Significance of abnormal expression of CD2-associated protein and phosphatidylinositol 3-kinase p85 in apoptosis of podocytes induced by puromycin aminonucleoside in vitro / 中华实用儿科临床杂志

ABSTRACT

Objective To observe the effect of puromycin aminonucleoside(PAN) on the expressions of CD2associated protein (CD2AP) and phosphatidylinositol 3-kinase (PI3 K) p85 in induced podocytes,and to explore the significance of abnormal expression of CD2AP in apoptosis of podocytes in vitro.Methods Mouse podocytes were injected into the control group and the PAN treatment group (PAN group).The cells were cultured for 8 h,24 h and 48 h respectively.The podocyte morphology was observed by phase-contrast microscope.The apoptosis ratio of podocytes was determined with flow cytometry.The mRNA expression of CD2AP was detected by real time fluorescent quantitative polymerase chain reaction.The protein expressions of CD2AP and PI3K p85 were detected by Western blot,respectively.The distributions and the relationships between CD2AP and PI3K p85 in the podocytes were detected by confocal fluorescence microscopy.Results PAN treatment led to significant shrinkage of podocytes with decreasing distribution tendency,podocyte foot process retraction as well as effacement and loss of cell contact.Compared with the control group,the apoptosis rate was increased at 24 h and 48 h[(7.52 ± 1.35)％,(17.09 ±2.53)％ vs (4.32 ±0.81％,(6.81 ± 1.34)％] in PAN group and the differences were significant (t =4.98,8.79,all P ＜ 0.05) ;CD2AP mRNA expression tended to decrease at each time point,and the differences were significant (0.34 ± 0.12,0.46 ±0.21,0.47 ± 0.10 vs 0.62 ± 0.23,0.97 ± 0.14,0.96 ± 0.23),and there were statistical differences (t =2.64,4.95,4.79,all P ＜ 0.05) ;CD2AP and PI3K p85 protein expressions tended to decrease at 24 h (0.36 ± 0.12,0.61 ± 0.20 vs 0.64 ±0.23,0.97 ±0.31) and 48 h(0.27 ± 0.15,0.48 ± 0.20 vs 0.51 ± 0.20,0.84 ± 0.35),and the differences were significant(t =2.64,2.31,2.35,2.40,all P ＜ 0.05).In the control group,the distribution of CD2AP was uniformly filamentary structure in cytoplasm and nucleus of podocytes,after PAN treatment the distribution of CD2AP changed to discontinuous coarse granular concentrated in the perinuclear.Immunocyte fluorescence showed that the distribution and the relationships between CD2AP and PI3K p85 in the podocytes became abnormal.Conclusions When the apoptosis of podocytes was induced by PAN,the expression and distribution of CD2AP were abnormal.CD2AP may play an important role in the survival of podocytes though the PI3K signaling pathway.