| Acute kidney injury（AKI）is defined as a sudden decline（hours to days）in glomerular filtration rate,contributing to the retention of nitrogenous waste products such as urea and creatinine in the plasma.Renal ischemia reperfusion injury（RIRI）is one of the leading causes that cause AKI.In addition,RIRI is one of the main causes resulting in delayed graft function.To date,AKI mediated by RIRI is a major clinical problem without effective therapeutics and is an important and frequent cause that leads to high morbidity and mortality during perioperative period.Therefore,it is of great clinical significance to explore the underlying molecular mechanisms and therapeutic targets of RIRI pathogenesis.Renal tubular epithelial cells are characterized by high oxygen consumption,abundant mitochondria and sensibility to oxidative stress injury mediated by ischemia or hypoxia.Renal tubular epithelial cell shedding and dysfunction,luminal disorder,and basement membrane disruption induced by RIRI constitute the pathological basis of RIRI-AKI.The main cause that leads to oxidative stress injury is massive reactive oxygen species（ROS）generating and accumulating in renal cells during RIRI process.Therefore,to identify the mechanism whereby modulates ROS generation affords cytoprotective role in RIRI.Histone lysine methyltransferase G9a is involved in the regulation of pathophysiological processes such as apoptosis and proliferation,immune response,and oxidative stress,and the above pathophysiological processes are also the main injury mechanisms of RIRI.G9a mainly forms H3K9me1/2 by transferring one or two methyl groups to H3K9,which enriches the promoters of target genes,thereby promoting transcriptional repression of target genes.G9a is involved in regulating the pathogenesis of various kidney-related diseases through histone methyltransferase activity,such as renal tumors and kidney fibrosis.However,whether G9a regulates oxidative stress injury and participate in RIRI-AKI has not yet been explored.SIRT1,belonging to the family of NAD+-dependent class III histone deacetylase,suppresses apoptosis,inflammation,and oxidative stress injury when cells are suffered from adverse stimuli.In kidney-related diseases such as obstructive renal fibrosis,RIRI-AKI,and diabetic nephropathy,SIRT1 activates antioxidant transcription factors and antioxidant enzymes that catabolize ROS to alleviate acute renal injury and inhibit the progression of chronic kidney disease.To date,little work is performed on G9a modulating RIRI through targeting SIRT1 and the underlying mechanism whereby G9a regulates SIRT1.Based on the study background and our previous research results,we hypothesized that G9a regulates ROS generation and accumulation by targeting SIRT1 during RIRI,mediating oxidative stress damage and disrupting tubular epithelial cell structure and function,ultimately leading to AKI.To test the above hypothesis,we deeply explored the specific role of the G9a/SIRT1 axis in RIRI-AKI and the molecular mechanism of G9a regulation of SIRT1 through the in vitro and in vivo RIRI model.To this end,we demonstrate and study from the following three-part experiments.Part Ⅰ The expression and role of G9a in renal ischemia reperfusion injuryObjective:To clarify the expression pattern of G9a in RIRI-AKI,and to explore the role of G9a in RIRI-AKI.Methods:1.In vitro and and in vivo study:（1）The RIRI-AKI model was established on C57BL/6J mice,and the mice were sacrificed at 6h,12h and 24h of reperfusion to collect mouse serum and kidney tissue samples;（2）Hypoxia and reoxygenation（H/R）model of HK2 cells was established,and cells were collected at1h,3h and 6h of reoxygenation respectively;（3）The overexpression plasmids of p-CMV-Flag（KLF16,KLF4,PLAG1,E2F6,STAT5b）and p RL-SV40（G9a-Promoter）were constructed and were transfected into 293T cells;（4）overexpression plasmids of p RL-SV40（G9a-Promoter mutant and wild type）were constructed and transfected into 293T cells;（5）si Stat5b and si KLF5 were designed and were transfected HK2cells,followed by establishment of H/R model.2.The role G9a of in RIRI-AKI:（1）Animal experiments:1)BIX01294（10mg/kg,30mg/kg,50mg/kg）was intraperitoneally injected once a day for 3 consecutive days,followed by establishment of RIRI-AKI and sham model,and serum and kidney tissue samples were obtained from mice after reperfusion 24h;2)flox/flox transgenic mice,G9a conditional