The Role And Mechanism Of Ferroptosis Mediated By Mitochondrial Damage In Obesity-related Kidney Injury

Background:Obesity,as a risk factor for many metabolic-related diseases,seriously threatens human life and health.Obesity-related nephropathy,a renal complication of obesity,has a relatively insidious onset and an increasing incidence.However,the mechanism of how obesity triggers renal injury is still unclear and require further investigation.Ferroptosis is an iron-dependent,non-apoptotic cell death mode characterized by increased iron ions and accumulation of lipid peroxidation.Studies have shown that ferroptosis is closely related to the pathological processes of many diseases,such as tumours,ischaemia-reperfusion injury,degenerative diseases and other disorders.However,there is limited research on whether ferroptosis is involved in obesity-related nephropathy and its mechanisms.Aims:To elucidate the role of ferroptosis in obesity-related kidney injury and its molecular mechanism through in vivo and in vitro experiments;to investigate the ameliorative effect of exenatide,a glucagon-like peptide-1（GLP-1）receptor agonist,on ferroptosis and obesity-related kidney injury.We hope to provide new ideas for the pathogenesis and treatment of obesity-related nephropathy.Methods:（1）High-fat fed obese mouse model:Six-week-old male C57BL/6J mice were randomly allocated into two groups:a control group fed with normal chow diet（NCD group）and a high-fat diet group（HFD group）.After 20 weeks of feeding,the mice were euthanized,and various parameters including blood glucose,blood lipids,urinary protein,and creatinine levels were measured.Renal histopathological changes were evaluated using HE staining for renal glomeruli and tubules.Lipid deposition in the kidneys was assessed by oil-red O staining;and the levels of renal tubular injury markers were detected by RT-q PCR and Western blot techniques.（2）High-fat-induced renal tubular epithelial cell injury model:A model of palmitic acid-induced renal tubular epithelial cell injury was constructed with different concentration gradients,and the effect of palmitic acid on the activity of renal tubular epithelial cells was detected using CCK-8;Intracellular triglyceride levels were measured by triglyceride kits,and lipid deposition was confirmed by Oil Red O and Bodipy 493/503 staining.（3）Detection of ferroptosis indicators:Malondialdehyde（MDA）levels were measured using an MDA assay kit,and iron ion levels were determined using an iron ion assay kit.Fluorescent probes were employed to measure reactive oxygen species levels in renal tubular epithelial cells,while RT-q PCR and Western blot techniques were used to detect the expression levels of glutathione peroxidase 4（GPX4）and prostaglandin-endoperoxide synthase 2（PTGS2）proteins.（4）Assessing mitochondrial function and morphology:Mitochondrial membrane potential changes were evaluated by flow cytometry and fluorescence probe staining（JC-1）.Transmission electron microscopy was utilized to observe mitochondrial morphological alterations in mouse kidney,and immunofluorescence was employed to visualize mitochondrial autophagy and morphology changes.Western blot analysis was conducted to detect the expression levels of proteins associated with mitochondrial dynamics and autophagy.（5）Evaluating the impact of AMPK/ULK1/FUNDC1 mediated mitochondrial autophagy on ferroptosis:Activation or knockdown of AMPK level in renal tubular epithelial cells,mitochondrial autophagy-related protein expression was detected by Western blot;changes in the level of mitochondrial membrane potential were detected by flow cytometry and in situ staining of fluorescent probe.Fluorescent probes were used to detect the level of reactive oxygen species in renal tubular epithelial cells;changes in intracellular MDA and ferrous ion levels were detected using kits;and the expression of ferroptosis-related proteins was detected by RT-q PCR and Western blot techniques.（6）Exenatide intervention experiment:In vivo experiments involved randomly assigning six-week-old male C57BL/6J mice to the NCD group,HFD group,and exenatide intervention group（Exenatide,EXE group）.Following 16 weeks of high-fat feeding,the EXE group received subcutaneous injections of exenatide,while the other two groups received saline injections as controls.After another 4 weeks of continued feeding,the mice were euthanized,and the renal lesions were detected as described previously.For in vitro experiments under palmitic acid modeling conditions,control,palmitic acid stimulation,exenatide and palmitic acid-treated group,and exenatide-treated group,and changes in the levels of cellular lipid