Sestrin2 Regulates High Glucose–induced Mitochondrial Dysfunction And Podocyte Apoptosis Through AMPK Signaling Pathway

Background and Objective:As one of the most common and serious microvascular complications among patients with diabetes mellitus,diabetic kidney disease(DKD)damages the renal function and leads to the end-stage of renal disease(ESRD).Associated with higher mortality and morbidity,the disease poses a substantial financial and health-care burden worldwide.The primary pathological changes associated with DKD include glomeruar cell hypertrophy,thickening of the glomerular basement membrane(GBM)and depletion of podocytes,which results in increased amount of urinary protein levels.By synthesizing extracellular matrix and cytoskeletal proteins,podocytes play an indispensable role in glomerular filtration barrier.Recently,there are increasing numbers of studies that indicate perturbed mitochondrial function contributes to the pathogenesis of DKD as mitochondria are involved in various cellular functions like oxidative damage and apoptosis.Hence,it is postulated that the identification of key molecules involved in mitochondrial dysfunction in podocytes may be a promising therapeutic strategy for patients with DKD.As members of the highly-conserved family of stress-responsive proteins with antioxidant properties,sestrins has three isoforms in mammals including sestrinl,sestrin2 and sestrin3,while invertebrates have only one form.Three important functions of sestrins are to suppress the formation of reactive oxygen species(ROS),to inhibit the activity of mTORC1 as well as to bind with leucine.The ROS-suppression function of sestrin2 partially depends on the inhibition of mTORCl,which can increase the degradation of damaged mitochondria or the Nrf2-inhibitor Keap1.When exposed to environmental and metabolic stresses,including DNA damage,oxidative stress,hypoxia,endoplasmic reticulum(ER)stress,energy deprivation and amino acid starvation,sestrin2 begins to accumulate,thus protecting cells from damage.In light of these important findings,sestrin2 was speculated to protect podocytes by maintaining mitochondrial homeostasis under HG stimulation.Interactions between sestrin2 and the AMP-activated protein kinase(AMPK)pathway have been shown to play a crucial role in the regulation of energy homeostasis,cellular proliferation and apoptosis.As a member of the serine-threonine protein kinase family,AMPK is an essential energy sensor which maintains cellular and whole-body energy homeostasis.Besides,there is increasing evidence to indicate the potential antioxidant properties of AMPK and its role in the preservation of mitochondrial function.Experiments conducted with streptozotocin(STZ)-induced diabetic mice revealed that renal damage was alleviated by improving mitochondrial function with an AMPK agonist.In the present study,it was proposed that sestrin2 may ameliorate mitochondrial dysfunction via activation of the AMPK pathway.Therefore,the purpose of the present study was to determine whether the anti oxidative effects of sestrin2 were active against HG treatment,which was achieved by analyzing the expression levels of sestrin2 in human podocytes after treatment with HG(to mimic diabetic conditions)and in rats,respectively.Experiments were also extended to dissect out the molecular role of sestrin2 in mitochondria and AMPK signaling.Methods:Part Ⅰ:At an environmental temperature of 21-24℃(55±12%humidity),12 eight-week-old male SD rats were raised in cages under a 12:12hr light/dark cycle.Rats had free access to food and sterile water throughout the experiment.Intraperitoneal injection of streptozotocin(STZ,80 mg/kg)dissolved in 0.1 M citrate buffer(pH 4.5)was used to induce diabetes,while control rats received citrate buffer alone.Glucose levels in urine and blood were measured regularly.Rats with glycemia levels exceeding 200mg/dl were considered diabetic.Blood was obtained from the tail vein and assayed using a blood glucose analyzer.After 12 weeks of diabetes,24h urine was collected in metabolic cages,and urinary albumin concentrations were measured by the Medical Laboratory Center at Renmin Hospital of Wuhan University.Kidney samples were collected under anesthesia.HE and PAS Staining were used to observe the histopathological changes;The expression of Sestrin2 in glomeruli was observed by immunofluorescence;The expressions