knockout mice（CKO）:RIRI-AKI and Sham models were established,and serum and kidney tissue samples were collected 24 hours after surgery;（2）In vitro model of H/R:1)HK2 cells were pretreated with BIX01294（0μM,1μM,5μM,10μM,30μM,40μM,50μM）for 6h,and the cells were collected;2)HK2 cells were pretreated with BIX01294（0μM,1μM,5μM,10μM,30μM,40μM,50μM）for 6h,followed by establishment of H/R model;3)si G9a was transfected with HK2 cells followed by establishment of H/R model.3.Experimental detection:renal tissue damages were assessed by H&E staining,kidney function was evaluated by detecting serum creatinine（Scr）and Blood urea nitrogen（BUN）,cell viability was assessed by CCK8,expression changes of G9a,KLF5,and Stat5b in kidney tissues and cells were detected by q PCR and WB,G9a expression and localization were detected by immunohistochemistry,the transcriptional activity of G9a promoter was detected by luciferase reporter assay,and the enrichment of KLF5 in G9a promoter was detected by Ch IP assay.Results:1.G9a was up-regulated in RIRI-AKI and H/R models,and further up-regulated with the increase of reperfusion or reoxygenation time;2.RIRI led to renal tissue damage,increased Scr and BUN concentrations,and further aggravated with the increase of reperfusion time;3.H/R decreased the cell viability of HK2,and further decreased with the prolongation of reoxygenation time;4.The results of immunohistochemistry were consistent with q PCR and WB,and G9a was mainly expressed and located in the nucleus of renal tubular cells;5.Luciferase reporter assay and Ch IP assay suggested that KLF5 regulated G9a transcriptional activity and mediated the up-regulation of G9a expression in RIRI;6.Suppression of G9a with BIX or G9a CKO protected against RIRI-AKI.Conclusion:1.The expression of G9a was significantly increased in RIRI and was positive correlated with renal tissue damage and dysfunction;2.The transcription factor KLF5 mediates the transcriptional activation of G9a promoter during the process of RIRI;3.inhibition of G9a or G9a dificiency alleviated ischemic AKI.Part Ⅱ Effects of G9a on oxidative stress and apoptosis in renal ischemia-reperfusion injuryObjective:To explore the role of G9a in RIRI-AKI.Methods:1.Animal RIRI-AKI model:（1）Wild-type C57BL/6J mice:mice were treated with various doses of BIX,followed by establishment of RIRI-AKI or Sham;（1）RIRI-AKI or Sham models were established on G9aflox/floxand G9a CKO mice.2.In vitro study:1)HK2 cells were treated with different concentration of BIX,followed by challenged by H/R stimuli;2)si G9a were transfected into HK2 cells and H/R model was established.3.Sample detection:Apoptotic cells were detected by TUNEL staining in vitro and in vivo,cell viability was measured by CCK8 assay,mitochondrial membrane potential was tested using JC-1 probe,Reactive oxygen species（ROS）was assessed by DHE probe,total ROS in HK2 cells was detected using DAFH-DA,Mito Sox probe was used to detect mitochondrial ROS,G9a,Catalase,SOD1,SOD2 and Bax/Bcl2 expression levels were measured by q PCR and WB.Results:1.In vivo study:（1）BIX01294 pretreatment or knockout of renal G9a significantly reversed the down-regulation of antioxidant enzymes Catalase,SOD1and SOD2 caused by RIRI-AKI;（2）BIX01294 pretreatment or knockout of kidney G9a reduced the number of apoptotic cells during RIRI-AKI process and down-regulated the expression of the pro-apoptotic gene Bax and up-regulated the expression of the anti-apoptotic gene Bcl2.2.In vitro study:（1）BIX01294pretreatment or silencing of G9a inhibited H/R-mediated apoptosis,down-regulated the expression of the pro-apoptotic gene Bax and up-regulated the expression of the anti-apoptotic gene Bcl2;（2）BIX01294 pretreatment or silencing of G9a up-regulated the expression of antioxidant enzymes Catalase,SOD1 and SOD2 in the H/R process;（4）BIX01294 pretreatment or silencing of G9a stabilized mitochondrial membrane potential in H/R injury model.Conclusion:Inhibition of G9a activity or knocking out G9a alleviated RIRI-AKI-mediated apoptosis,stabilized mitochondrial membrane potential,increased mitochondrial respiratory capacity and reduced ROS accumulation in tissue cells.Part Ⅲ G9a regulates renal ischemia-reperfusion injury through epigenetic modification of SIRT1 transcriptionObjective:To screen the downstream target gene SIRT1 regulated by G9a in the process of RIRI-AKI,to verify the role of SIRT1 in