Myo-inositol Oxygenase Modulates Oxidative Stress And Necroptosis In Cadmium Induced Kidney Injury

OBJECTIVE: To investigate the role and potential mechanism(s)by which Myo-inositol oxygenase(MIOX),an enzyme specifically localized in the renal proximal tubules,potentiates cadmium induced renal tubular damage.This may yield novel potential therapeutic targets for amelioration of cadmium induced kidney injury.METHODS: In in vitro experiments,two types of renal tubular epithelial cells i.e.,HK-2 and LLC-PK1 cells,were applied.The renal tubular cells were treated with cadmium(15 μM)for 4-20 h to detect changes of MIOX expression profile.Besides,to demonstrate the regulation of cadmium induced tubular cell injury,oxidative stress and necroptosis by MIOX expression profile,MIOX knockdown and overexpression tubular cells were treated with cadmium,followed by the evaluation of cell death,morphology,reactive oxygen species(ROS)level and necroptosis sensors.Furthermore,the role of necroptosis in cadmiuminduced renal tubular cell injury was elaborated by using necroptosis inhibitor Nec-1(50 μM).In in vivo studies,CD1 mice and C57BL/6J mice were treated with cadmium(2 mg/kg/day and 3 m/kg/day for 14 days)to further clarify the changes of MIOX expression profile compared to in vitro strudies.Subsequently,C57BL/6J mice with different MIOX expression levels i.e.,wild-type mice(WT),renal proximal tubules MIOX conditional overexpression mice(MIOX-TG)and renal proximal tubules MIOX conditional knockout mice(MIOX-KO),were treated with Cd(2mg/kg/day for 14 days).Mice were sacrificed and perturbations in renal patholocial morphology,ROS level and necroptosis sensors were evaluated to elaborate the regulation of cadmium induced tubular injury,oxidative stress and necroptosis by MIOX expression profile in vivo.Finally,necroptosis inhibitor Nec-1(1.65 mg/kg/day for 14 days)was applied to CD1 mice aimed to probe the role of necroptosis in cadmium induced renal tubular injury.RESULTS: 1.In vitro and in vivo studies showed cadmium induced dynamic changes of MIOX expression.The highest level of MIOX expression in renal tubular cells was observed after treated with cadmium for 4h,and then began to decline.In vivo studies,MIOX expression was increased in the low dose cadmium treated group and down-regulated in the high dose group;2.Overexpression of MIOX promoted cadmium induced kidney injury,ROS generation and necroptosis,while knockdown or knockout MIOX attenuated these conditions;3.Nec-1 partially alleviated cadmium induced renal tubular cells injury,which was accompanied by altered oxidative stress levels.CONCLUSION: MIOX potentiated redox injury and necroptosis are intricately involved in the pathogenesis of cadmium induced nephropathy.