Role Of SIRT1/PGC-1α In Mitochondrial Oxidative Damage And Apoptosis Of Podocyte In Diabetic Mice And Effects Of Resveratrol

Objectives:Diabetic nephropathy (DN) has been the focus in researchesof kidney disease for years due to its high morbidity,high disability and highmortality.Studies found that, podocyte plays an important role in maintainingthe integrity of the glomerular filtration barrier and preventing proteinuria.Thepodocyte damage affect the development and prognosis of diabeticnephropathy. Podocyte is a kind of highly specialized cell,whose regeneratedability is poor. Podocyte need large amount of energy consumption tomaintain its normal function of multi-level structure of processes,which issensitive to oxidative stress. All of these characteristics led to the vulnerabilityof podocyte. Some researches reported that podocyte was the earliest damagedcell in diabetic nephropathy, which appeared apoptosis and fall off in the earlystage of DN, then aggravated the process of proteinuria and glomerularsclerosis.The pathogenesis of diabetic nephropathy is very complicated, Previousstudies have shown that, four major molecular mechanisms were implicated inhyperglycemia-induced tissue damage：activation of protein kinase C(PKC)isoforms via de novo synthesis of the lipid second messengerdiaeylglycerol(DAG)， increased hexosamine pathway flux， increasedadvanced glycation endproduct(AGE)formation, and increased polyol pathwayflux． However, there was no apparent common element linking thesemechanisms. In2002， Professor Michael Brownlee pointed out thathyperglycemia-induced mitochondrial overproduction of reactive oxygenspecies(ROS) is the primary initial mechaniam of diatbeticcomplications．ROS overexpression can activate the above four biochemicalpathways, and these pathways can also cause the accumulation of ROS. Through direct peroxidation and activation of related signaling pathways,ROS can lead to cellular damage and apoptosis, then cause multiple organcomplications of diabetes. Through experimental researches for many years,this unified theory has been accepted by most scholars. Mitochondrial ROSoverexpression may play a beginning and key role in the pathogenesis ofdiabetic nephropathy.The overexpression of mitochondrial ROS can target itself and lead tomitochondrial damage, including the respiratory chains, inner and outermembranes and matrix proteins. That lead to produce more ROS, while ATPsynthesis is reduced, and then the mitochondrial membrane permeabilitychanges. A series of pro-apoptotic factors such as cytochrome C, AIF andsmac-diablo outflowing into cytoplasm induces the activation ofmitochondrial apoptosis pathway. Therefore, mitochondrial oxidative damageand cell apoptosis may be the important pathogenic mechanism of diabetes,which is also expected to become the new target for treatment of diabeticnephropathy.Mitochondrial biogenesis is the physiological activities for maintainingand repairing the Mitochondrial structure. It is the important bridge to mediatregulation of nuclear genes to Mitochondrial genes. The study found that,PGC-1α had been identified as a master regulator of mitochondrial biogenesis.Its main biological characteristics was to stimulate mitochondrial biosynthesisand enhance the capacity of oxidative metabolism in different organizations.PGC-1α was found initially as a transcriptional coactivator of nuclearhormone receptor Peroxisome Proliferator Activated Receptor gamma (PPARγ) in mice. A series of studies have shown that it plays an important role inenergy balance, mitochondrial oxidative metabolism, glucose and lipidmetabolism. PGC-lα stimulates the expression of nuclear respiratory factor(NRF) and mitochondrial transcriptional factor A (TFAM), and then activatesexpression of nuclear and mitochondrial genes encoding mitochondrialproteins．