RIRI-AKI,to clarify the specific molecular mechanism of G9a’s regulation of SIRT1,and to determine that G9a’s regulation of RIRI-AKI is dependent on SIRT1.Methods:1.Identification of the downstream gene targeted by G9a:（1）RIR-AKI model was established on flox/flox and G9a knockout mice,followed by RNA-Seq sequencing on renal tissues to analyze differentially expressed genes regulated by G9a;（2）In vitro and in vivo experimental models were established to detect the expression of SIRT1 by q PCR and WB.2.To verify the role of SIRT1:（1）Animal model:1)A RIRI model was established in wild-type C57BL/6J mice,and kidney tissue samples were collected at 6h,12h,and 24h after reperfusion;2)AAV9-SIRT1 overexpression vector and AAV9 empty vector were constructed and injected into wild-type C57BL/6J mice via tail vein,and RIRI-AKI model was established two weeks later.（2）In vitro model:1)H/R model was established,and cells were collected at 1h,3h and 6h of reoxygenation;2)Ad-SIRT1 overexpression vector and Ad empty vector were constructed,and were transfected into HK2 cells,followed by establishment of H/R model;3)HK2 cells were pretreated with SIRT1small molecule activator SRT2183（5μM）for 6 h to establish H/R model.（3）Experimental analysis:q PCR and WB were used to detect the expression of SIRT1,Catalase,SOD1,SOD2,Bax/Bcl2,Co IP to detect the effect of BIX01294 on H3K9me1/2,H&E staining to assess tissue damage,kit to detect Scr and BUN concentrations,TUNEL staining to detect apoptotic cells,JC-1 probe to detect mitochondrial membrane potential of HK2 cells,DHE probe to detect ROS in kidney tissue,DAFH-DA probe to detect total ROS of HK2 cells,and Mito Sox probe to detect mitochondrial ROS.3.The molecular mechanism whereby G9a regulated SIRT1:（1）In vivo study:The RIRI-AKI and Sham models were established in flox/flox and G9a knockout mice;2)The RIRI-AKI and Sham models were established in wild type mice pretreated with BIX01294.（2）In vitro study:1)The H/R model was established in HK2 cells pretreated with BIX01294 or transfected with si G9a;2)p-CMV-Flag（HP1α,HP1β,HP1γ）and p RL-SV40（SIRT1-Promoter）overexpression plasmids were constructed and were transfected into 293T cells;3)The H/R model was established in HK2 cells transfected with si HP1α、si HP1βor si HP1γ.4.G9a modulated RIRI by targeting SIRT1:（1）Animal study:1)AAV9-SIRT1 overexpression vector and AAV9 empty vector were constructed and injected into flox/flox and CKO mice,followed by establishment of RIRI model;2)AAV9-SIRT1 overexpression vector and AAV9 empty vector were constructed and injected into wild-type C57BL/6J mice pretreated with BIX01294,followed by establishment of RIRI model.（2）Cell experiments:（1）si SIRT1 was transfected with HK2 cells pretreated with BIX01294,followed by exposure to H/R stimuli.5.Experimental analysis:（1）WB and q PCR were used to assessed the expression of G9a,SIRT1,Catalase,SOD1,SOD2,HP1α,HP1β,and HP1;（2）Luciferase reporter and Ch IP experiments were employed to confirm that G9a,H3K9me1,H3K9me2,HP1α,HP1β,HP1γinteracted with SIRT1 promoter chromatin;（3）H&E staining was used to assess tissue damage,kits were used to detect Scr and BUN concentrations,TUNEL staining was used to detect apoptosis in kidney tissue and HK2 cells,JC-1probe was used to detect mitochondrial membrane potential of HK2 cells,DHE probe was used to detect renal tissue ROS,and DAFH-DA probes were used to detect total ROS in HK2 cells,and Mito Sox probes were used to detect mitochondrial ROS.Results:1.The downstream gene targeted by G9a:（1）The results RNA-Seq sequencing indicated that SIRT1 might be a potential downstream gene target of G9a;（2）SIRT1 expression was down-regulated in in vitro and in vivo models,and further down-regulated with prolonged reperfusion or reoxygenation;2.The role of SIRT1 in in vitro and in vivo models:（1）H/R cell model:1)SRT2183 activation of SIRT1 or Ad-SIRT1 transfection into HK2 cells reversed the H/R-mediated down-regulated expression of antioxidant enzymes Catalase,SOD1,and SOD2;2)Activation or overexpression of SIRT1 inhibited cellular and mitochondrial ROS accumulation during H/R;3)Activation or overexpression of SIRT1 stabilized the membrane potential in the H/R process;4)Activation or overexpression of SIRT1 inhibited apoptosis,downregulates the pro-apoptotic gene Bax and up-regulates the anti-apoptotic gene Bcl2.