High-fat Diet Induces Renal Injury By Promoting Oxidative Stress And Mitochondrial Dysfunction

Objectives: With the change of dietary habits and lifestyle,the obesity rate shows an increasing trend.At present,about 33% of adults in the world are overweight or obese,and the obese and overweight populations have exceeded 60% of the total population in the United States.In China,the number of obese people is 250 million,ranking the first in the world.Obese subjects face serious health problems,which cause huge medical expenses.Obesity is a core manifestation of metabolic syndrome,which is characterized by concurrent existence of hyperglycemia,diabetes,dyslipidemia and cardiovascular disease.In the obese state,the enlarged white adipose tissue not only serves to store triglycerides but also secrets a large amount of adipokines,pro-inflammatory cytokines and chemokines,which make obesity a chronic inflammatory state.Chronic inflammation contributes to the development of insulin resistance.Under obesity lipolysis is increased in the adipose tissue that releases a large amount of free fatty acids(FFAs)into the bloodstream,which influx into peripheral tissues such as the liver and skeletal muscle causing lipotoxicity to the tissues.Recent epidemiological studies have demonstrated obesity as a major and independent risk factor for the development of chronic kidney disease(CKD)and end stage renal disease.In the chronic hyperlipidemic state excessive lipid accumulation also occurs in the kidney,and lipotoxicity drives the activation of pro-inflammatory,pro-fibrogenic and pro-apoptotic pathways,causing cell injury and renal dysfunction.Long term high fat diet(HFD)feeding,which results in obesity and produces metabolic syndrome,has been shown to alter renal lipid metabolism and induce lysosomal dysfunction leading to renal injury in mice.Lipotoxicity induces mitochondrial dysfunction and promotes oxidative stress and endoplasmic reticulum stress in tissues.Mitochondria play an essential role in cellular metabolism and energy production that are required for all cellular functions.Mitochondria are the primary source of intracellular reactive oxygen species(ROS)production and contain a self-destructive arsenal of apoptogenic factors that can be unleashed to promote programmed cell death.It is now well recognized that mitochondria are a dynamic subcellular organelle that undergoes constant fusion and fission in response to metabolic and environmental stresses(such as excess nutrient intakes).Mitochondrial fusion and fission play important roles in maintaining mitochondrial functions.Fusion helps reduce stress by mixing the contents of partially damaged mitochondria,whereas fission is needed to create new mitochondria.Fission also contributes to quality control by removing damaged mitochondria and facilitating apoptosis during high levels of cellular stress.Fission is mediated by a cytosolic dynamin family member dynamin-related protein 1(Drp1),which is recruited from the cytosol to form spirals around mitochondria that constrict to sever both inner and outer membranes.The loss of mitochondrial membranes or increased mitochondrial membrane permeability during mitochondrial fission releases cytochrome c into the cytoplasm,where cytochrome c binds to apoptotic protease-activating factor 1 to form apoptosome,which activates downstream caspases to drive apoptosis.Drp1 is regulated by multiple posttranslational modifications(such as phosphorylation)that are induced by increases in intracellular Ca2+ or ROS.Previous studies have linked ROS and mitochondrial dysfunction to renal injury in experimental models of both acute kidney injury and CKD,but the specific mechanism of kidney injury caused by long-term high-fat diet is not clear.Chronic hyperglycemia and hyperlipidemia associated with obesity drive excess nutrient influx to the kidney.We have recently reported that kidney injury occurs under nutrient overload as a result of ectopic lipid accumulation and epigenetic activation of prolipogenic and pro-fibrotic pathways within the kidney.In this report we presented evidence that HFD feeding promotes renal injury by inducing oxidative stress and mitochondrial dysfunction that lead to excess renal tubular apoptosis.Methods: 1.Wild type C57BL/6 mice were fed HFD for 16 weeks to induce obesity,diabetes and renal dysfunction.The following parameters were measured in the mice:plasma cholesterol,triglycerides and free fatty acids,urinary albumin and creatinine,and plasma BUN and creatinine.Glucose tolerance test and insulin tolerance test were performed.2.The morphology of liver was assessed by H&E staining.The expression of lipogenic genes in the liver and kidney were measured using Western blot and real-time RT-PCR.The contents of cholesterol and triglyceride in kidney were assayed using commercial assay kits.The m RNA levels of pro-inflammatory cytokines,chemokines and leptin in abdominal fat were measured by real-time RT-PCR.3.PAS staining was used to assess the changes of renal structure and the degree of glomerular fibrosis.The thickness of glomerular basement membrane,morphological changes of podocytes and mitochondria were assessed by transmission electron microscope(TEM).4.Dichlorodihydrofluorescein diacetate(DCFH-DA)probe was used to detect ROS,TUNEL was used to detect apoptosis of renal tubular epithelium,and pro-apoptotic factors were detected by Western blotting.5.Renal tissue from HFD-induced obese mice was cultured ex vivo in the presence of a ROS inhibitor and the changes in oxidative stress,mitochondrial fission and pro-apoptotic factors were assessed.HK-2 cells and SV40 MES 13 mesangial cells were treated with high glucose(HG),tumor necrosis factor ?(TNF?)(30 ng/ml)and/or palmitic acid(PA)(100 ?M),and ROS production was measured using DCFH-DA probe to evaluate the level of oxidative stress.Mitotrack red fluorescence staining and JC-1 staining were used to evaluate the changes of mitochondrial membrane potential and the function of mitochondria.Key proteins involved in fatty acid synthesis,oxidative stress,mitochondrial dysfunction and apoptosis were detected by Western blotting.Results: 1.Obesity,diabetes mellitus and renal insufficiency were observed in C57BL/6 mice fed HFD for 16 weeks,including increased plasma cholesterol,triglycerides and free fatty acids,glucose intolerance and insulin resistance,and accumulation of blood BUN and creatinine,and proteinuria.2.HFD feeding significantly increased the contents of triglyceride and cholesterol in the kidney,and activated the lipogenic pathways for cholesterol and triglyceride synthesis.3.The damage of renal tubules and glomeruli in HFD fed mice was obvious,including defects in glomerular filtration barrier and increases in apoptosis of renal tubular cells.4.HFD feeding also increased oxidative stress in renal tubular cells and induced mitochondrial fission,thus activating the pro-apoptotic pathway.5.ROS inhibitors reduced oxidative stress in renal tubular cells,prevented mitochondrial dysfunction and inhibited apoptosis.6.In HK-2 and mesangial cell cultures,high glucose,palmitic acid and TNF? activated the lipogenic pathways,increased oxidative stress,promoted mitochondrial fission and induced the proapoptotic pathway,all of which were ameliorated by inhibitors that block ROS production.Conclusion: Long-term HFD feeding can lead to kidney damage,which is caused by tissue lipid accumulation,increased oxidative stress and mitochondrial dysfunction that triggers programmed cell death of renal tubular cells.Blockade of ROS production protects the mitochondria and prevents kidney injury caused by HFD feeding.Our results have therapeutic implications for the management of obesity-induced kidney injury.