In recent years, lots of researches have found the down-regulation ofPGC-1α expression in a variety of diabetic complications, with close relationship with mitochondrial damage, which may be one of the importantmechanisms of diabetic complications. Further studies found that PGC-1α wasactivated by deacetylation of NAD+dependented deacetylatise silentinformation factor2(sir2) related enzyme (SIRT1). Resveratrol is thestrongest SIRT1activator found by now, which has many important biologicalefficacies such as anti-inflammation, antioxidant, anticancer, regulatingglucose and lipid metabolism. Most studies suggested that thepharmacological effects of resveratrol were depended on the activation ofSIRT1. Recently, the role of resveratrol in the treatment of kidney disease getsmore and more scholars’ attention. Researches about animal models of diabeticnephropathy have showed that resveratrol has renal protective effects, such asreducing proteinuria and extracellular matrix deposition, inhibiting oxidativestress and apoptosis. In the studies about myocardial and skeletal muscles,resveratrol was reported to promote mitochondrial biogenesis and protect cellsfrom metabolic disease through SIRT1-dependented deacetylation of PGC-1αand FOXO. In diabetic nephropathy, whether resveratrol has renal protectiveeffect by the improvement of mitochondrial function through its activation ofSIRT1/PGC-1α, and then reducing oxidative stress and apoptosis still needmore studies to confirm.The present research will discuss possible mechanisms of mitochondrialoxidative damage and cell apoptosis in diabetic podocyte lesions throughestablishing mouse models of type1diabetes and stimulating podocytes withhigh glucose, and interventing with resveratrol and PGC-1α siRNAtransfection, in order to provide theoretical basis for early prevention ofdiabetic nephropathy.Methods：1The expression of SIRT1/PGC-1α in the renal tissues of STZ-induceddiabetic mice and the effects of resveratrolMale CD-1mice were randomly divided into three groups: normal controlgroup(NC), diabetic group (DM) and diabetic+Resveratrol group(DR).Diabetes were induced by intraperitoneal injection of streptozotocin (STZ, 150mg/kg). Individual animals with blood glucose concentrations more than16.7mM and the glucose in urine positive for3consecutive days afterinjection were confirmed as diabetes. The control mice were received0.1Mcitrate buffer solution only. After the diabetic model was affirmed to besuccessful, the mice of diabetic+resveratrol group were administered dailywith resveratrol (30mg/kg) by gavage. The mice of control group and diabeticgroup were only administered daily with the same volume of solvent bygavage. At4,8and12weeks after treatment, mice from every group wererespectively sacrificed. Blood and24h urine samples were collected forbiochemical indicators such as blood glucose, serum creatinine, totalcholesterol and urinary protein. Partial renal tissures were fixed in4%neutralformalin for histochemical, TUNEL and immunohistochemical staining.Partial renal cortices were fixed in4%glutaraldehyde for electron microscopicobservation. Protein extracted from partial renal cortices were used to detectedthe expression of SIRT1, PGC-1α, NRF1, TFAM, nephrin and cleave caspase3by Western blot.The other Protein was used to evaluated the MDA contentand Mn-SOD activity in renal cortex by colorimetry.2The effects of high glucose on SIRT1/PGC-1αexpression, mitochondrial oxidative damage and apoptosis in podocytesTo induce proliferation, the conditionally immortalized mouse podocyteswere cultured at33℃with γ-INF under growth permissive conditions, andthen cells were cultured at37℃without γ-INF under growth restrictiveconditions for10-14days to induce quiescence and the differentiatedphenotype. The podocytes were divided into3groups: Normal glucose group(NG), mannitol group (MG), high glucose group (HG). The three groups werecultured for12,24,48and72h respectively, and then podocytes wereharvested.The expression of SIRT1, PGC1α, NRF1and TFAM were detectedby immunocytochemistry and Western blot. The mRNA levels of SIRT1,PGC1α, NRF1and TFAM were evaluated by Real-time PCR. The expressionof SIRT1, PGC-1α, NRF1, TFAM, Nephrin, cleaved caspase-3, Cyto C andDIABLO was respectively evaluated by Western blot. The cell ROS production were detected by DCHF-DA staining.The mitochondrial ROSproduction was detected by MitoSOX