The Effect Of Ox-LDL On Contrast-Induced Renal Tubular Cell Injury

Objective:1.To investigate the effect of ox-LDL on contrast-induced renal tubular cells apoptosis and the molecular mechanism.2.To establish in vitro model of ox-LDL aggravates contrast-induced acute renal injury.3.Based on the in vitro model of ox-LDL aggravates CI-AKI,the effect of mitophagy 、 mitochondrial injury and oxidative stress on ox-LDL aggravates contrast-induced renal tubular cells apoptosis were discussed.Methods:1.NRK-52 E cells were cultured as the research object in vitro.2.NRK-52 E cells were exposed to iohexol(100 mgI/mL)and different concentrations(0,25,50 m g/mL)of ox-LDL.Cells Apoptosis was detected by flow cytometry,and explore the optimal ox-LDL concentration in the vitro model of ox-LDL aggravates CI-AKI.3.The optimal ox-LDL concentration in the above experiments was used to establish an in vitro model of ox-LDL aggravates CI-AKI,and subsequent experiments were conducted.NRK-52 E cell were divided into four groups,Control group:cells were normal cultured in serum-free medium,Iohexol group: cells were treated with 100 mgI/mL iohexol,ox-LDL group: cells were treated with 50 mg/mL ox-LDL,Iohexol+ox-LDLgroup: cells were treaed with 100mgI/mL iohexol and50ug/mL ox-LDL.Apoptosis and intracellular ROS were detected by flow cytometry.Cyt c was detected by western blot.JC-1 method was used to detected MMP.To investigate the molecular mechanism of ox-LDL aggravates contrast-inducing renal tubular cells apoptosis.Colocalization expressions of Lyso-Tracker Red labeled lysosomes and Mito-Tracker Green labeled mitochondria were observed by fluorescence microscope.To investigate whether mitophagy is involved in the ox-LDL aggravates contrast-induced renal tubular cell apoptosis.Results:1.Compared with control gruop,ox-LDL aggravated iohexol-induced cell apoptosis in a concentration-dependent manner(P<0.05).The apoptosis rate was the highest with ox-ldl at a concentration of 50 ug/ml.(P<0.05).2.Compare with control group,apoptosis rate increased in iohexol group,ox-LDL group,iohexol and ox-LDL group(P<0.05).Compare with iohexol group,cell apoptosis was significantly increase in the ox-LDL and iohexol group(P<0.05).3.Compared with control group,the expression of ROS production increased in iohexol group、ox-LDL group、iohexol and ox-LDL group(P<0.05).Compare with iohexol group,ROS production was significantly increase in the ox-LDL and iohexol group(P<0.05).4.Compared with control group,iohexol group、ox-LDL group、iohexol and ox-LDL group induced a increased expression in cytosolic/mitochondrial Cyt c(P<0.05).Compare with iohexol group,cytosolic/mitochondrial Cyt c was significantly increase in the ox-LDL and iohexol group(P<0.05).5.Compared with control group,MMP were decrease in iohexol group、ox-LDL group、iohexol and ox-LDL group(P<0.05).Compare with iohexol group,MMP was significantly decrease in the ox-LDL and iohexol group(P<0.05).6.Apoptosis rate was positively correlated with ROS expression(r=0.9167,P<0.01)and cytoplasmic/mitochondrial Cyt c expression(r=0.8566,P<0.01),and negatively correlated with MMP(r = 0.8276,P < 0.01).7.Compared with the control group,the co-localization expressions of Lyso-Tracker Red labeled lysosomes and Mito-Tracker Green labeled mitochondria in the iohexol group were increased,compared with the iohexol group the co-localization expressions of Lyso-Tracker Red labeled lysosomes and Mito-Tracker Green labeled mitochondria was increased in iohexol and ox-LDL group.Conclusions:1.Ox-LDL aggravates contrast-induced renal tubular cells apoptosis.2.An in vitro model of ox-LDL aggravates CI-AKI is successfully established.3.Oxidative stress and mitochondrial injury are one of the mechanisms by which ox-LDL aggravates contrast-induced renal tubular cells apoptosis.4.Mitophagy may be involved in the mechanism of ox-LDL aggravating contrast-induced renal tubular cells apoptosis.