| Background:In humans,diabetic nephropathy(DN)is characterized by increased urinary albumin excretion(microalbuminuria);this often progresses to proteinuria,which is one of the most important prognostic crisk factors for kidney disease progression.Studies in humans and DN animal models reveal podocyte injury,detachment,apoptosis,and loss.Although some stimuli induce podocyte necrosis in vitro,more in vivo evidence suggests podocyte loss in DN isassociated with apoptosis.More specifically,podocyte apoptosis coincides with albuminuriaonset and precedes podocyto peniain different mouse models of diabetes.Although these observations identify podocyte apoptosis as one of the earliest cellular features of diabetic kidney disease,the mechanisms underlying podocyte loss in DN remain poorly understood.Thus,investigating the mechanisms of podocyte apoptosis in diabetic conditions will be critical for thedevelopmentof novel preventive and therapeutic approaches to DN.Autophagy is a catabolic process that degrades damaged proteins and organelles in mammalian cells and plays a critical rolein maintaining cellular homeostasis.It has been reported that autophagy has a protective function against renal damage.Normally,high level of autophagy has been observed in podocyte of the kidneys.However,autophagy activity declines in kidneys under diabetic conditions.Resveratrol,a natural polyphenolic phytoalexin that is found abundant in grapes,mulberries,peanuts,and many other plants.The beneficial bioactivities of resveratrol,such as anti-inflammation,antioxidant,cardioprotection,lipid modification and anticancer effect,have attracted more and more attentionResveratrol,a natural polyphenolic phytoalexin that is found abundant in grapes,mulberries,peanuts,and many other plants.The beneficial bioactivities of resveratrol,such as anti-inflammation,antioxidant,cardioprotection,lipid modification and anticancer effect,have attracted more and more attention.In streptozotocin-induced diabetic rats,db/db mice,resveratrol can reduce proteinuria,improve renal function,but the role of resveratrol and target the mechanism of action are not complete clear.Until date,the mechanism of autophagy in podocytes has not been completely revealed and the study on resveratrol-mediated autophagy in podocytes also not been conducted.Here,we investigated whether resveratrol could relieve the apoptosis of podocytes via the activation of autophagy.Objective:To investigate whether resveratrol could relieve the apoptosis of podocytes via the activation of autophagy and reveal the mechanism of resveratrol action.Methods:part 1:8 weeks female mice,including C57BL/KsJ db/db mice 12,C57BL/KsJ db/m mice 6.All the mice were fed adaptively for 1 week,12 db/db mice and 6 db/m mice were randomly divided into three groups with six animals of each group,as follows:normal control group,diabetic nephropathy group and diabetic nephropathy with resveratrol.Resveratrol was administrated to diabetic mice at a dosage of 10mg/(kg·d)for 12 successive weeks.After administration,all the mice were sacrificed,body weight,kidney weight,fasting glucose,24 h microalbuminuria,serum creatinine and urea nitrogen were measured.Part 2:(1)db/m mice as normal control group,db/db mice as model group,db/db mice + resveratrol as treatment group.After 12 weeks,the kidney tissues were collected and kidney tissue was stained by HE and PAS,podocyte and glomerular basement membrane were examined by transmission electron microscopy.The expression of nephrin,LC3 II,synaptopodin and cleaved caspase-3 in the kidney was detected by immunohistochemistry and immunofluorescence.(2)Human podocytes(HPC)were treated with NG(5.6 mM),HG,HG plus resveratrol(15 ?M),HG plus resveratrol(15?M)and bafilomycin A(10nM)for 48 h.The effect of resveratrol on HG-induced autophagy and apoptosis of podocytes were further evaluated by flow-cytometric analysis of Annexin V-FITC and propidium iodide(PI)-dual-stained cells,immunofluorescence staining,transmission electron microscopy,and evaluation of green fluorescent protein(GFP)-microtubule-associated protein 1 light chain 3(LC3)punctate structures by fluorescence microscopy.Part 3:Resveratrol may reduce high glucose induced-podocyte apoptosis by increasing podocyte autophagy pathway.Human podocytes were pretreated with 3-MA for 2h followed by HG plus resveratrol treatments for 48h.The expression of Cleaved caspase-3,Bax,Atg5 and LC3-? were assessed by western blotting.Next,cells were transfected with Atg5 shRNA and subjected to the same HG plus resveratrol treatments for 48h.The effects were also evaluated by western blotting,flow-cytometric analysis of Annexin V-FITC and PI-dual-stained cells and transmission electron microscopy.Results:Part 1:To investigate the potential therapeutic effect of resveratrol on DN in a db/db diabetic mouse model.Three groups of mice were used:control db/m mice,db/db mice,and resveratrol-treated db/db mice(db/db+Res)(n = 6 per group).The body and kidney weights were significantly higher in db/db mice than in db/m mice throughout the experiment.In db/db mice,treatment with resveratrol tended to lead to lower body and kidney weights by the end of the experiment.However,there was no difference in the kidney index(body weight/kidney weight)between untreated and resveratrol-treated db/db mice.Additionally,the fasting blood glucose(FBS)was higher in db/db than in db/m mice;however,no significant difference was noted in the level of fasting blood glucose(FBS)of db/db mice with and without resveratrol treatment.The Systolic blood pressure(SBP)was higher in db/db mice than in db/m mice,however,SBP was not significantly different between db/db mice and resveratrol-treated db/db mice.In addition,24-h microalbuminuria(UAER),serum creatinine(Cr),and urea nitrogen(BUN)were all higher in the db/db than in the control mice,while these parameters were significantly decreased by treatment with resveratrol,indicating that resveratrol ameliorates the DN-associated functional abnormalities in db/db mice.Part 2:Resveratrol induces autophagy and attenuates HG-induced apoptosis in both db/db mice and human podocytes.