Effects Of SIRT1/FOXO1-mediated Autophagy On Diabetic Nephropathy And The Mechanism Of Metformin Therapy

Background:Diabetic nephropathy(DN)is one of the main chronic microvascular complications of diabetes mellitus(DM),which is the primary cause of dialysis in developed countries,and the main cause of dialysis in patients with kidney disease in China.With the acceleration of aging,significant changes in diet and lifestyle in china,the prevalence of DM increased significantly(10.9%),and more than 1/3 of patients with type 1 diabetes mellitus(T1DM)and 20%of patients with type 2 diabetes mellitus(T2DM)eventually develop to DN.The typical characteristics of DN are glomerular hypertrophy,proteinuria,progressive decrease of glomerular filtration rate and renal fibrosis,which leads to the loss of renal function.Intraglomerular hypertension and high filtration are caused by abnormal glucose metabolism,hemodynamics,genetic susceptibility and other abnormalities.However,the specific molecular mechanism leading to DN is not fully understood.Due to the complex background of metabolic diseases,DN is more challenging than other kidney diseases in prevention and treatment.DN has become an important scientific and social problem in China.Autophagy plays a key regulatory and protective role in many aspects of the normal and disease status of the kidney.In some cases,the changes of autophagic activity of the kidney also affect the progress of kidney disease.The increase of autophagic degradation substrate protein p62 was detected in kidney biopsy tissues of patients with T2DM and kidney tissues of rats with T2DM,indicating that autophagic activity of kidney was inhibited in DN.In the case of excess energy,such as high concentration of glucose,amino acids and insulin can activate mTOR effectively and inhibit cell autophagy;on the contrary,in the case of energy deficiency,the increase of AMP and NAD+levels in cells can activate AMPK and SIRT1 effectively,thus inducing autophagy.Metformin is the preferred oral hypoglycemic drug for T2DM.Recent studies have found that metformin has significant effects on polycystic ovary syndrome,cancer,heart and cardiovascular diseases,non-alcoholic fatty liver disease and puberty precocity in addition to hypoglycemic effects,and has protective effects on DN.However,the molecular mechanism of its effect on protecting kidney is still unclear.SIRT1 is mainly expressed in the renal medulla and renal interstitium.In human and DN animal models,the expression and activity of SIRT1 were decreased,and the activation of SIRT1 could protect the kidney from hyperglycemia injury.Silencing SIRT1 can inhibit the induction of autophagy by resveratrol and nutrient deprivation,while overexpression of SIRT1 can promote autophagy of these cells.SIRT1 can activate autophagy by deacetylating a series of transcription factors to promote or enhance the expression of autophagy mechanism components.The most prominent one is the members of the FOXO family.After removing glucose,in vitro cultured cardiac myocytes induced autophagy by SIRT1 and FOXO1 dependent manner.Activation of FOXO1 can increase the expression of Rab7,which is a small GTPase that regulates late autophagic lysosome fusion.Increased expression of Rab7 can promote FOXO1-induced autophagy,while silence Rab7 can inhibit FOXO1 induced autophagy.In conclusion,we speculate that metformin may promote autophagy and alleviate renal injury by regulating SIRT1/FOXO1-mediated autophagy pathway in DN.In this study,we established a diabetic rat model with high-fat diet and STZ treatment to observe whether metformin exerts renal protective effect by regulating autophagy.The role of metformin in the proliferation,inflammation and ECM accumulation of glomerular mesangial cells induced by high glucose was further observed at the cellular level.The potential mechanism of metformin in inducing autophagy through regulating SIRT1/FOXO1-mediated autophagy pathway was clarified,which provided new insights into the development of DN and the molecular mechanism of metformin protecting kidney.Part 1 Metformin alleviates renal injury in diabetic rats by inducing SIRT1/FOXO1-mediated autophagyObjective:Metformin is the first-line hypoglycemic drug for T2DM.Recent studies have shown that besides lowering blood glucose,metformin can also inhibit endoplasmic reticulum stress,epithelial-mesenchymal transition and oxidative stress in kidney tissues of DN patients,promote the expression of hypoxia-inducible factor(HIF)and autophagy,and protect renal function.However,its molecular mechanism is still unclear.In this study,diabetic rat model was established by high fat feeding combined with intraperitoneal injection of STZ.Metformin