Role of prolyl hydroxylases 2 in the cellular response to hypoxia activated autophagy in human renal epithelial cell model / 中华肾脏病杂志

ABSTRACT

Objective To test the hypothesis of autophagy that silencing PHD2 gene could increase hypoxia inducible factor (HIF)-1α levels in the renal medulla and attenuate hypoxia injury in cultured human renal proximal tubular epithelial cell (HK-2) under cobalt dichloride (CoCl2) exposure.Methods HK-2 cells were harvested at hour 0,6,12,24,36 and 48 after exposure to CoC12 (200 μmol/L).The role of HIF/PHD pathway in CoCl2-induced cell apoptosis/autophagy was studied by employing small-interfering RNA (siRNA).Dynamic profiles of apoptosis markers (Bax,Bcl-xl) and autophagy marker (LC3) of HK-2 cells within 48 h after exposing to CoCl2 were recorded.Alamar Blue assay was used for quantitative analysis of cellular growth and viability.Electron microscopy analysis was employed to evaluate the changes in autophagic structures.Results The protein expressions of PHD2 were gradually increased after exposing to CoCl2 (200 μmol/L),with statistics significance at 24 h and reached the peak at 48 h (both P ＜ 0.01).PHD2 siRNA reduced PHD2 levels by ＞ 60％ and significantly increased HIF-1α protein levels (P ＜ 0.01),but had little effect on HIF-2α.The protein expression of Bcl-xl was significantly up-regulated,while the level of Bax and LC3-Ⅱ/LC3-Ⅰ were down-regulated in PHD2 siRNA group (all P ＜ 0.01),compared with the negative control group.Meanwhile,either 3-Methyladenine (an autophagy inhibitor) treatment or PHD2 knockdown rescued cell death and increased cell viability through autophagy inactivation.The ratio of LC3-Ⅱ/LC3-Ⅰ and the quantity of autophagosomes were decreased,and the cell ultrastructure was also relatively intacter than the negative control group.Of interest,co-administration of HIF-1α siRNA with PHD2 siRNA abrogated renoprotective effect conveyed by PHD2 siRNA alone,suggesting that activation of endogenous HIF-1α-dependent pathways mediated the autophagy inactivation effects of PHD2 silencing.Conclusions Direct inhibition of PHD2 promotes renal epithelia cell survival against CoCl2-induced cell apoptosis/autophagy.Activation of the HIF-1α signaling pathway is required to reduce apoptosis and autophagy via up-regulating the expression of Bcl-xl protein.