(1)In comparison with db/m mice,renal tissues stained with HE and PAS show that glomerulus hypertrophy,capillary basement membrane thickening and mesangial matrix expansion in db/db mice.After treatment with resveratrol,glomerular lesion was remarkable alleviated.(2)Compared with db/m mice,under transmission electron microscope,fusion of foot process and glomerular basement membrane were observed in db/db mice,while the damage of podocyte was alleviated after treatment with resveratrol.(3)In renal tissues of db/db mice,the expression level of nephrin protein significantly decreased,while treatment with resveratrol reduced the decrease.(4)In comparison with db/m mice,the expression of LC3 II and synaptopodin increased accompanied by a decrease of cleaved caspase-3,while treatment with resveratrol reverse the change.(5)Human podocytes were incubated with 30mM glucose for various periods.LC3-II and Atg5 were up-regulated as early as 6 h,and both declined dramatically at 48 h,on the contrary,p62 was increased significantly at 48 h.Cells were treated with different concentrations of resveratrol(0,5,10,and 15?M),followed by HG(30mM)treatment for 48 h.Resveratrol induced a dose-dependent accumulation of LC3-II and Atg5 in podocytes as indicated by western blotting,on the contrary,treatment with resveratrol strongly decreased expression of p62.Next,cells were divided into four groups:normal glucose(NG:5.6mM),HG(30mM),HG plus resveratrol(15?M),HG plus resveratrol(15?M)and bafilomycin A(10nM).Autophagosomes(sta:ined with the anti-LC3-? antibody,red)were localized in the cytoplasm,and in cells treated with HG plus resveratrol and bafilomycin A(10nM),the number of red fluorescent spots were highest among all the groups.Transmission electron microscopy is the gold standard for detecting autophagy;the number of autophagosomes represents autophagic activity.HG could induce autophagy and resveratrol further increased the number of autophagosomes.Finally,the widely used fusion protein GFP-LC3 was employed to monitor autophagy.Podocytes were transfected with GFP-LC3 plasmid and treated as mentioned above.GFP-LC3 green dots showed the highest accumulation after exposure to bafilomycin A although resveratrol also increased the number of green dots.Conversely,the fluorescence was predominantly difffuse in the cytoplasm of control cells.(6)Cleaved caspase-3 and Bax proteins were upregulated in podocytes with the treatment of high glucose.Then,treatment with resveratrol strongly decreased expression of these proteins.What's more,the number of early apoptotic cells was increased by HG,and normalized by resveratrol treatment.Immunofluorescence microscopy confirmed that the expression of Cleaved caspase-3 protein was markedly elevated after HG stimulation,while treatment with resveratrol inhibited these effects.Part 3:Inhibition of autophagy by 3-MA and Atg5 shRNA reverses the protective effects of resveratrol on human podocytes.To examine the role of autophagy in podocytes after exposure to HG and resveratrol,autophagy was inhibited chemically with 3-MA and withAtg5 shRNA.Cells were pretreated with 3-MA for 2 h followed by HG plus resveratrol for 48 h.As expected,3-MA attenuated LC3-II expression and led to increases in p62,Cleaved caspase-3 and Bax expressionin the presence or absence of resveratrol(Figure 6a).To further confirm these results,cells were transfected with Atg5 shRNA and subjected to the same treatment.Transfection with Atg5 shRNA reduced Atg5 and LC3-II expression as indicated by immunoblot analysis.In addition,Atg5 inhibition enhanced caspase-3 cleavage and Bax expression.Similarly,flow cytometric analysis showed that Atg5 inhibition significantly increased apoptotic cells in the case of resveratrol treatment.Meanwhile,immunofluorescence showed that suppression of Atg5 enhanced Cleaved caspase-3 expression.Taken together,these findings confirmed that resveratrol attenuates HG-induced apoptosis in podocytes via the activation of autophagy.Conclusion:Part 1:Resveratrol decreased significantly serum creatinine and urea nitrogen,24 h microalbuminuria,which could attenuate development of diabetic nephropathy mice.Part 2:Human podocytes were incubated with 30mM glucose for various periods.LC3-? and Atg5 were up-regulated as early as 6 h,and both declined dramatically at 48 h,on the contrary,p62 was increased significantly at 48 h.resveratrol ameliorates the abnormal autophagy level in db/db mice and induces autophagy in human podocytes.Furthermore,resveratrol attenuates HG-induced apoptosis in human podocytes.Part 3:Resveratrol could attenuate diabetic nephropathy mice through the activation of autophagy and inhibition of apoptosis in podocyte. |
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