Expression Of SIRT6 In Renal Fibrosis In UUO Mouse

Background and Objective:Renal fibrosis is caused by trauma,infection,inflammation,blood microcirculation disorder,autoimmune response and other factors.Characterized by extracellular matrix deposition of the kidney,renal fibrosis is an important cause of end-stage renal failure.Sirtuin 6(SIRT6)is a homologous protein of silencing information regulator 2,which is involved in various signal regulation pathways and plays a very important role in regulating the lifespan of cells.Therefore,it is of great significance for the occurrence and development of organ fibrosis diseases.However,the intrinsic relationship between SIRT6 and renal fibrosis remains to be further studied.A typical mice renal fibrosis model–unilateral ureteral obstruction(UUO)–was constructed and was given renal pelvis injection SIRT6 adenovirus in situ.The expression level of SIRT6 and the probable regulating mechanism in renal fibrosis mice was researched,aiming to provide new ideas for the prevention and control of renal fibrosis.Methods:30 healthy,clean grade male wild-type C57BL/6J mice were chosen and randomly divided into three groups,10 in each group,namely Sham group,overexpressed control enhanced green fluorescent protein(EGFP)group(UUO+EGFP group),and overexpressed SIRT6 group(UUO+SIRT6 group).The left ureter was dissociated but not ligated in Sham group.In UUO+EGFP group and UUO+SIRT6group,the left ureter was dissociated and ligated to build renal fibrosis mice model.UUO+SIRT6 group was injected with 50?l(6x10~9 PFU/ml)SIRT6 over-expression adenovirus in renal pelvis,but UUO+EGFP group was given EGFP adenovirus50?l(6x10~9 PFU/ml)in the same way.Animals were harvested 7 days after the the surgery with the left kidney tissue.HE staining,Masson staining,Sirius red staining and immunohistochemical staining were used to observe the pathological changes of kidney.Detection of hydroxyproline(HYP)content was used to evaluate fibrosis.Western blot and real-time fluorescent quantitative PCR were used to observe the expression of renal fibrosis markers(?-SMA,Collegan-?),fibrosis factor(TGF-?1)and SIRT6 in mice kidney.Correlation analysis was applied to confirm the relationship among SIRT6,?-SMA,Collegan-I and TGF-?1.Results:1.SIRT6 expression in kidney tissues of mice in each groupSIRT6 was normally expressed in renal mesenchyme and tubular epithelial cells in Sham group.SIRT6 protein and gene expression in UUO+EGFP group decreased compared with Sham group(P<0.05).After SIRT6 overexpression adenovirus injection,SIRT6 protein and gene expression level in UUO+SIRT6 group increased significantly compared with that in UUO+EGFP group(P<0.01).This result was the basis of the whole experiment.2.Effect of SIRT6 on renal structureKidney pathological sections of mice in each group were prepared,and HE staining,Masson staining,Sirius red staining and immunohistochemical staining were performed.(1)HE staining:no abnormalities were observed in the renal tissues of mice in Sham group;Compared with Sham group,the degree of renal injury in UUO+EGFP group was significantly increased(P<0.01).After SIRT6 overexpression treatment,the injury degree of UUO+SIRT6 group decreased(P<0.05)compared with UUO+EGFP group.(2)Masson staining:UUO+EGFP group showed significant renal fibrosis compared with Sham group,and a large amount of blue collagen fibers accumulated in the interstitium(P<0.01).After SIRT6 overexpression,UUO+SIRT6 group showed less fibrosis than UUO+EGFP group(P<0.01).(3)Sirius red staining:compared with Sham group,UUO+EGFP group showed significant renal fibrosis,with a large amount of red collagen fibers deposited in the renal interstitium(P<0.01).After SIRT6 overexpression,the lesion degree of UUO+SIRT6 group was less than UUO+EGFP group(P<0.01).(4)Immunohistochemical staining:In UUO+EGFP group,renal interstitial and glomerular basement membrane was obviously stained on a large scale,the expression level of?-SMA and Collegan-I increased compared with Sham group(P<0.01).After SIRT6overexpression,the expression level of?-SMA and Collegan-?in UUO+SIRT6 group decreased significantly than UUO+EGFP group(P<0.01).3.Effect of SIRT6 on HYP content in kidney tissuesThe HYP content measurement results showed that the HYP content in UUO+EGFP group(0.22±0.03?g/mg)was increased compared with Sham group(0.12±0.02?g/mg)(P<0.05),but the content in UUO+SIRT6 group(0.18±0.01?g/mg)was lower than that in UUO+EGFP group(P<0.05).4.Effect of SIRT6 on markers of renal fibrosisCompared with Sham group,protein and mRNA expression levels of markers of renal fibrosis including?-SMA,Collegan-I and TGF-?1 in UUO+EGFP group were increased(P<0.05).The relative protein and mRNA expression levels of markers of renal fibrosis in UUO+SIRT6 group with SIRT6 overexpression treatment were decreased compared with UUO+EGFP group(P<0.05).In UUO model,the expression level of SIRT6 was negatively correlated with?-SMA(r=-0.474,P=0.027),Collegan-I(r=-0.516,P=0.024)and TGF-?1(r=-0.507,P=0.040).Conclusions:1.Overexpression of SIRT6 can reduce renal fibrosis of UUO model in mice,with a protective effect on the kidney.2.Its mechanism may be related to inhibiting the expression of?-SMA,Collegan-I and TGF-?1.And SIRT6 may be a new therapeutic target of renal fibrosis.