Dexamethasone Againsts Apoptosis Of Podocytes Via Stabilizing Activation Of CD2AP -PI3K/Akt Signaling Pathway

Objective To investigate whether dexamethasone protects podocytes against PAN-induced apoptosis requiring activation of the CD2AP-PI3K/GSK3βsignaling pathway.Methods Mouse podocytes were cultured in 0.02% DMSO in control group, subjected to puromycin aminonucleoside (PAN,50μg/ml) treatment alone or with dexamethasone(DEX,1μmol/L) in other two groups. The mRNA expression and protein expression of CD2AP,PI3K p85 and Bad were respectively detected by indirect semi-quantitative RT-PCR and Western blot. Double immunofluorescence was used to stain the molecules:CD2AP/PI3K p85, A laser scanning confocal microscopy was applied to study the distributions and the relationships of CD2AP and PI3K p85 in the Mouse podocytes. Mouse Podocytes were incubated in media containing either buffer(as control) or increasing concentrations of PAN (12.5,25,50,75,100μg/ml) for variable time periods. The podocytes were also treated with increasing concentrations of DEX(10-1-101μmol/L) or both with LY294002 (inhibitor of PI3K,25mmol/L).At the end of the incubation period, apoptosis was evaluated by flow cytometric analyses.Activation of Akt and GSK3βphosphorylation of cultured mouse podocytes was evaluated by Western blot.Results (1)Semi-quantitative RT-PCR revealed that the mRNA and protein expressions of CD2AP and the protein of PI3K p85 significantly decreased in response to PAN stimulation, and the colocalization between CD2AP and PI3K p85 changed to abnormal, but the mRNA and protein expressions of Bad markedly increased in PAN group, apoptosis of cultured podocytes was induced by PAN in a dose- and time-dependent manner. The mRNA and protein expressions of CD2AP ang the protein expression of PI3K p85 increased in DEX group while compared with PAN group, DEX inhibited the abnormal distribution of colocalization between CD2AP and PI3K p85,which was induced by PAN, both the mRNA and protein expressions of Bad and apoptosis of podocytes markedly decreased in DEX group. (2) PAN inhibited the activity of Akt and GSK3βin a dose-dependent manner at 15min or 30min. This was accompanied by a significant increase in apoptosis, treatment with DEX not only attenuated the proapoptotic effect of PAN, but also reversed its effects on Akt and GSK3βsignaling pathway, which in a DEX dose-dependent manner. (3) Akt and GSK3βactivity was further inhibited by the addition of LY294002 1h before the podocytes in the presence of PAN, and treatment with DEX neither can attenuated the proapoptotic effect of PAN nor reversed its effects on Akt and GSK3βsignaling pathway.Conclusion DEX exerts a direct action to podocytes, so againsts podocyte injury and alleviates proteinuria via stabilizing expressions and distribution of CD2AP and activation of PI3K/Akt/GSK3βsignaling pathway.