The Role Of Autophagy Mediated By Mitochondria-produced ROS In Renal Tubular Epithelial Cell Injury Induced By Albumin Overload

Objective: Urinary protein is not only a sign of clinical diagnosis of kidney disease,but also can aggravate renal injury.As the most important component of urinary protein,albumin has been proved to cause apoptosis,mitochondrial dysfunction and oxidative stress in renal tubular cells.Previous studies have shown that ROS mediated the activation of autophagy induced by urinary protein overload,and autophagy alleviated renal tubular epithelial cell damage.However,the specific mechanisms of autophagy induced by urinary albumin in renal tubular epithelial cells and cell damage reduced by autophagy are unclear.Mitochondria are the organelles that regulate energy metabolism in eukaryotic cells.Various stimuli can cause mitochondrial damage and injured mitochondria can release excessive ROS,which can activate autophagy through different mechanisms.Autophagy can further scavenge damaged mitochondria and excessive ROS to promote cell survival.Therefore,we speculate that ROS derived from mitochondria may mediate the autophagy activation in renal tubular epithelial cell injury induced by albumin overload,and autophagy activation may protects against renal tubular injury induced by albumin overload by promoting removal of damaged mitochondria and reducing ROS.In this study,we used a variety of molecular biology technology to investigate the role of autophagy mediated by mitochondria-produced ROS in renal tubular epithelial cell injury induced by albumin overload.Methods:(1)Establishment of urinary albumin overload model in vitro: We treated human proximal tubular epithelial cells(HK-2 cell)with human serum albumin at a concentration of 8mg/ml for 8h in Albumin group(ALB group).(2)Groups of the effect of antioxidant intervention on autophagy induced by albumin in HK-2 cells: we established control group,ALB group,ALB+NAC group,NAC group;Groups of the effects of mitochondrial function intervention on the ROS production and autophagy of HK-2 cells induced by albumin: we established control group,ALB group,ALB+CCCP group,ALB+ALC group;Groups of The effects of autophagy intervention on mitochondrial damage,ROS,apoptosis and cell viability of HK-2 cells treated with albumin: we established control group,ALB group,ALB+RAP group,ALB+CQ group.(3)LC3,p62,and cytoplasmic cytochrome-c expression in HK-2 cells were evaluated by immunofluorescence and immunoblotting.ROS levels were examined by DCF immunofluorescence.Mitochondrial membrane potential was tested by JC-1 kit staining.TUNEL assay was applied to measure the apoptosis rate of HK-2 cells and MTT was used to assay viability of HK-2 cells.Results:(1)The effects of albumin on autophagy,ROS level and mitochondrial function in HK-2 cells: Immunofluorescence and Western blotting showed that the expression of LC3 and cytoplasmic cytochrome-c in HK-2 cells in ALB group was significantly higher than that in control group,and the level of p62 in ALB group decreased compared with control group(P<0.05).The results of DCF immunofluorescence showed that the expression of ROS in ALB group was more than that of control group(P<0.05).JC-1 staining showed that the mitochondrial membrane potential of the ALB group decreased significantly compared with the control group(P<0.05).(2)The effect of antioxidant intervention on autophagy induced by albumin in HK-2 cells: Western blotting showed that compared with ALB group,the expression of LC3 protein in ALB+NAC group was down regulated(P<0.05),but there was no significant difference in LC3 protein level between NAC group and control group(P>0.05).(3)The effects of mitochondrial function intervention on the ROS production and autophagy of HK-2 cells induced by albumin: The results of DCF assay showed that compared with ALB group,the expression of ROS in the HK-2 cells in ALB+CCCP group obviously increased,which markedly decreased in ALB+ALC group(P<0.05).Immunoblotting showed that the level of LC3 and cytoplasmic cytochrome-c protein in ALB+CCCP group was further increased than that in ALB group(P<0.05),and the p62 level was further decreased than that in ALB group(P<0.05),while ALC addition exerted opposite results.(4)The effects of autophagy intervention on mitochondrial damage,ROS,apoptosis and cell viability of HK-2 cells treated with albumin: Western blotting showed that the cytoplasmic cytochrome-c level in ALB+RAP group was further reduced than that of ALB group(P<0.05),while the cytochrome-c level of ALB+CQ group was significantly higher than that of ALB group(P<0.05).JC-1 staining immunofluorescence showed that the mitochondrial membrane potential in ALB+RAP group was significantly higher than that of ALB group(P<0.05),and the mitochondrial membrane potential in ALB+CQ group was significantly lower than that of ALB group(P<0.05).DCF immunofluorescence showed that compared with ALB group,the ROS level of HK-2 cells decreased in ALB+RAP group(P<0.05),while ROS level in ALB+CQ group was further increased than ALB group(P<0.05).The results of TUNEL kit and MTT showed that cell apoptosis rate increased and cell viability in ALB group decreased than control group,which were aggravated in ALB+CQ group and alleviated in ALB+RAP group(P<0.05).Conclusion: This study proved that(1)Albumin treatment can lead to autophagy activation,increased ROS and mitochondrial injury.(2)Antioxidation intervention can reduce the expression of autophagy induced by albumin.(3)Inhibition of mitochondrial function can further promote ROS release and autophagy in HK-2 cells,while stabilization of mitochondria function can inhibit ROS production and autophagy in HK-2 cells.(4)Autophagy inducer can mitigate mitochondrial damage,reduce ROS level and cell apoptosis,and enhance cell viability in HK-2 cells,which were reverted by autophagy inhibitor.In conclusion,ROS produced from injured mitochondria mediated albumin overload induced autophagy activation in renal tubular epithelial cells,and autophagy alleviated renal tubular epithelial cell damage by clearing damaged mitochondria and reducing ROS.