Effect Of RHuEPO Pretreatment On PI3K/Akt/GSK-3β Signaling Pathway In Human Renal Tubular Epithelial Cells Apoptosis Induced By Ischemia-reperfusion Injury

Objectives To determine the relationship between PI3K/Akt/GSK-3βsignaling pathway and ischemia-reperfusion induced apoptosis in human renal tubular epithelial cells , and to explore the cell protective mechanism of rHuEPO pretreatment.Methods The human kidney tubular epithelial cells(HK-2)were cultured in vitro in different conditions as①control group received serum,②ischemia-reperfusion(I/R)group received 10umol/L Antimycin A and 1umol/L A23187,③LY294002 group received LY294002(Akt inhibitor) 10 umol/L 30 minutes before I/R treated,④Licl group received Licl(GSK-3βinhibitor) 20 umol/L 30 minutes before I/R treated,⑤rHuEPO group received EPO 20 U/ml 30 minutes before I/R treated,⑥rHuEPO +LY294002 group received EPO 20 U/ml and in the presence of LY294002 (10 umol/L) 30 minutes before I/R treated,⑦rHuEPO +Licl group received EPO 20 U/ml and in the presence of Licl (20 umol/L) 30 minutes before I/R treated. Akt,GSK-3βand Caspase-3mRNA were measured by RT-PCR and Akt,GSK-3βand Caspase-3 activation were measured by Western blot.The apoptotic ratio of HK-2 cells was monitored by flow cytometry. Cell viability was monitored by MTT.Results In comparison with the control group, the apoptotic ratio raised up to(15.2±1.43%),the expression of Akt activity decreased, GSK-3βactivity increased, Caspase-3 mRNA and activity markedly elevated in I/R group(P<0.05). However, Akt and GSK-3βmRNA remained unchanged. LY294002 group up-regulated the apoptotic ratio(18.2±2.06%), decreased the expression of Akt activity ,increased GSK-3βactivity and Caspase-3 activity . However, Licl group down-regulated the apoptotic ratio(12.3±0.85%), increased the expression of Akt activity , decreased GSK-3βactivity and Caspase-3 activity compared with I/R group ( P<0.05). rHuEPO group remarkably decreased the apoptotic ratio(11.1±1.62%), increased the expression of Akt activity, decreased GSK-3βactivity and Caspase-3 activity compared with I/R group ( P<0.05). rHuEPO +LY294002 group elevated the apoptotic ratio(13.4±1.94%), decreased the expression of Akt activity , increased GSK-3βactivity and Caspase-3 activity ,meanwhile, rHuEPO +Licl group down-regulated the apoptotic ratio(7.5±1.31%), increased the expression of Akt activity , decreased GSK-3βactivity and Caspase-3 activity compared with rHuEPO group ( P<0.05).Conclusions PI3K/Akt/GSK-3βsignaling pathway is involved in HK-2 cells apoptosis induced by ischemia-reperfusion injury and may be the key pathway by which rHuEPO pretreatment makes cell-protective effect.