was used to treat diabetes mellitus and to observe whether metformin could alleviate renal injury by autophagy Methods:1)To observe the protective effect of MET on diabetic kidney,50 SD rats were divided into 5 groups:control group(NC group)fed with conventional diet,DM group(DM group)fed with high-fat diet and intraperitoneal injection of STZ at 30 mg/kg body weight on fasting state,DM+low dose MET group(MET150 group),DM+medium dose MET group(MET300 group),and DM+high-dose MET group(MET500 group).MET150,MET300 and MET500 groups were given MET 150,300 and 500 mg/kg.d by gavage for 8 weeks.2)To observe the effect of SIRT1/FOXO1-mediated autophagy in the treatment of MET,another 40 SD rats were randomly divided into four groups:control group(NC group),DM group(DM group),DM+high dose MET intervention group(MET group),DM+ MET+SIRT1 inhibitor EX527 intervention group(EX527 group).MET group rats were given MET 500mg/kg.d intragastric administration,EX527 group rats were given MET 500mg/kg.d intragastric administration and 5 mg/kg.d intraperitoneal injection of EX527 for 8 weeks.After 8 weeks,urine samples were collected to detect urinary albumin and creatinine on automatic biochemical analyzer,and the ratio of urinary albumin to creatinine was calculated.After anesthesia,blood and bilateral kidney tissues were taken from rats in each group,and some right renal cortex tissues were fixed with 4%neutral formaldehyde to prepare paraffin sections for HE,Masson and TUNEL staining.Some right renal cortex was fixed with 4%glutaraldehyde and observed under electron microscope.Left kidney tissues were used to extract protein for Western blot analysis and biochemical detection.Blood was used to detect blood glucose(BG),blood urea nitrogen(BUN)and creatinine(Scr)and so on.Results:After 8 weeks of MET treatment,levels of BG,Scr,BUN and UACR in MET150,MET300 and MET500 groups were significantly lower than those in DM group(P<0.01).levels of SOD in kidney tissues were increased(P<0.01),levels of MDA were decreased(P<0.01),and the effect of metformin was dose-dependent.HE and Mason staining results showed that metformin could significantly reduce renal tubular injury(P<0.01)and collagen accumulation in renal tissue(P<0.01),TUNEL staining results showed that metformin could significantly reduce the apoptosis rate of renal tissue in diabetic rats(P＜0.01).Electron microscopic observation showed that metformin could significantly reduce the thickness of glomerular basement membrane and foot process fusion in diabetic rats(P<0.01).Western blot analysis showed that the expression levels of SIRT1 and FOXO1 in kidney tissue of diabetic rats decreased,while high dose metformin could significantly up-regulate the expression of SIRT1 and FOXO1,increase the ratio of LC3-Ⅱ/LC3-I,promote the expression of Beclin-1,inhibit the expression of p62,promote autophagy,and significantly inhibit the expression of collagen I,collagen IV and fibronectin.There were no significant changes in the levels of BG,Scr,BUN,UACR,SOD and MDA in renal tissue of diabetic rats treated with metformin and EX527,compared with untreated diabetic rats.EX527 could inhibit the effects of metformin on renal tubular injury score,collagen volume fraction and apoptotic ratio in diabetic rats.Western blot results showed that EX527 could inhibit the up-regulation of SIRT1 and FOXO1 expression by metformin,decrease the ratio of LC3-Ⅱ/LC3-Ⅰ,inhibit the expression of Beclin-1,promote the expression of p62,inhibit autophagy,and significantly promote the expression of collagen I,collagen IV and fibronectin.Conclusions:The results of this in vivo study showed that MET could protect renal function by up-regulating autophagy,alleviating oxidative stress and glomerular pathological and structural changes,and inhibiting the expression of extracellular matrix.SIRT1 inhibitor EX527 can block the protective effect of MET on diabetic rat kidney,suggesting that MET can alleviate renal injury in type 2 diabetic rats by inducing SIRT1/FOXO1-mediated autophagy.Part 2 Metformin inhibits high glucose-induced mesangial cell proliferation,inflammation and ECM expression through SIRT1/FOXO1-mediated autophagy Objective:As the preferred hypoglycemic drug for T2DM,the effect of MET on rat mesangial cells(RMC)and its molecular mechanism are not fully understood.This study explored the effects of MET on the proliferation,inflammation and extracellular matrix(ECM)accumulation of RMC induced by high glucose,and further analyzed the role of SIRT1/FOXO1 autophagic signaling pathway in order to provide a theoretical basis for the treatment of DN by MET.Methods:To observe the effect of MET on RMC induced by high glucose,RMC was randomly divided