The Role And Molecular Mechanism Of SIRT3 In Mouse Hypertensive Kidney Injury

BackgroundHypertension is a major cause of chronic kidney disease(CKD).Hypertensive kidney damage,as a major complication of hypertension,which is characterized by proteinuria,progressive renal fibrosis and inflammation.Angiotensin ?(Ang ?)is a key mediator in hypertension-induced renal injury.It plays a key role in the progression of chronic kidney disease.Proteinuria,as an independent risk factor of hypertensive kidney damage,can worsen renal fibrosis.Renal fibrosis exhibits excessive production and deposition of extracellular matrix(ECM),which leads to the destruction of renal parenchyma and progressive loss of renal function.The degree of renal fibrosis closely relates with the prognosis of CKD.Podocytes are the terminally differentiated visceral epithelial cells.They are the outer surface of the glomerular capillaries.As a major component of the glomerular ultrafiltration apparatus,podocytes have a complex cellular structure consisting of cell body and foot processes.The interdigitated foot processes(FPs)are formed by major processes that extend outward from their cell body.Podocytes,as the terminally differentiated cells of glomerular filtration barrier,play an essential role in maintenance of the glomerular filtration barrier so as to block proteinuria in CKD.In addition,podocytes damage or loss can result in the accumulation of fibrotic factors,followed by renal fibrosis and CKD.Reduced fibrosis factors in kidneys,such as fibronectin and collagen type IV,could improve and even reverse renal fibrosis in hypertensive kidney injury.SIRT3 is a member of the evolutionary conserved family of NAD+-dependent deacetylase.It plays a key role in various processes.SIRT3 is localized in the mitochondria matrix.It can regulate various processes through deacetylating its substrates at posttranslational modification.Recently,a study reported that SIRT3 blocks TGF(31 signaling and protects aging-associated tissue from fibrosis by deacetylating GSK3?.In kidney,SIRT3 has been reported to exhibit a protective role in cisplatin-induced acute kidney injury(AKI).Furthermore,Ang? downregulates SIRT3 mRNA expression in cultured tubular epithelial cells,and the effect might be prevented by an AT1 antagonist.In addition,SIRT3 is upregulated by nicorandil and reduced by glibenclamide in dose-dependent manner in cultured podocytes.However,the function of SIRT3 in hypertension-induced kidney damage and podocyte injury remains unclear.Aims:1.To explore the expression of SIRT3 in hypertensive renal cortex;2.To explore whether SIRT3 can involve in the process of renal damage in hypertensive mice;3.To explore whether SIRT3 can regulate podocyte injury caused by hypertension.Experiment and methods:1.Animals:SIRT3-knockout(SIRT3-KO,129S6/SvEvTac)mice,were purchased from the Jackson Laboratory(USA).129 wild-type(WT)male mice of 8 weeks old were purchased from Department of Laboratory Animal Science of Peking University(Beijing,China).SIRT3-overexpression(SIRT3-LV)mice were obtained by injection to the 129 WT mice SIRT3-overexpression-lentivirus(JIKAI GENE,Shanghai,China)via caudal vein.The animal experimental protocol and animal care procedures complied with the Animal Management Rules of the Ministry of Public Health,People's Republic of China(documentation No 55,2001)and were approved by the Animal Care Committee of Shandong University.Together,the experimental animals(90 mice)were randomly assigned to six groups:WT+saline group,WT+AngII group,SIRT3-KO+saline group,SIRT3-KO+Ang? group,SIRT3-IV+saline group,SIRT3-LV+AngII group.2.Animal model:WT mice,SIRT3-KO mice and SIRT3-LV mice were all implanted subcutaneously corresponding osmotic pumps beforehand filled with Ang? or saline for 42 days(2000ng/Kg/min).Mice were sacrificed at the end of experiment and the kidney cortex were used to study.In addition,before modeling and death,blood pressure were measured by Tail-Cuff way(BP2010A,softron,Japan).To collect mouse urine and blood to detect renal function.3.Renal function:The supernatant of urine and blood were cryopreserved into the-80?refrigerator.The renal function indexes were detected by the ELISA kit according to the direction of the kit.4.Histology and immunohistochemistry:Periodic acid Schiff(PAS)staining and Masson trichrome staining were performed to observe the level of glomerulosclerosis and renal interstitial fibrosis.The immunohistochemical staining of the fibrotic factors were used to evaluate the degree of renal fibrosis.These analysis of the results were quantified using Image-Pro Plus 6.0.5.Transmission Electron Microscopy:Renal cortex were fixed in 2.5%glutaraldehyde for electron microscopy to observe foot process.The images were observed using a transmission electron microscope(TEM,H-7000FA,Hitachi,Tokyo,Japan).6.Cell culture and treatment:Conditionally immortalized mouse podocytes(MPC-5)were cultured at 33? and 5%CO2 to propagate and were incubated at 37? to induce differentiation.Podocytes were verified by the immunofluorescence of WT-1 and synaptopodin,which are the markers of podocytes.The mature podocytes were used for experiments,include transfection and Ang?