身免疫原性， 还原和非还原的单个亚基的致肾炎性 HN抗原gp 600 用分离的亚单位免疫大鼠， 将检查制备聚丙烯酰胺凝胶电泳， HN的发展。 将分析血清中的自身抗体， 天然的、还原的和非还原的gp 600和单个亚基， 间接免疫荧光，酶联免疫吸附测定 （ELISA）和蛋白质印迹上的免疫反应性。 肾脏会 通过免疫荧光和免疫电镜检查， HN的典型特征，以及鉴别和超微结构 与沉积的自身抗体反应的表位的定位 使用大鼠抗GP 600的单克隆抗体。 数据将提供 关于是否存在必需的疾病产生表位的信息， 是否存在完整的二硫键， 亚基是致肾炎所必需的; 2）检验假设 一些致肾炎表位在亚基之间共享， 有些则不然。 这将使用大鼠单克隆抗体进行 显示出与表位反应性相同的针对gp 600的抗体， 致肾炎自身抗体 方法包括竞争性 抑制免疫测定法，Western印迹上的酶免疫检测法， 和放射自显影; 3）进一步表征生物化学和 免疫学上，由有限的 蛋白水解裂解在肽组成中的相关性，性质 致肾炎表位，自身免疫原性和致肾炎 潜力 这将涉及个体的肽图谱 亚基，用2-D凝胶电泳，以确定 如果有的话，子单元的相关性。 部分特性 致肾炎表位的性质将使用 在抗体存在下的蛋白水解技术 蛋白水解片段的电泳和Western分析。 的 将进一步分析仍然结合抗体的片段， 致肾炎和自身免疫原性潜力; 4）确定 gp 600中糖残基对自身抗体的意义 结合自身免疫原性和致肾炎性：这将是 通过用exo-和 内切糖基化酶，然后检测去糖基化制剂 ELISA法测定自身抗体结合， 免疫刘易斯大鼠。 拟议的研究将提供有关 gp 600的生物化学和免疫学结构， 涉及GP 600的致肾炎性。 这些知识将导致 进一步加深对MGN发病机制的认识。

The long term objective of this project is to understand the pathogenesis of membranous glomerulonephropathy (MGN), a common kidney disease in humans. Heymann Nephritis (HN) of rat, an accepted model of MGN, will be used to gain insight into the mechanisms of this disease. The specific aims are: 1) Test the autoimmunogencity and nephritogenicity of reduced and nonreduced individual subunits of the HN antigen, gp600. Rats immunized with subunits isolated by preparative polyacrylamide gel electrophoresis will be examined for development of HN. Sera will be analyzed for autoantibodies to native, reduced and nonreduced gp600 and individual subunits by indirect immunofluorescence, enzyme linked immunosorbent assay (ELISA) and immunoreactivity on Western blots. Kidneys will be examined by immunofluorescence and immunoelectron microscopy for typical features of HN, and the identity and ultrastructural location of the epitopes reacting with deposited autoantibodies using rat monoclonal antibodies to gp600. Data will provide information on whether the necessary disease producing epitopes are present on all subunits and whether intact disulfide bonds in subunits are essential for nephritogenicity; 2) Test the hypothesis that some nephritogenic epitopes are shared among subunits while others are not. This will be performed with rat monoclonal antibodies to gp600 which show identity for epitope reactivity with the nephritogenic autoantibodies. Methods will include competitive inhibition immunoassays, enzyme immunodetection on Western blots, and autoradiography; 3) Further characterize biochemically and immunologically the peptides of subunits produced by limited proteolytic cleavage for relatedness in peptide composition, nature of nephritogenic epitopes, and autoimmunogenic and nephritogenic potential. This will involve peptide mapping of individual subunits using 2-D gel electrophoresis to determine the relatedness, if any, of the subunits. Partial characterization of the nature of the nephritogenic epitopes will be done using the proteolytic technique in the presence of antibody followed by gel electrophoretic and Western analyses of proteolysed fragments. The fragments which still bind antibody will be further analyzed for nephritogenic and autoimmunogenic potential; 4) Determine the significance of carbohydrate residues in gp600 towards autoantibody binding autoimmunogenicity and nephritogenicity: this will be examined by performing deglycosylation of gp600 with exo- and endoglycosidases and then testing the deglycosylated preparation for-autoantibody binding by ELISA and for nephritogenicity by immunizing Lewis rats. The proposed studies will provide new information regarding the biochemical and immunological structure of gp600 and how that relates to the nephritogenicity of gp600. This knowledge will lead to further progress in the understanding of the mechanism of MGN.