Roles of Intercellular junction of podocytes in surviving injuries

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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.