deposition,changes in mitochondrial autophagy,and changes in ferroptosis-related indexes were detected by the same methods as described previously.Results:（1）Mice in the HFD group had significantly higher body weight,higher levels of urinary protein,creatinine,and urea nitrogen,and higher levels of triglycerides and cholesterol in serum and renal tissues compared with mice in the NCD group.Oil Red O staining revealed increased lipid deposition in the renal tissues of the HFD group.Additionally,HE staining indicated glomerular hypertrophy and prominent vacuolar degeneration in renal tubules,with elevated levels of renal tubular injury markers kidney injury molecule-1（KIM-1）and neutrophil gelatinase-associated lipocalin（NGAL）.（2）As the concentration of palmitic acid increased,the viability of renal tubular epithelial cells gradually decreased,accompanied by increased intracellular lipid deposition.This was characterized by an increase in Oil Red O staining for lipid droplets,enhanced in situ fluorescence staining with Bodipy 493/503,and elevated intracellular triglyceride levels.（3）Ferroptosis is involved in obesity-related kidney injury.In kidney tissues of HDF mice and high-fat-induced renal tubular epithelial cell injury model,the level of key regulator of ferroptosis GPX4 was decreased,the level of PTGS2,MDA and ferrous ions was increased,and transmission electron microscopy revealed a reduction in mitochondrial volume,a decrease in the number of mitochondrial cristae,impaired intracellular mitochondrial membrane potential was detected by JC-1,and the results of DCFH-DA fluorescent probe suggested increased intracellular reactive oxygen species content.（4）Mitochondrial damage contributes to ferroptosis in obesity-related nephropathy.In the high-fat-induced renal tubular epithelial cell injury model,the expression of mitochondrial fusion protein MFN2 increased,while the expression of fusion protein DRP1^Ser616/DRP1 decreased.Meanwhile,the results of fluorescent labelling suggested that the morphology of mitochondria in HK-2 cells of the obesity-related nephropathy group showed fragmentation,with a decrease in the mitochondrial membrane potential and an increase in the accumulation of superoxide,and that the Ad-m Cherry-GFP-LC3B dual-colour fluorescence detection system labelled intracellular autophagosomes and found that a large number of yellow aggregated particles were visible in the cells,proving that mitochondrial autophagy was inhibited.Application of autophagy activator（Rapamycin）or superoxide scavenger（Mito-TEMPO）improved mitochondrial damage and alleviated ferroptosis.（5）The AMPK/ULK1/FUNDC1 pathway is involved in the regulation of mitochondrial autophagy and ferroptosis.Application of AICAR activated AMPK expression,and compared with the palmitate stimulation group alone,AICAR pretreatment inhibited p62 expression,up-regulated LC3II and FUNDC1 expression,and improved mitochondrial autophagy impairment.In situ fluorescence labelling revealed that AICAR could rescue mitochondrial membrane potential damage and reduce reactive oxygen species deposition in HK-2 cells.In addition,AICAR treatment increased GPX4 level and decreased PTGS2 expression,while AICAR reduced intracellular MDA and iron ion levels,suggesting that AICAR could rescue cell activity by alleviating mitochondrial damage and mitigating ferroptosis.On the contrary,application of AMPK si RNA to knock down AMPK expression aggravated mitochondrial autophagy impairment and ferroptosis.（6）Exenatide inhibits ferroptosis by activating the AMPK/ULK1/FUNDC1pathway.Exenatide intervention alleviated lipid deposition in obesity-related nephropathy.Triglyceride and cholesterol content in mouse kidney tissue were significantly reduced,and oil red O staining confirmed the reduction of renal lipid deposition.In vitro experiments showed that exenatide could attenuate the palmitic acid-induced elevation of intracellular triglyceride level in HK-2 cells.In addition,exenatide could increase the expression levels of p-AMPK,p-ULK1 and FUNDC1,reduce the inhibition of mitochondrial autophagy and ferroptosis,and alleviate the kidney injury.Conclusions:（1）Ferroptosis is an important pathogenic mechanism in obesity-related kidney injury.（2）The AMPK/ULK1/FUNDC1 pathway can lead to the development of ferroptosis by affecting mitochondrial autophagic and inducing superoxide accumulation,ultimately leading to obesity-associated kidney injury.（3）Exenatide can alleviate mitochondrial autophagy,reduce the accumulation of reactive oxygen species,and suppress ferroptosis through the AMPK/ULK1/FUNDC1pathway,and ultimately alleviate the obesity-induced kidney injury.