Effect of erythropoietin on mesenchymal stem cells proliferation in vitro under acute kidney injury microenvironment and its mechanism / 中华肾脏病杂志

ABSTRACT

Objective To investigate the effect of erythropoietin (EPO) on mesenchymal stem cells (mMSCs) proliferation under acute kidney injury (AKI) microenvironment,and to study its possible mechanism.Methods C57BL/6 mice's MSCs (mMSCs) were isolated by Percoll density gradient centrifugation and adherence cultivation.Surface markers were identified by flow cytometry.AKI mice models were made by clamping bilateral renal pedicles for 30 minutes and reopening for 30 minutes.Then both renal cortex was drew immediately to make IR kidney homogenate supernatant.P3-mMSCs were divided into different groups Group A low glucose DMEM medium with 10% fetal bovine serum; Group B low glucose DMEM medium with 10% fetal bovine serum plus IR kidney homogenate supernatant; Group C low glucose DMEM medium with 10% fetal bovine serum plus IR kidney homogenate supernatant and different concentrations of EPO (1,5,10,50 U/ml).Each group was incubated for 1 d,3 d,5 d,7 d.Proliferation of mMSCs was detected by CCK-8,and apoptosis was detected by TUNEL.The protein expression of erythropoietin receptor(EPOR) and the proteins of proliferation/apoptosis related signal pathway were examined by Western blotting.Results Under IR kidney homogenate supernatant,the proliferation ability of mMSCs decreased significantly (P＜0.01),while the apoptoic percentage was significantly higher than that of Group A (P＜0.01).After intervention of EPO,mMSCs proliferation enhanced,at the same time,the apoptoic percentage decreased,in a dose-dependent manner.EPOR was positive in P3-mMSCs by Western blotting.EPO decreased the expression of caspase-3 in mMSCs under AKI microenvironment in a dose- and time-dependent manner,but increased the expression of Bcl-2.Cultured for 5 d,the expression of phosphor-Janus kinase2(p-JAK2) [(0.641 ±0.028) vs (0.456±0.012)] and phosphor-signal transducer and activator of transcription(p-STAT5)[(0.398±0.016) vs (0.209±0.020)] was significantly higher in 10 U/ml EPO group compared to group B.Conclusion Erythropoietin can promote proliferation of mMSCs in vitro under AKI microenvironment,which is mediated by EPOR and related with proliferation/apoptosis signal pathway.