-treatment(10-6)in this study.7.Western blot:Isolated kidney cortex or cultured cells were lysed by the addition of lysis buffer.Protein samples were subjected to electrophorese,and then transferred the disconnected proteins to PVDF membrane in a designated time before blocked with 5%skim milk in 1X TBST for 1h.Next,the blocked membrane were incubated with corresponding antibodies at 4? overnight.The membrane were incubated with the secondary antibody for 1h and the protein band were showed by enhanced chemiluminescence(Millipore)and an Image Quant LAS4000 chemiluminescence reader(GE,USA).The analysis of the results were quantified using Image J.8.Immunofluorescence:Cultured podocytes were fixed with 4%paraformaldehyde and then treated with Triton X-100 followed by blocked,and then incubated with antibodies at 4? overnight.All imaging analyses were used in laser scanning confocal microscope after the incubating with the secondary antibodies marked with FITC or TRITC.9.Statistical analysis:Data were shown as mean ± SEM.Statistical analysis was performed with unpaired t test to analyze data between two groups or ANOVA to assess differences among groups,using GraphPad Prism 6(La Jolla,CA,USA).P<0.05 was considered significant.Results:1.The decrease of SIRT3 in mouse hypertensive kidney cortexWe detected the blood pressure so as to ensure the success of the hypertensive model.We detected SIRT3 expression in mouse kidney cortex.Western blot showed that SIRT3 decreased in mice subjected to chronic Ang? infusion compared with the controls infused with saline.Moreover,immunofluorescence also showed the reducing of SIRT3 expression in hypertensive glomerulus.Altogether,the results revealed that SIRT3 decreased in mouse hypertensive kidney cortex.2.SIRT3 regulated renal function in hypertensive miceWe detected renal function and found that the ratio of urine creatinine to albumin,BUN and serum creatinine were all increased in mice with Ang? infusion,and especially in the SIRT3-KO group.However,the overexpression of SIRT3 improved hypertensive renal function.It is suggested that SIRT3 might involve in the regulation of renal function in hypertensive kidney injury.3.SIRT3 regulated renal fibrosis in hypertensive nephropathyPAS and MASSON staining results showed that the level of glomerulosclerosis and interstitial fibrosis increased in Ang?-infused mice,of which the extent in SIRT3-KO mice was the greatest.However,SIRT3-overexpression improved renal fibrosis.Furthermore,Western blot and immunohistochemistry showed that the expressions of fibronectin and collagen type IV were elevated in mice with chronic Ang? treatment.However,SIRT3-overexpression mice exhibited lower expression of fibrosis markers than WT and SIRT3-KO mice.Altogether,SIRT3 may play a key role in renal fibrosis in hypertensive nephropathy.4.SIRT3 participated in the process of hypertensive podocyte injuryTransmission electron microscope showed that foot processes were fused or effaced in Ang?-infused mice and SIRT3 ablation aggravated podocyte injury.Furthermore,the electron density of glomerular basement membrane increased in hypertensive kidney without SIIRT3.The overexpression of SIRT3 improved hypertension-induced podocyte injury.In a word,SIRT3 may regulate podocyte damage in hypertensive mice.5.SIRT3 reduced the expression of fibrotic factors in podocytesWe knocked down SIRT3 through siRNA transfection in cultured podocytes and they were stimulated by Angll(10-6 mol/L,48h).Western blot showed that the expressions of fibronectin and collagen type IV were increased with siRNA transfection and elevated more following Ang? stimulation.In sum,the results revealed that SIRT3 could decrease the expression of fibronectin and collagen type IV in podocytes.Conclusions:1.SIRT3 can be decreased in hypertensive kidney cortex;2.SIRT3 can regulate renal function and fibrosis in hypertensive nephropathy;3.The decrease of SIRT3 can involve in the process of podocyte