Effect And Mechanism Of Sirt1 On The Exosome Release Of Podocytes In Diabetic Kidney Disease

Objective:Podocyte injury is one of the crucial pathological characteristics of diabetic kidney disease(DKD).Exosomes are extracellular vesicles with diameters ranging from50 to 150nm.They contain several biologically active molecules and participate in various pathophysiological processes.Previous studies showed that exosome secretion increased in high glucose-treated podocytes.Nevertheless,the precise mechanisms of dysfunctional exosome secretion by podocytes in DKD remain unclear.Sirtuin1(Sirt1)is a member of deacetylase family that participates in maintaining kidney homeostasis.Several lines of evidence suggest that Sirt1 ameliorates the progression of DKD by regulating oxidative stress,apoptosis,inflammation,and renal fibrosis.Nevertheless,it remains unclear whether Sirt1 affects podocyte exosome release in DKD.This study aims to investigate the effect and mechanism of Sirt1 on the exosome release of podocytes in DKD.These findings provide a novel therapeutic target for preventing of DKD progression.Methods:6-week-old C57BL/6J male mice were randomly divided into control group(n=5)and experimental group(n=15).Diabetes mellitus(DM)model was constructed by intraperitoneal injection of streptozotocin.Mice with fasting blood glucose levels above16.7mmol/L were considered as successful DM models.The experimental mice were then divided randomly into a diabetic group(DM,n=5)and diabetic transfected with empty lentivirus(DM+NC-LV,n=5)or Sirt1 overexpressed lentivirus(DM+Sirt1-LV,n=5)group.At week 12 after the successful construction of the model,urine,serum and kidney specimens were collected,and renal pathological changes were observed.Immunofluorescence staining was used to observe the expression and localization of CD63 and Alix,exosome-specific markers.Multivesicular bodies(MVBs)were imaged using transmission electron microscopy(TEM).Western blotting was used to analyze the protein levels of Nephrin,Podocin,Sirt1 and A subunit of the lysosomal vacuolar-type H~+ATPase proton pump(ATP6V1A).RT-q PCR was performed to detect the expression of Sirt1 and ATP6V1A m RNA.The immortalized mouse podocytes were divided into normal glucose(NG),high mannitol(HM),high glucose(HG),HG+NC-LV,HG+Sirt1-LV and HG+ATP6V1A-LV(ATP6V1A overexpressed lentivirus)groups.The particle size and concentration were detected by nanoparticle tracking analysis.Lysosomal probes were used to detect lysosomal p H alterations.Western blotting was used to analyze the expression of Nephrin,Podocin,Sirt1,ATP6V1A,CD63,CD81 and Alix.Immunofluorescence staining was used to observe the localization relationship between lysosome-associated membrane protein 1(LAMP1)and MVBs-specific marker VPS16.RT-q PCR was used to detect the expression of Sirt1 and ATP6V1A m RNA.Results:Compared with the control group,the urinary albumin to creatinine ratio(ACR),fasting blood glucose levels and kidney weight-to-body weight ratio in diabetic mice were significantly elevated(all P<0.01).Renal pathology revealed a significant increase in glomerular volume,mesangial expansion,podocyte injury,and tubulointerstitial fibrosis in diabetic mice.In addition,increased expression of glomerular CD63 and Alix as well as decreased expression of Nephrin,Podocin,Sirt1 and ATP6V1A showed in kidney tissues of diabetic mice(all P<0.01).Increased podocyte MVBs volume could be observed by TEM.Above pathological changes were attenuated after Sirt1 overexpression.Compared with DM+NC-LV group,ACR was reduced in mice of DM+Sirt1-LV group(P<0.01).The expression of Nephrin,Podocin,Sirt1 and ATP6V1A was increased and glomerular CD63 and Alix expression was reduced(all P<0.05)after Sirt1 overexpression.In addition,the volume of podocyte MVBs became smaller.There were no statistical differences between the NG and HM groups(P>0.05).The HG group showed increased exosome secretion,impaired lysosomal acidification,reduced MVBs and lysosomal fusion,and decreased expression of Nephrin,Podocin,Sirt1 and ATP6V1A in podocytes(all P<0.01).After Sirt1 overexpression,increased expression of Nephrin,Podocin,Sirt1and ATP6V1A(all P<0.05),increased MVBs and lysosomal fusion,and decreased exosome secretion were observed.GW4869(an exosome secretion inhibitor)increased Nephrin and Podocin expression and attenuated podocyte injury.Compared with HG+NC-LV group,HG+ATP6V1A-LV group showed increased expression of ATP6V1A,increased fusion of MVBs and lysosomes,and decreased secretion of exosomes.Conclusions:Loss of Sirt1 inhibits the lysosomal acidification by regulating ATP6V1A and reduces the fusion of MVBs and lysosomes in podocytes in DKD,resulting in increased exosome secretion and podocyte injury.These findings constitute a previously undiscovered pathophysiological mechanism of podocyte injury and might provide a new therapeutic target for preventing DKD progression.