staining. The mitochondrial membranepotential was measured by JC-1staining. Complex Ⅰ and Ⅲ enzymeactivities in mitochondria were measured by colorimetry. Podocyte apoptosiswas detected by AnnexinⅤ/PI staining.3The effects of resveratrol and PGC-1α siRNA on mitochondrialoxidative damage and apoptosis in podocytes stimulated by high glucose.Podocytes were randomly divided into six groups: normal glucose group(5.5mmol/L glucose, NG), high glucose group (30mmol/L glucose, HG),high glucose+resveratrol group(30mmol/L glucose+resveratrol10μmol/L，HG+Res), high glucose+EX527group(30mmol/L glucose+EX52710μmol/L,HG+EX527), high glucose+PGC1-siRNA group (30mmol/L glucose+PGC1siRNA, HG+PGC1siRNA) and high glucose+PGC1.siRNA+resveratrolgroup (30mmol/L glucose+PGC1siRNA+resveratrol10μmol/L,HG+PGC1siRNA+Res). The six groups were cultured for48hours, and thenpodocytes were harvested. The expression of SIRT1, PGC1α, NRF1andTFAM were detected by immunocytochemistry and Western blot. The mRNAlevels of them were evaluated by Real-time PCR. The expression of SIRT1,PGC-1α, NRF1, TFAM, Nephrin, cleaved caspase3, Cyto C and DIABLOwas respectively evaluated by Western blot. The cell ROS production weredetected by DCHF-DA staining. The mitochondrial ROS production wasdetected by MitoSOX staining. The mitochondrial membrane potential wasmeasured by JC-1staining. ComplexⅠ and Ⅲ enzyme activities inmitochondria were measured by colorimetry.Podocyte apoptosis was detectedby AnnexinⅤ/PI staining.Results：1The expression of SIRT1/PGC-1α in the renal tissues of STZ-induceddiabetic mice and the effects of resveratrol①Compared with control group,24h urine protein upregulated in atime-dependent manner in diabetic group. The BUN and TC levels wereincreased from week8in diabetic group than control group. Compared with diabetic group,24h urine protein and the BUN,TC levels ofdiabetic+Resveratrol group were significantly decreased.②Diabetic miceshowed significant glomeruli hypertrophy and expansion of the mesangialmatrix in glomeruli by light microscope. Uneven increase in thickness ofglomerular basement membrane, the fusion of podocyte foot processes,andthe decreased number and Morphological abnormalities of mitochondria inpodocytes of diabetic rats were observed by transmission electron microscopy.These lesions were improved in diabetic+Resveratrol group.③Colorimetrytest results showed that the MDA content in renal cortex of DM groupincreased than NC group, and upregulated in a time-dependent manner. TheMnSOD activity were reduced than NC group. Compared with DM group,The two indicators in DR group were improved.④Apoptotic cells byTUNEL were observed in renal tissues of the diabetic kidney. Apoptotic ratewas significantly lower in the diabetic+resveratrol group than that in diabeticgroup.⑤By immunohistochemical staining, the protein expression of SIRT1,PGC-1α, NRF1and TFAM were reduced in glomeruli and tubule cells ofdiabetic group than that in control group. Compared with control group,Western blot indicated that the protein expression of SIRT1in diabetic groupbegan to reduced at week4, and then down-regulated in a time-dependentmanner. The expression of PGC-1α, NRF1and TFAM were reduced fromweek8, while resveratrol increased their protein expression in diabetic mice.The expression of nephrin reduced,while the expression of cleaved caspase3were increased in diabetic group at week12, The two indicators in DR groupwere improved.2The effects of high glucose on SIRT1/PGC-1αexpression, mitochondrial oxidative damage and apoptosis in podocytes①Immunocytochemical staining showed that the expression of SIRT1,PGC-1α, NRF1and TFAM reduced in podocytes induced by high glucose.②By Western blot, the protein expression of SIRT1began to reduce after thestimulation of HG for12h. PGC-1α expression at12h increased slightly than0h, there was no difference between the expression of NRF1and TFAM at 12h with that of0h. They all reduced at24h, and down-regulated intime-dependent manner.③The effects of HG on mRNA levels of SIRT1wererevealed at12h and peaked at48h. HG also reduced PGC-1α, NRF1andTFAM mRNA expression from24h.