The Effects Of Ox-LDL On Contrast-induced Renal Tubular Cell Injury:the Protective Role Of Mitophagy

Objective:Contrast-induced acute kidney injury is one of the main causes of hospitalacquired acute kidney injury.Oxidized low-density lipoprotein is an important lipid product in hyperlipidemia.Previous studies have shown that hyperlipidemia aggravates contrast-induced acute kidney injury.Mitophagy timely clearing dysfunctional mitochondria reduces mitochondrial oxidative stress,which is of great significance for maintaining normal cell growth and metabolism.However,the role of mitophagy in hyperlipidemia aggravating contrast-induced kidney injury is still unclear.In this study,we aimed to determine effects of ox-LDL on contrast-induced renal tubular epithelial cells injury and the potential role of mitophagy in ox-LDL aggravated contrast-induced renal tubular epithelial cells injury.Methods:(1)Human renal proximal tubular epithelial cell(HK-2)were cultured as the research object in vitro.(2)HK2 cells were treated with ox-LDL concentration gradient(0,25,50 μg/ml)for 2 hours and then treated with iohexol(100 mg I / ml)for 4 hours.Ox-LDL(50 μg/ml)pretreated HK2 in time gradient(0h,2h,4h)then treated with iohexol(100mg I/ml)for 4hours.Cell viability was measured by cell counting kit-8 to explore the optimal ox-LDL concentration treatment time in the vitro model of ox-LDL aggravates CI-AKI.(3)Groups of the effects of ox-LDL on contrast-induced renal tubular cell injury:Control group,ox-LDL group,Iohexol group,Iohexol+ox-LDL group;(4)Groups of the effects of RAP in ox-LDL aggravating contrast-induced renal tubular cell injury: Control group,RAP group,Iohexol+ox-LDL group,Iohexol+ox-LDL+RAP group;(5)Groups of si RNA transfection:Control group,Iohexol + ox-LDL group,Parkin si RNA group,Parkin si RNA+Iohexol+ox-LDL group;(6)Detection index: Cell apoptosis were detected by TUNEL assay and Western Blot;Mitochondrial function were detected by JC-1 kit staining and Mito SOX fluorescent staining;Immunofluorescence staining technology was used to detect the fluorescence intensity of LC3II;Immunoblotting was used to detect the expression of autophagy and mitophagy related proteins LC3,P62,Beclin-1,PINK1,Parkin;Lyso Tracker and Mito Tracker Colocalize was used to detect mitophagy.Results: Ox-LDL 50umol/L pretreat HK2 cells for 2 hours,and co-treated with Iohexol 100 mg I/ml for 4 hours,cells vitality was significantly reduced(P<0.05).Cell apoptosis increased in rest of groups compared with control group and was significantly increased in Iohexol+ox-LDL group(P<0.05).MMP decreased in rest of groups compared with control group,and was significantly decreased in Iohexol+ox-LDL group(P<0.05).Compared with control group,the mitochondrial ROS level increased in ox-LDL group,Iohexol group ox-LDL+Iohexol group and was significantly increased in Iohexol+oxLDL group(P<0.05).Active autophagy demonstrated by the overexpression of LC3 II and Beclin1 and downexpression of P62(P<0.05)and increased fluorescence intensity of LC3 II in in rest of groups compared with control group(P<0.05).Mitochondrial and lysosomal fluorescence co-localization increased in in rest of groups compared with control group and was increased in Iohexol+ox-LDL+RAP group compared with Iohexol+ox-LDL group(P<0.05).The expression of LC3 II,PINK1,Parkin increased in RAP group,Iohexol group,Iohexol+ox-LDL group and Iohexol+ox-LDL+RAP group while P62 decreased compaired with control group,and was increased in Iohexol+ox-LDL+RAP group while P62 decreased compaired with Iohexol+ox-LDL group(P<0.05);Cell apoptosis of Iohexol+ox-LDL+RAP group was reduced compaired with Iohexol+ox-LDL group(P<0.05).Mitochondrial ROS release was significantly reduced in Iohexol+ox-LDL+RAP group compared with Iohexol+ox-LDL group(P<0.05).Parkin gene silencing significantly downregulated expression of LC3 II,parkin and upregulated P62 expression in Parkin si RNA + Iohexol + ox-LDL group compared with Iohexol + ox-LDL group(P<0.05);Western Blot results showed that the expression level of Cleared-caspase3 in Parkin si RNA + Iohexol + ox-LDL group was significantly increased compared with Iohexol + ox-LDL group(P <0.05).Conclusion: Ox-LDL aggravates contrast-induced HK2 cell apoptosis,mitochondrial dysfunction mitochondrial oxidative stress and autophagy.Mitophagy plays a protective role in HK2 cell injury induced by ox-LDL and iohexol co-treatment.