into six groups:normal glucose group(5.5 mol/L glucose,NG group),high glucose group(30 mmol/L glucose,HG group),high mannitol group(5.6 mmol/L glucose+24.4 mmol/L mannitol,HM group),high glucose+10 micromol/L MET(MET-10 group),high glucose+50 micromol/L MET(MET-50 micromol/L mannitol,HM group).Group B)and high glucose+100 micromol/L MET(MET-100 group).To observe the effect of autophagy on high-glucose-induced RMC,we randomly divided RMC into four groups:normal glucose group(5.5mmol/L glucose,NG group),high-glucose group(30 mmol/L glucose,HG group),high-glucose+50 micromol/L MET(MET group)and high-glucose+50 micromol/L MET+10 mmol/L 3-MA(MET+3-MA group).To observe the effect of SIRT1/FOXO1 on autophagy of RMC induced by high glucose in MET treatment,RMC was randomly divided into three groups:high glucose+MET+siRNA control group(si-Ctrl),high glucose+MET+SIRT siRNA group(si-SIRT1)and high glucose+MET+FOXO1 siRNA group(si-FOXO1).The proliferation ability of cells in different treatment groups was detected by MMT assay.The expression levels of TNF-a,IL-6 and TGF-β1 in supernatants of different treatment groups were detected by ELISA.The expression levels of fibronectin,collagen IV,Beclin-1,LC3-Ⅰ,LC3-Ⅱ,SIRT 1 and FOXO1 in different treatment groups were detected by Western blot.The ratio of LC3-II/LC3-I was analyzed,and the number of autophagic bodies in cells of different treatment groups was observed by laser confocal microscopy.Results:In RMC cells cultured with high glucose,the proliferation of RMC cells treated with 50 or 100μmol/L metformin could be reduced by more than two times.ELISA results showed that levels of TNF-a,IL-6 and TGF-β1 stimulated by high glucose were significantly decreased by 50 or 100μmol/L metformin treatment.High glucose can inhibit RMC autophagy,resulting in a reduction in the number of autophagic bodies in cells.Western blot showed that high glucose could inhibit the expression of SIRT1 in RMC,increase the acetylation level of FOXO1,increase the expression level of LC3-I,and decrease the expression levels of LC3-Ⅱ and Beclin-1.Metformin can promote autophagy of high glucose-induced RMC,inhibit cell proliferation,reduce the expression levels of TNF-a,IL-6 and TGF-β1 in cell culture supernatant,and reduce the expression levels of fibronectin and collagen IV in cells.Autophagy inhibitors 3-MA,SIRT1 siRNA and FOXO1 siRNA can block the effects of metformin on promotes high glucose-induced RMC autophagy,inhibits cell proliferation,inflammatory response and extracellular matrix expression.Similarly,the expressions of fibronectin and collagen IV in metformin treated cells were significantly lower than those in high glucose treated cells.Metformin at 50 and 100 pmol/L was more effective than metformin at 10pμmol/L,while metformin at 50μmol/L and 1000μmol/L had no significant difference.The number of green fluorescent spots in RMC pretreated with metformin increased significantly compared with RMC only treated with high glucose by fluorescence microscopy.Western blot analysis showed that metformin treatment could significantly increase the expression of Beclin-1 and the ratio of LC3-I/LC3-II in ECM induced by high glucose.MTT analysis showed that cell proliferation was significantly increased in the presence of autophagy inhibitors 3-MA compared with metformin alone.ELISA results showed that after 3-MA treatment,the levels of TNF-a,IL-6 and TGF-β1 in cell culture supernatant increased significantly.The expression of fibronectin and collagen IV increased significantly after 3-MA treatment compared with metformin alone.In order to determine the molecular mechanism of metformin activating autophagy,the expression levels of SIRT1 and FOXO1 protein and FOXO1 acetylation were detected by Western blot.High glucose significantly inhibited the expression of SIRT1 and FOXO1 protein,but metformin could up-regulate the expression of SIRT1 and FOXO1 protein and down-regulate the acetylation of FOXO1.Transfection of small interfering RNA targeting SIRT1 and FOXO1 could counteract the inhibition of RMC proliferation by metformin,significantly increase the expression of TNF-a,IL-6 and TGF-beta,and reduce the number of autophagic bodies in RMC.Small interfering RNA targeting SIRT1 and FOXO1 significantly decreased the expression of Beclin-1 and the ratio of LC3-Ⅰ/LC3-Ⅱ,but increased the expression of fibronectin and collagen Ⅳ.Conclusions:In vitro results suggest that metformin inhibits the proliferation,inflammation and extracellular matrix expression of RMC induced by high glucose through SIRT1/FOXO1-mediated autophagy.SIRT1/FOXO1-mediated autophagic signaling pathway is a potential target for metformin in the treatment of diabetic nephropathy.