ABSTRACT
Objective To monitor reactive oxygen species(ROS) levels in renal proximal tubular epithelial cells cultured in vitro under albumin overload and their effect on autophagy activation,and to investigate the underlying mechanisms.Methods HK-2 cells cultured in vitro were divided into a normal control group (NC),an albumin (ALB) group,an ALB + N-acetyl-L-cysteine (NAC) group,an NAC group,an ALB+chloroquine(CQ)group and an ALB+ rapamycin(RAP)group.The expression of molecular markers for autophagy,Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3),were detected by Western blotting.ROS levels were measured by a dichloro-dihydro-fluorescein diacetate(DCFH-DA) immunofluorescence method.Results Albumin overload-induced autophagy was activated in HK-2 cells as assessed by the significant upregulation of Beclin-1 and LC3-Ⅱ levels,compared with the control group(both P＜0.05).Albumin overload triggered oxidative stress in HK-2 cells as revealed by the increased production of ROS and the enhancement of green fluorescence brightness,compared with the control group [(22.47 ± 0.79) vs.(10.15 ± 0.57),P ＜ 0.05].The antioxidant NAC significantly inhibited albumin-induced autophagy(P ＜0.05).Moreover,the increase in ROS levels caused by albumin overload was promoted by chloroquine and blocked by rapamycin (both P＜0.05).Conclusions The mechanisms for albumin overload-induced autophagy in HK-2 cells were related to oxidative stress.