| Objective In order to demonstrate the possible anti-proteinuria mechanisms of dexamethasone, a study was designed to explore the podocyte apoptosis rate and expression and distribution of podocin and CD2AP 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 apoptosis rate was detected by flow cytometry,the distribution, mRNA expression and protein expression level of podocin and CD2AP 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<0.01), together with podocyte foot processe retraction, effacement and loss of cell contact. The apoptosis rate was increased at 48h(P<0.05).RT-PCR found podocin and CD2AP mRNA expression prone to decrease, Western blot showed podocin protein expression significant decrease and immunocytochemistry suggested podocin and CD2AP disappeared in the cellular membrane after PAN treatment.(2) DEX significantly prevented the shrinkage and apoptosis of podcyte,increased podocin and CD2AP mRNA expression,and increased podocin protein expression level at 48h(P< 0.05). The abnormal distriution of podocin and CD2AP were also alleviated by the protection of DEX.Conclusion DEX exerts a direct action to podocyte which retains the integrity of slit diaphragm, againsts podocyte injury and alleviates proteinuria via decreased apoptosis rate and stabilized mRNA, protein expression and distribution of podocin and CD2AP. |
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