injury in hypertensive mice.BackgroundChronic kidney disease(CKD)is a global public health problem.Hypertension is the second leading cause of CKD.Renal fibrosis is the main pathological basis of hypertension-induced kidney damage,such as glomerulosclerosis and renal interstitial fibrosis.However,so far,the mechanism of renal fibrosis has not been fully understood.Renal fibrosis lead to the progressive loss of kidney function to end-stage renal disease.At present,therapeutic options for this devastating disorder are scarce and often ineffective,except for dialysis or kidney transplantation.Hence,an improved understanding of the molecular mechanisms involved in the initiation and development of CKD may play a key role in the development of rational strategies to treat kidney disease and prevent its progression.SIRT3 is a member of sirtuins,located in mitochondria.It is a key mediator in varieties processes include metabolism and oxidative stress.At present,many targets of SIRT3 have been reported.SIRT3 plays a role through regulating the activity of its targets.However,the molecular mechanism of SIRT3 in hypertensive kidney damage remains unclear.KLF15,as a transcription factor,is rich in kidney and liver.KLF 15 could prevent cardiac fibrosis induced by stress through reducing the expression of extracellular matrix(ECM).Furthermore,KLF15 improves renal interstitial fibrosis in the renal interstitium of 5/6 nephrectomized rats.However,the study on the interaction between SIRT3 and KLF15 requires further research.The evidence that supplementation of NAD and the administration of AICAR could elevate SIRT3 expression,so as to protect from organic injury.It provides a new therapeutic manipulation aimed at increasing SIRT3.However,the activator of SIRT3 has been found little.Honokiol,as a low molecular weight polyphenolic compound,is isolated from the traditional Chinese herb Magnolia officinalis(Houpo).It has been suggested that honokiol exhibits a variety of pharmacological activities,include anti-inflammatory,antiangiogenic,antioxidant,and antitumor properties.It has been reported to activate SIRT3 and prevent cardiac hypertrophy.In addition,honokiol could attenuate interstitial damage and progressive tubule fibrosis in UUO kidneys.In renal cells,honokiol also blocked TGF-? 1-induced expression of pro-fibrotic factors and ECM accumulation.Hence,in our study,we would explore the role and molecular mechanism of activation of SIRT3 by honokiol in hypertensive kidney damage.Aims:1.To explore the molecular mechanism of SIRT3 in hypertensive kidney injury;2.To explore the role of honokiol in regulating SIRT3 in kidney cortex and podocyte;3.To explore the function of activated SIRT3 in kidney injury caused by hypertension.Experiment and methods:1.Animals:129 wild-type(WT)mice(8 weeks,male)were purchased from Department of Laboratory Animal Science of Peking University(Beijing,China).The animal experimental protocol and animal care procedures complied with the Animal Management Rules of the Ministry of Public Health,People's Republic of China(documentation No 55,2001)and were approved by the Animal Care Committee of Shandong University.Together,the experimental animals(60 mice)were randomly assigned to four groups:WT+saline group,WT+Ang? group,WT+HKL+saline group,WT+HKL+Ang? group.2.Animal model:The HKL treatment model was obtained by intraperitoneally injecting HKL in 129 WT mice(25mg/Kg,once a day).All experimental mice were implanted subcutaneously corresponding osmotic pumps beforehand filled with Ang?or saline for 42 days(2000ng/Kg/min).Mice were sacrificed at the end of the experiment,and the kidney cortex were used to assay for the study on renal fibrosis.In addition,Blood pressure were measured by Tail-Cuff way before modeling and death.Urine and blood were also collected in the end to detect renal function.3.Renal function:Urine albumin and creatinine,BUN,and serum creatinine were detected by ELISA kit according to the direction of the kit,so as to assess the renal function.4.Histology and immunohistochemistry:Periodic acid Schiff(PAS)staining and MASSON trichrome staining were performed to assess the level of glomerulosclerosis and interstitial fibrosis.The fibrosis factors were evaluated by immunohistochemical staining.The photomicrographs were quantified also using Image-Pro Plus 6.0.5.Cell culture and the treatment:Conditionally immortalized mouse podocytes(MPC-5)were cultured at 33? and 5%CO2 to propagate.Podocytes were incubated at 37? to induce differentiation used for experiments,include transfection of siRNA-SIRT3 and Ang?