of Sestrin2、p-AMPK and t-AMPK in glomeruli were detected by Western Blotting;Podocyte ultrastructure and mitochondrial morphology were observed under transmission electron microscopic analysis.Part Ⅱ:In vitro,the differentiated cultured human podocytes were stimulated by high glucose(35mM)for variable times(0h,6h,12h,18h and 24h),and by variable concentrations of glucose(5mM,15mM,25mM and 35mM)for 24h.The expressions of Sestrin2 were analyzed by Western Blotting.Then the differentiated cultured human podocytes were cultured in the normal group(5mM glucose),the mannitol group(5mM glucose+25mM mannitol)and the high glucose group(35mM glucose)for 24h.The expression of Sestrin2、p-AMPK and t-AMPK in podocytes were analyzed by Western Blotting and immunofluorescence assay.The podocyte apoptosis rate was assessed by flow cytometry.The mitochondrial morphology in podocytes was observed under transmission electron microscopic analysis.The level of intracellular ROS was assessed using the dichlorofluorescein diacetate fluorescent probe.JC-1 staining was used to evaluate the mitochondrial membrane potential.Part Ⅲ:Sestrin2 specific small interfering RNA(Sestrin2 siRNA),scramble siRNA,Sestrin2 specific high-expression plasmid(pcDNA3.1 Sestrin2)and negative control with pcDNA3.1 were transfected into cultured human podocytes to downregulate or upregulate the expression of Sestrin2.The expression of Sestrin2、p-AMPK and t-AMPK in podocytes were analyzed by Western Blotting.The expression levels of ROS in different groups were assessed using the dichlorofluorescein diacetate fluorescent probe.JC-1 staining was used to evaluate the mitochondrial membrane potential.The AMPK activator,AICAR,as well as the AMPK inhibitor,Compound.C,were added to the culture medium to regulate the phosphorylation level of AMPK in podocytes respectively.The expression of p-AMPK and t-AMPK were analyzed by Western blotting.The expression level of intracellular ROS was assessed using the dichlorofluorescein diacetate fluorescent probe.JC-1 staining was used to evaluate the mitochondrial membrane potential.Results:Part Ⅰ:Compared with the control group,diabetic rats exhibited higher blood glucose levels,lower body weight,elevated levels of albumin creatinine ratio and 24h urine total proteins.HE staining and PAS staining revealed histological features of DKD,including mesangial expansion,extracellular matrix deposition and glomerulosclerosis from 12 weeks after modeling.Besides,transmission electron microscopy analysis showed evident diffuse foot process fusion and abnormalities in mitochondria such as swelling and disorganization of cristae.Western blotting and immunofluorescence showed that the expressions of Sestrin2 and p-AMPK in glomeruli of diabetic rats were decreased(p<0.05).Part Ⅱ:Western Blotting showed that HG stimulation significantly decreased the Sestrin2 expression in cultured podocytes(p<0.05).HG-treated podocytes presented a decreased staining of Sestrin2 in the cytoplasm via immunofluorescent assays.Consistent with the results in vivo,HG-treated podocytes presented a decrease in the p-AMPK/AMPK protein ratio(p<0.05).Electron microscopy showed severe mitochondrial swelling,mitochondrial cristae loss and decreased matrix density in podocytes treated with HG.The apoptosis rate of podocytes was evidently aggravated under HG conditions.Meanwhile,the intracellular ROS production was markedly increased while the mitochondrial membrane potential was decreased(p<0.05).Part Ⅲ:Sestrin2 siRNA transfection resulted in the down-expression of Sestrin2 compared with the control and scramble siRNA group.The p-AMPK/AMPK protein ratio was also declined(p<0.05).Sestrin2 siRNA transfection exacerbated the apoptosis and mitochondrial defects in podocytes by flow cytometry,the dichlorofluorescein diacetate fluorescent probe and JC-1 staining.By contrast,compared with the HG stimulation group,transfection of pc-DNA3.1 Sestrin2 reversed the downregulation of Sestrin2 and dephosphorylation of AMPK under high glucose.Sestrin2 overexpression evidently ameliorated the mitochondrial defects and apoptosis in HG-treated podocytesConclusion:The present study suggests that Sestrin2 can alleviate HG-stimulated podocyte apoptosis,which is attributed to the protection of the mitochondria via activating AMPK,reducing ROS production,maintaining mitochondrial morphology and stabilizing MMP.