The Ketogenesis/SIRT3 Pathway Mediates Renoprotection By Green Tea Polyphenols Against The Oxidative Stress Induced By A High-fat Diet

Part I The effects of green tea polyphenols on the oxidative stress inrenalcortex and renal function induced by HFDObjective:Oxidative stress plays a crucial role in functional and pathological renal damage induced by high-fat diet(HFD).And phytochemicals can reduce the oxidative damage via various mechanisms.This part will observe the effects of green tea polyphneols(GTPs)on oxidative stress and renal damage induced by high-fat diet.Methods:Sixty male Wistar rats weighting 160-180g were fed standard.After one week of acclimation,all the animals were randomly divided into 6 groups.The CON group(n=10)was fed standard as the control.The CON+GTPs group(n=10)was fed standard,to which was added GTPs based on the food intake and weight(200mg/kg.bw).The HFD group was fed a modified HFD(60%standard diet,12%lard,10%sugar,8%yolk powder.6%peanut powder,3%casein and 1%milk powder,w/w).The HFD+GTPs group was treated with 200mg/kg.bw GTPs,which was added in HFD,based on the food intake and weight.CR group(n=10)was treated with 30%calorie restriction of CON.CR + GTPs group(n=10)was treated with 30%calorie restriction of CON which was added GTPs(200mg/kg.bw).At the 16th week,the animals were subjected to an intraperitoneal glucose tolerance test(IPGTT)or an insulin tolerance test(ITT)after 12 or 6 hours of fasting.At 17th and 18th weeks,all of the animals were housed individually in metabolic cages to collect 24-hoururine.The urinary microalbuminlevels.creatinine level and activity of urinary N-acetyl-?-D-glucosaminidase(NAG)were measured by commercial assay.Sixty rats were decapitated at 18th week.and serum was isolated for measurement of glucose,lipid,insulin level,creatinine and cyctatin c by commercial kits.The fat deposition of kidney and liver were detected by Oil red O staining on frozen sections.The renal struction changes were reflected by hematoxylin-eosin staining on paraffin sections.The level of MDA and the activities of MnSOD,Cu/ZnSOD,total SOD and catalase in kidney were measured by commercial kits.The 4-Hydroxy-2-nonenal level was measured by immunohistochemical method.Results:1)GTPs treatment could reduce the body weight and visceral fat coefficient which were increased by HFD treatment.2)Lower level of blood glucose,triglyceride,total cholesterol and LDL-C,and higher level of HDL-C were observed in HFD+GTPs group.3)Compared to vehicle,the area under IPGTT curve was larger in HFD group.And after intraperitoneal insulin injection,the serum glucose level at 60 min was much higher in HFD group than that in CON group.HFD increased the serum insulin concentration,which seemly was reduced by GTPs treatment,the difference had no statistical significance yet.4)The levels of 4-HNE and MDA in renal cortex were dramatically increased by an HFD,while both indices could be brought back to the normal level by GTPs treatment.5)The activities of MnSOD,catalase,Cu/ZnSOD and total SOD in rat renal cortex was lower in HFD group than those in CON group.And those were increased significantly in GTPs+HFD group.6)Notable difference between CON group and HFD group was observed in microalbuminuria/creatatine ratio,urinary NAG activity and serum cystatin C level.Addition of GTPs could normalize urinary NAG activity and serum cystatin C level,which were increased by high-fat diet.Calorie restriction could increase Ccr and decrease the activity of NAG.7)Rich Oil Red O-positive vacuoles were observed in the liver of HFD group.No positive vacuole was observed in kidney.No significant change of renal structure was observed according to hematoxylin-eosin staining in each group.Conclusion:GTPs ameliorated the impairments of renal function induced by HFD,and the antioxidation of GTPs contributed to the renoprotection.Part ? The effect of GTPs on renal ketogenesis of rats fed an HFDObjective:Several reports suggested the renoprotection of ketone body and green tea polyphenols(GTPs).Our previous study found GTPs consumption could elevate the renal expression of ketogenic rate-limting enzyme,which was decreased by high-fat diet in rats.We here study whether ketogenesis can mediate the renoprotection of GTPs against a high-fat diet.Methods:The commercial kits were used to detect the ?-hydroxybutytate in serum and renal cortex,and the total NAD in renal cortex.The proteins expression of renal HMGCS2,SIRT3,Nampt,FOXO3a,MnSOD,acetyl-MnSOD and Catalase were detected by Western blot.Results:High-fat diet consumption could increase the serum ?-hydroxybutytate concentration and decrease the renal(3-hydroxybutytate level and the expression of SIRT3 and Nampt.GTPs treatment could elevate the renal P-hydroxybutytate level and the expression of HMGCS2,SIRT3,Nampt and deactylation of MnSOD of rats fed with HFD.Calorie restriction could upregulate the(3-hydroxybutytate concentration in serum and kidney,and the renal expression of HMGCS2,SIRT3,Nampt and deactylation of MnSOD.Conclusion:The renal ketogenesis,and the expression and activity of SIRT3 were increased by GTPs supplementation in context of HFD.Part III Ketogenesis elevated the activity of SIRT3 and subsequently reduced the oxidative damage.Objective:It was demonstrated that GTPs treatment could increased the renal ketogenesis and reduced oxidative stress in rats fed with HFD.This part will verify the antioxidation of ketogenesis in vitro.Methods:Human embryonic kidney cells(HEK293)were cultured in high glucose DMEM medium.When the celluar growth density reached 60-70%,HEK293 cells were transfected with the eukaryotic expression plasmid pcDNA HMGCS2.And 24 hours later,the tranfected HEK293 cells were treated separately by GTPs(4?g/ml,24 hours),EGCG(4?g/ml,24 hours),EGC(4?g/ml,24 hours),H2O2(0.1 mM,6 hours)and ?-hydroxybutytate(0.5mM,5 mM,25 mM for 24 hours).Results:HMGCS2 expression in HEK293 cells could be upregulated by GTPs,EGCG and EGC.and reduced by ?-hydroxybutytate treatment.SIRT3 expression was elevated by GTPs and EGCG.Although,the expression of SIRT3 was not affected by HMGCS2 transfection,the lower 4-Hydroxy-2-nonenal(4-HNE)level and acetyl-MnSOD(K122)/MnSOD ratio were detected in HMGCS2 transfected cells treated by H2O2(0.1 mM)for 6 hours.Conclusion:Transfected with plasmid pcDNA HMGCS2 could increase the activity of SIRT3.subsequently decrease the oxidative damage.