The Influence Of Dexamethasone On Expression And Distribution Of CD2AP/TRPC6 In Mouse Podocyte

Objective Slit diaphragm is the most important part in the glomerular filtration function. Some important slit diaphragm proteins such as CD2AP,TRPC6, nephrin and podocin, compose slit diaphragm complex, maintaining the completeness of foot process and slit diaphragm. In order to demonstrate the possible anti-proteinuria mechanisms of dexamethasone, a study was designed to explore the expression and distribution of CD2AP and TRPC6 in a time series scale in the case of podocyte injury and protection in vitro.Methods Mouse podocyte cell line (MPC5) were cultured in 0.02% DMSO in control group, subjected to puromycin aminonucleoside (PAN) treatment alone or with dexamethasone(DEX) in other two groups.The podocyte morphology was observed and took pictures by phase-contrast microscope, then the differences of morphology and areas between each groups were analyzed by ImageJ. The distribution, mRNA expression and protein expression level of CD2AP and TRPC6 were detected by indirect immunocyto-fluorescence, semi-quantitative RT-PCR and Western blot, respectively.Results(1)The well-developed podocyte arborization and interconnection was formed in control group, but PAN treatment led to significant shrinkage of podocytes(P<0.05), together with podocyte foot processe retraction, effacement and loss of cell contact. DEX significantly prevented the shrinkage and apoptosis of podcyte.(2) RT-PCR and Western blot found CD2AP mRNA and protein expression prone to decrease in PAN group while compared with control group. The distribution of CD2AP becames abnormal in PAN group. DEX can increased CD2AP mRNA and protein expression significantly and alleviate the abnormal distriution while compared with PAN goup.(3) Western blot showed TRPC6 protein expression significant increase while Immunocytochemistry suggested TRPC6 disappeared in the cellular membrane after PAN treatment. DEX decreased TRPC6 protein expression level at 48h(P< 0.01). The abnormal distriution of TRPC6 were also alleviated by the protection of DEX.Conclusions PAN damages podocyte. The expression and distribution of CD2AP and TRPC6 become abnormal. DEX exerts a direct action to podocyte which retains the integrity of slit diaphragm, againsts podocyte injury and alleviates proteinuria via stabilized mRNA, protein expression and distribution of CD2AP and TRPC6.