Mitochondria-derived Reactive Oxygen Species Are Involved In Renal Cell Ferroptosis During Lipopolysaccharide-induced Acute Kidney Injury

Objective This study focused on the effects of LPS exposure on kinedy quality in male mice,and explore the role of mitochondria-derived ROS on renal cell ferroptosis during LPS-induced AKI.Method This study included in vivo and in vitro experiments,and the in vivo experiment was composed of three independent experiments.The in vitro experiment consisted of two independent experiments.In experiment 1,48 male mice were randomly divided into control group and LPS group.The control group was injected with normal saline,and the LPS group was injected with 2.0mg/kg LPS.The kidneys of mice were collected at 6h and 24 h.BUN,UA and Scr were detected by biochemical apparatus.The contents of GSH and MDA in mouse kidney tissue were detected.The content of total iron in mouse kidney tissue was detected.TUNEL assay was used to detect renal cell death.GPX4,4HNE and other indexes were used to detect iron death in mouse kidney cells.Mitochondrial morphology was observed by TEM.In experiment 2,48 mice were randomly divided into4 groups: the control group was intraperitoneally injected with normal saline,the LPS group was intraperitoneally injected with 2mg/kg LPS,the Fer-1 randomly divided into4 groups.The control group was injected with normal saline,the LPS and LPS + Mito Q groups were injected with 2mg/kg LPS,and the Mito Q and LPS + Mito Q groups were injected with 5mg/kg Mito Q.Mito Q was injected 1 hour before,and kidney was collected24 hours later.BUN,UA and Scr were detected by biochemical apparatus.The contents of GSH and MDA in mouse kidney tissue were detected.TUNEL assay was used to detect renal cell death.GPX4,4HNE and other indexes were used to detect iron death in mouse kidney cells.Mitochondrial morphology was observed by TEM.In vitro experiment 1.5ug/ml LPS was added into HK-2 cell culture medium to detect mitochondrial reactive oxygen fluorescence;Intracellular lipid reactive oxygen species were detected.Detection of cell death;Mitochondrial membrane potential was detected.Mitochondrial ROS and mitochondrial membrane potential were detected by flow cytometry.Western blotting was used to detect GPX4 protein levels.In vitro experiment ii Mito Q intervention to detect mitochondrial reactive oxygen species,cell death and other indicators.Results Male CD-1 mice were intraperitoneally injected with LPS（2.0 mg/kg）.Renal MDA and 4HNE residue,two markers of lipid peroxidation,were increased in LPSexposed mice.Oxidized lipids were detected in LPS-treated human HK-2 cells.In vivo,ferroptosis-characteristic ultrastructure changes,including cell volume reduction,nuclear pyknosis and smaller mitochondria,were shown in renal cortex.In vitro,abnormal alteration of mitochondrial membrane potential was observed in LPS-treated human HK-2 cells.Ferrostatin-1,a specific inhibitor of ferroptosis,attenuated LPS-evoked renal lipid peroxidation,ferroptosis-characteristic mitochondrial damage and renal cell death.Mechanistically,mitochondria-derived ROS were elevated in LPS-stimulated HK-2 cells.Mito Q,a mitochondria-targeted antioxidant,almost completely scavenged LPSstimulated mitochondrial ROS in human HK-2 cells.Moreover,pretreatment with Mito Q attenuated LPS-induced GSH depletion and lipid peroxidation in mouse kidney.Finally,pretreatment with Mito Q alleviated LPS-induced renal cell death and AKI.Taken together,these results suggest that mitochondria-derived ROS contribute,at least partially,to renal cell ferroptosis during LPS-induced AKI.Mitochondria-targeted antioxidants may be potential therapeutic agents for sepsis-induced AKI.Conclusion mitochondria-derived ROS contribute,at least partially,to renal cell ferroptosis during LPS-induced AKI.Mitochondria-targeted antioxidants may be potential therapeutic agents for sepsis-induced AKI.