（2）Animal models:1)AAV9-mediated overexpression of SIRT1 significantly improved the pathological damage of renal tissue caused by RIRI-AKI and reduced the concentration of Scr and BUN;2)Overexpression of SIRT1 inhibited the down-regulation of Catalase,SOD1 and SOD2 in the process of RIRI-AKI,and reduced the accumulation of ROS in renal tissue;3)AAV9overexpression of SIRT1 reversed RIRI-AKI-mediated Bax upregulation and Bcl2downregulation,and reduced apoptotic cells in kidney tissue.3.The molecular mechanism of G9a regulating SIRT1:（1）Cell experiments:1)The results of 293T cell experiments suggest that H3K9me2 catalyzed by G9a bound to the-657bp/1263bp region of the SIRT1 promoter,while H3K9me1 hardly enriched in SIRT1 promoter;2)In the H/R model,G9a and H3K9me2 were recruited to the SIRT1 promoter,and H3K9Ac enrichment on the SIRT1 promoter was significantly reduced;3)During the H/R process,G9a,H3K9me2,H3K9me1 and H3K9Ac were not enriched in GAPDH and SOD1 promoters;4)BIX01294 inhibition or si RNA silencing of G9a significantly inhibited the enrichment of H3K9me2 into SIRT1 promoter chromatin during H/R process;5)During the H/R process,the expression of HP1βwas up-regulated and further increased with the prolongation of reoxygenation time,while the expressions of HP1αand HP1γremained unchanged;6)HP1βcooperated with G9a to form the H3K9me2/HP1β/G9a transcriptional repression complex in the chromatin region of the SIRT1 promoter;7)Silencing HP1βreversed the H/R-mediated down-regulation of antioxidant enzymes Catalase,SOD1,SOD2 and SIRT1;while silencing HP1αor HP1γhardly affected the expressions of Catalase,SOD1,SOD2 and SIRT1;8)Silencing HP1βinhibited the up-regulation of Bax and down-regulation of Bcl2 in H/R process,while silencing HP1αor HP1γhad no significant effect on Bax/Bcl2.（2）Animal experiments:1)During the RIRI process,G9a and H3K9me2 were recruited to the SIRT1 promoter,and the enrichment of H3K9Ac on the SIRT1 promoter was significantly reduced;2)During the RIRI process,G9a,H3K9me2,H3K9me1 and H3K9Ac hardly enriched in GAPDH and SOD1 promoters;3)Inhibition of G9a by BIX01294 or knockout of G9a significantly inhibited the enrichment of H3K9me2 in SIRT1 promoter chromatin;4)RIRI caused up-regulation of HP1βexpression,but had no significant effect on HP1αand HP1γexpression;5)During the RIRI process,HP1βcooperated with G9a to form the H3K9me2/HP1β/G9a transcriptional repression complex around the chromatin region of the SIRT1 promoter;6)Inhibition of G9aBy BIX01294 or knockout of G9a promoted SIRT1 transcription during RIRI.4.The regulation of RIRI-AKI by G9a is dependent on SIRT1:（1）In vitro model:1)In the H/R process,BIX01294 combined with si SIRT1 inhibited the BIX01294-mediated up-regulation of Catalase,SOD1,SOD2 expression;2)Transfection of si SIRT1 into HK2 cells on the basis of BIX01294 pretreatment counteracted the scavenging effect of BIX01294 on cytoplasmic and mitochondrial ROS;3)In the H/R process,BIX01294 combined with si SIRT1 treatment significantly reduced the number of JC-1 polymers and increased the number of JC-1 monomers;4)BIX01294 combined with si SIRT1 treatment up-regulated the expression of Bax,down-regulated the expression of Bcl2,and increased the number of apoptotic cells.（2）In vivo model:1)In the RIRI model,BIX01294 combined with AAV9-sh SIRT1 promotes renal tissue damage and renal dysfunction;2)BIX01294 combined with AAV9-sh SIRT1treatment inhibited the expression of Catalase,SOD1,SOD2,and increased the accumulation of ROS in renal tissue;3)In the RIRI model,G9a knockout combined with AAV9-sh SIRT1 treatment resulted in obvious pathological damage and kidney dysfunction;4)G9a knockout combined with AAV9-sh SIRT1 treatment of mice subjected to RIRI showed that the expressions of Catalase,SOD1 and SOD2 in the kidney tissue were significantly down-regulated,and the accumulation of ROS in the kidney tissue was increased.Conclusions:1.The downstream gene directly target by G9a was SIRT1;2.Overexpression or activation of SIRT1 alleviated renal tissue damage and renal dysfunction caused by RIRI-AKI through regulating mitochondrial function and ROS generation;3.In in vitro and in vivo models,HP1βcooperated with G9a to form the H3K9me2/HP1β/G9a transcriptional repression complex on the SIRT1 promoter to inhibit SIRT1 transcription,thereby regulating SIRT1 expression;4.In vivo and in vitro experiments,G9a modulated RIRI-AKI dependent on SIRT1. |
PDF Full Text Request