-treatment in this experiment.6.Western blot:Cultured cells or isolated kidney cortex were lysed to collect the total proteins.Protein samples were subjected to electrophorese on 10%SDS/PAGE gels,and then the disconnected proteins transferring to PVDF membrane in a designated time.Blocked with 5%skim milk in 1X TBST for 1h.The blocked membrane were incubated with corresponding antibodies at 4? overnight.The membrane were incubated with the secondary antibody and the protein band were showed by enhanced chemiluminescence(Millipore)and an Image Quant LAS4000 chemiluminescence reader(GE,USA).7.Immunoprecipitation:After the preparation of protein samples,to remove nonspecific binding according to manufacturer's instructions.SIRT3 or KLF15 was immunoprecipitated with their corresponding antibodies and then the samples were performed immunoblotting according to the above.8.Immunofluorescence:Cultured podocytes were fixed with 4%paraformaldehyde,treated with Triton X-100,blocked with 1%BSA and then incubated with antibodies.All imaging analyses were used in laser scanning confocal microscope after the incubating with the secondary antibodies.9.Statistical analysis:Data were shown as mean ? SEM.Statistical analysis was performed with GraphPad Prism 6(La Jolla,CA,USA)and using unpaired t test to analyze data between two groups.To assess differences among groups by using ANOVA.P<0.05 was considered significant.Results:1.SIRT3 blocked the expression of fibrotic factors via deacetylating KLF15In this study,immunofluorescence showed a co-localization of SIRT3 and KLF15 in podocytes and they could interact directly with each other.Moreover,immunoprecipitation confirmed SIRT3 could deacetylate KLF15 at posttranslational modification in vitro.However,the level of KLF15 acetylation was increased and the interaction between SIRT3 and KLF15 weakened by Angll stimulation.Furthermore,we knocked down KLF15 in podocytes,and found that the expressions of both fibronectin and collagen type IV were increased compared with the controls.It is suggested that SIRT3 activated the anti-fibrotic function of KLF15 via deacetylating KLF15.2.HKL increased SIRT3 expression in podocyteWe found that SIRT3 was increased in podocyte with HKL treatment compared with the controls by Western blot.Furthermore,the group with HKL treatment in Ang?-stimulated group also showed increased SIRT3 in podocyte compared with the Ang?-stimulated group.The results suggested that HKL could increase SIRT3 expression in podocyte.3.The activating of SIRT3-KLF15 signaling blocked Ang?-induced fibrosisTo further validate the activity of SIRT3-KLF15 signaling affected by HKL treatment,immunoprecipitation and Western blot confirmed the expression and the deacetylation status of KLF15 were increased after HKL treatment.In addition,HKL treatment decreased fibrotic factors.These results suggested that the activating of the SIRT3-KLF15 signaling by HKL can alleviate Ang?-induced fibrosis in podocytes.4.The activating of SIRT3-KLF15 signaling improved renal function in hypertensive miceWe detected urine albumin and creatinine,BUN,serum creatinine.It is found that the ratio of urine creatinine to albumin,blood urea nitrogen(BUN)and serum creatinine(Scr)were all reduced in hypertensive mice with HKL treatment.It is suggested that the activating of SIRT3-KLF15 signaling might play a key protective role in renal function in hypertensive mice.5.The activating of-SIRT3-KLF15 signaling suppressed glomerulosclerosis and interstitial fibrosis in hypertensive nephropathyPAS and MASSON staining results showed that the areas of mesangial matrix and interstitial fibrosis reduced in HKL-treatment hypertensive mice.The results indicated that the activating of SIRT3-KLF15 signaling may protect against glomerulosclerosis and interstitial fibrosis in hypertensive kidney injury.6.The activating of SIRT3-KLF15 signaling decreased fibronectin and collagen type IV in hypertensive miceWestern blot and immunohistochemistry confirmed that the expressions of fibronectin and collagen type IV were all decreased in hypertensive mice with HKL treatment.Whereas,SIRT3 was increased with HKL treatment in kidney cortex.The results indicated that the activating of SIRT3-KLF15 signaling may decrease fibronectin and collagen type IV in hypertensive miceConclusions:1.SIRT3 can block renal fibrosis by activating KLF15;2.The activating of SIRT3-KLF15 signaling can alleviate hypertension-induced kidney injury;3.HKL can activate SIRT3-KLF15 signaling so as to be a novel target in the therapy on hypertensive kidney damage.