Abnormal Glycolysis Metabolism In Proximal Tubular Epithelial Cells Engaged In The Development Of Renal Interstitial Fibrosis

Objections:Renal proximal tubule is susceptible to hypoxic injury,because of the reliance on aerobic oxidative metabolism.Dysfunctional mitochondria participate in the progression of chronic renal disease.However,the profile of the energy metabolism of the proximal tubule is presently uncertain in renal fibrosis,and the relationship between the change of energy metabolism and tubular epithelial cell damage is not clear.In this paper,we investigate the profile of the energy metabolism of the proximal epithelial cell in fibrotic kidney,and evaluate the role of anaerobic metabolism in renal fibrosis.Methods:8 weeks old male CD1 mice weighing approximately 18-22 g were acquired from the Specific Pathogen-Free Laboratory Animal Center of Nanjing Medical University and maintained according to the guidelines of the Institutional Animal Care and Use Committee at Nanjing Medical University.2-deoxyglucose(2-DG)was administered orally at a dose of 20mg/Kg or 100mg/Kg body weight three days before unilateral ureter obstruction surgery and 2-DG was executed for consecutive 7 days.Shikonin was oral performed at a dose of 1 mg/Kg or 5 mg/Kg body weight three days prior to unilateral ureter obstruction surgery and Shikonin was administered for successive7 days.UUO was administered using an established protocol,as described anteriorly.Sham-operated mice were used as normal controls.Human Recombinant TGF-β1(5ng/ml)and lactic(10mmol/L)were added to the serum-free medium for indicated time periods and concentration.2-Deoxyglucose(2-DG,0.2 or 1mmol/L),shikonin(1 or 5nmol/L),dichloroacetic acid(DCA,5nmol/L),BroPA(5nmol/L)and oxamate(100nmol/L)were added to the serum-free medium 30 minutes for indicated time periods and concentration before TGF-β1 treatment,then to examine the expression of mRNA and protein of key enzymes of glycolysis.Results:The protein expression of key enzymes of glycolysis such as hexokinase2(HK2),pyruvate dehydrogenase4(PDK4),phosphorylase-pyruvate kinase of type M2(p-PKM2)were markedly increased in a time depended in UUO induced renal interstitial fibrosis mice model.There was a similar result in primary renal tubular epithelial cells after disposed with TGF-β1.In addition,the fibrotic matrix was reduced and marks of collagen fibrils such as a-smooth muscle actin(α-SMA),fibronectin(FN)were down-regulated after mice or primary renal tubular epithelial cells(PTC)deal with inhibitors of glycolytic pathway.At the same time,we found the expression of TGF-β1 receptor and p-smad3 were decreased.In the meantime,lactic acid,the production of glycolysis pathway,was involve in the activation of TGF-β/Smad pathway and changed renal tubular cells phenotype and contributed to the development of renal interstitial fibrosis.The results above all had statistic differences.Conclusions:The results above demonstrated that metabolic reprogramming involved in the development of UUO induced renal fibrosis or TGF-β1 induced PTC fibrosis.Suppressing aerobic glycolysis in renal tubular epithelial cells(PTC)could significantly reduce renal fibrosis.Our findings thus provide a novel potential therapeutic strategy for treating renal fibrosis.