④Western blot result showed that, theexpression of nephrin was down-regulated with HG stimulated intime-dependent manner.whereas cleaved caspase3, Cyto C and Diablo wereincreased at the same time.⑤Compared with normal glucose group, the ROScontent in podocyte treated with HG for12h was increased, and thenup-regulated over time.⑥The increase of mitochondrial ROS production inpodocyte treated with HG were observed by fluorescence microscope.⑦ComplexⅠ and Ⅲ enzyme activities in mitochondria were reduced by HGtreatment for24h, the reduce was more apparent at48h and72h.⑧Themitochondrial membrane potential was down-regulated in HG groupcompared with NG group.⑨Apoptosis rate of podocyte exposed to HG wasincreased gradually than that of NG group.3The effects of resveratrol and PGC-1α siRNA on mitochondriaoxidative damage and apoptosis of podocytes stimulated by high glucose.①Western blot and Real time PCR resulte showed that, the mRNA andprotein expression of SIRT1, PGC-1α, NRF1and TFAM reduced in HG groupthan those of NG group.They were up-regulated in HG+Res group,while theywere lower in HG+EX527group than HG group.The protein expression ofnephrin in HG group was reduced than NG group.Nephrin expression inHG+EX527group was down-regulated more remarkably than that of HGgroup, while that in HG+Res group was up-regulated than HG group. Theprotein expression of cleaved Caspase3，Cyto C and Diablo in HG group wereall increased than those of NG group.Those in HG+EX527increased morethan HG group，while those in HG+Res group were reduced than HG group.③There was no difference in SIRT1expression of HG+PGC1siRNAgroup and that of HG group.SIRT1expression in HG+PGC1siRNA+Resgroup was increased than HG group, but there was no obvious changes withthat of HG+Res group. The expression of PGC-1α, NRF1and TFAM were reduced than those of HG group, those in HG+PGC1siRNA+Res group werereduced than that of HG+Res group.④The expression of Nephrin inHG+PGC1siRNA group was more lower than that of HG group.That ofHG+PGC1siRNA+Res group was also lower than that of HG+Res group. Theexpression of cleaved caspase3in HG+PGC1siRNA group was higher thanthat of HG group.That of HG+PGC1siRNA+Res group was up-regulated thanthat of HG+Res group.⑤flow cytometry test results showed that, comparedwith HG group, the mitochondrial ROS production in HG+Res group wasdecreased significantly, while that of HG+EX527group was increased.⑥theComplexⅠ and Ⅲ enzyme activities and the mitochondrial membranepotential in HG+Res group were increased,whereas that of HG+EX527groupwere decreased.⑦The total ROS content and apoptosis rate of podocyte inHG+Res group were reduced than that of HG group. Those of HG+PGC1siRNA group were more higher than those of HG group. Those of HG+PGC1siRNA+Res group were higher than HG+Res group.Conclusions：1The expression of SIRT1, PGC-1α in renel tissues from diabetic miceor in the mouse podocytes stimulated by high glucose were all reduced,accompanied with enhanced oxidative stress and increased cell apoptosis.2Abnormal mitochondria morphology, fusion of podocyte foot andapoptosis in the glomerular podocytes were detected in diabetic mice.Mitochondrial ROS synthesis was increased, mitochondrial respiratoryfunction was decreased, mitochondrial apoptotic pathways was activated inpodocyte treated with high glucose, which suggested that mitochondrialdysfunction might participate in the oxidative damage and apoptosis ofpodocyte in diabetic nephropathy.3Resveratrol increased the expression of SIRT1,PGC-1α and nephrin,and improved mitochondrial morphology abnormalities and foot processfusion in podocyte, and reduced cell apoptosis in renal cortex of diabetic mice.4Resveratrol inceased the expression of SIRT1and PGC-1α,andreduced mitochondrial ROS synthesis, improved mitochondrial respiratory function and inhibited the activation of the mitochondrial apoptosis pathwayin podocytes stimulated by high glucose.5Resveratrol could relieve the podocyte oxidative damage and apoptosisin diabetic mice maybe through the mitochondrial protective mechanismsmediated by the activation of SIRT1/PGC-1α pathway.