Trimetazidine Inhibits Renal Tubular Epithelial Cells To Mesenchymal Transition In Diabetic Rats Via Upregulation Of SIRT1

Background and objective:Trimetazidine(TMZ)is a piperazine derivative drug,which has a good protective effect on ischemic heart disease by inhibiting the activity of 3-ketoacyl-Co A thiolase(3-KAT)during mitochondrial?oxidation.Previous studies have reported that TMZ also has protective effect on acute kidney injury(AKI),however,the effect of TMZ on diabetic nephropathy(DN)remains unknown.The purpose of this study is to explore the roles and mechanisms of TMZ in streptozotocin(STZ)induced DN.Methods and results:Part 1:the effects of TMZ on renal dysfunction and pathological remodeling in DNType 1 diabetes model was induced in SD rats by intraperitoneal injection of STZ at a large dose(55mg/kg).Successfully induced diabetic rats were randomly divided into DN group and DN+TMZ group.Metabolic parameters,renal function indexes and renal pathological changes were detected after oral administration of TMZ at a dose of 5 mg/kg/day for 20weeks.The experimental results showed that rats in DN group had obvious metabolic dysfunction,including declined weight,increased blood glucose and blood lipids,but TMZ intervention had no obvious effect on these parameters.The renal function indexes showed that plasma creatinine,blood urea nitrogen,the ratio of urine albumin to urine creatinine(UACR)and the ratio of urine?-N-acetyl-glucosaminidase(?-NAG)to urine creatinine increased significantly in DN group,showing obvious renal insufficiency.However,treatment with TMZ improved these situations.Renal pathological examination showed obvious glomerular and renal tubular injuries in DN group.TMZ alleviated these pathological changes,especially tubulointerstitial fibrosis.Epithelial-to-mesenchymal transition(EMT)of renal tubular epithelial cells is an important cause of tubulointerstitial fibrosis during the progression of DN.This study investigated the influence of TMZ on EMT,and the results showed that TMZ could significantly inhibit it.In vitro experiments,High fat and glucose(HFG)were used to imitate diabetic environment in human proximal tubule epithelial cells(HK-2),which induced obvious EMT phenotype.Part 2:the underlying mechanisms of TMZ inhibiting diabetic environment induced EMT in renal tubular epithelial cellsOxidative stress pathway and TGF?/SMAD pathway are two core pathways to induce EMT.In order to study whether TMZ plays a role in them,this study examined these two pathways respectively.The experimental results showed that TMZ inhibited the levels of reactive oxygen species(ROS)in kidney cortex of DN and HK-2 cells after HFG intervention.NAD~+dependent protein deacetylase Sirtuin 1(SIRT1)is a protein sensitive to metabolic changes,and has the function of resisting oxidative stress.In this study,western blots showed that SIRT1 expression increased after TMZ intervention.By silencing SIRT1 expression with si RNA,it was observed that TMZ played an antioxidant role depending on SIRT1/FOXO1/SOD2 pathway.On the other hand,we examined the effect of TMZ on TGF?1/SMAD4 pathway,and the results showed that TMZ could alleviate TGF?1 induced EMT through SIRT1/SMAD4 pathway.Part 3:the underlying mechanisms of TMZ upregulating SIRT1expressionTo explore the mechanism of TMZ regulating SIRT1 expression,this study detected nicotinamide adenine dinucleotide(NAD~+)content and the protein expression levels of nicotinamide phosphoribosyl transferase(NAMPT)and p-AMPK in renal cortex and HK-2 cells.The results showed TMZ increased NAD~+content and upregulated the expression of NAMPT and p-AMPK.Furthermore,FK866,a NAMPT inhibitor and compound c(CC),an AMPK inhibitor were used respectively in HK-2 cells,and the results showed that TMZ maintained the NAD~+content in HK-2 cells under diabetic environment depending on the activities of NMPT and AMPK.Interestingly,FK866,but not CC,inhibited the regulatory effect of TMZ on SIRT1expression.In addition,the inhibitory effect of FK866 on SIRT1 expression could be eliminated by NAD~+supplementation,suggesting that TMZ promotes SIRT1 expression through NAMPT/NAD~+pathway rather than AMPK/NAD~+pathway.Conclusion:1.TMZ can relieve renal insufficiency and pathological remodeling of DN;2.TMZ attenuates tubulointerstitial fibrosis by inhibiting tubular EMT in DN;3.TMZ inhibits tubular EMT through SIRT1/FOXO1/SOD2 pathway and SIRT1/TGF?1/SMAD4 pathway;4.TMZ upregulates the expression of SIRT1 through NAMPT/NAD~+pathway.This study provides a theoretical basis for the application of TMZ in DN,and provides a new candidate drug for clinical prevention and treatment of DN,which has important clinical significance.