Roles of Intercellular junction of podocytes in surviving injuries

为了阐明足细胞损伤后存活的机制，我们从细胞间连接的角度分析了足细胞对损伤的反应。1.足细胞损伤体外检测方法的建立：我们尝试了磁珠结合胶原酶的肾小球分离法，成功地获得了大量适合于足细胞原代培养的肾小球。用此方法每只大鼠可获得5×10^5个足细胞。甲萘醌在体外产生自由基引起足细胞损伤。50μM甲萘醌在30分钟内损伤足细胞。缝隙连接蛋白43（Cx43）基因是足细胞损伤后最早反应的基因之一，通过siRNA技术敲低Cx43基因。遗憾的是，Cx43基因敲除的足细胞与对照足细胞在甲萘醌作用下的存活率无显著差异。2.足细胞细胞间连接成分的鉴定：足细胞损伤后，足细胞内新形成紧密连接和缝隙连接。分离肾小球的细胞质膜根据通过Western印迹检测的Cx43的存在进行分级。在一维凝胶电泳中，切出含有密封蛋白的部分并通过LC-MSMS分析。因此，在级分中检测到密蛋白-5、密蛋白-6、密蛋白-12和密蛋白-15。免疫组化结果显示，claudin-6主要定位于足细胞，其余定位于内皮细胞。本研究建立了足细胞损伤的体外检测方法，并首次鉴定了足细胞紧密连接的跨膜成分。

To clarify the mechanism of survival of podocytes after injuries, we analyzed the podocyte responses to injuries focusing on the intercellular junctions.1.Establishment of in vitro assay for podocyte injury : We tried a glomerular isolation method with magnetic beads and collagenase, and succeeded in getting a number of glomeruli suitable for primary podocyte culture. By this method, we could get 5×10^5 podocytes per rat. Podocyte injury was caused in vitro by menadione producing free radicals. 50μM of menadione damaged podocytes within 30min exposure. Connexin43 (Cx43) gene, one of earliest response genes responding to podocyte injury, was knocked down in cultured podocytes by siRNA. Unfortunately there was no significant difference of survival rate under the menadione exposure between the Cx43-knockdown podocytes and control podocytes.2.Identification of components of intercellular junctions of podocytes : When podocytes are injured, tight junctions and gap junctions are newly formed in podocytes. Cytoplasmic membranes of isolated glomeruli were fractionated according to the presence of Cx43 detected by Western blotting. In one-dimensional gel electrophoresis, the portion which contained claudins was cut out and analyzed by LC-MSMS. Consequently, claudin-5,-6,-12 and -15 were detected in the fraction. Immunohistochemistry demonstrated claudin-6 localized in podocytes and the others in endothelial cells.In this study, we could establish the in vitro assay for podocyte injury, and identified the transmembrane component of the tight junction of podocytes for the first time.