ABSTRACT
Objective To investigate the effect of Sirt1 gene knockout on chronic kidney disease induced by 5/6 nephrectomy in mice and vascular endothelial growth factor (VEGF)/fetal liver kinase-1 (Flk-1) signaling pathway.Methods Twenty four male Sirt1 +/+ and Sirt1 +/-mice wererandomly divided into four groups:Sirt1+/+ mice with sham-operation (WT-Sham,n=6),Sirt1+/-mice with sham-operation (KO-Sham,n=6),Sirt1 +/+ mice with 5/6 nephrectomy (WT-Nx,n=6) and Sirt1 +/-mice with 5/6 nephrectomy (KO-Nx,n=6).Proteinuria was determined by urine collection from 8:00 to 8:00 the next day at 20 weeks.Serum creatinine (Scr),urea nitrogen (BUN) and the renal pathological changes were measured after 20 weeks.Expressions of Sirt1,collagen Ⅰ and transforming growth factor β(TGF-β) were used to analyze the changes of renal fibrosis by immunohistochemistry staining.Real-time PCR and Western blotting were used to measure the mRNA and protein expressions of Sirt1,fibronectin,collagen Ⅰ,VEGF and Flk-1 in kidney.Results Sirt1 expressed in glomernlar endothelial cells,podocytes,mesangial cells and renal tubular epithelial cells in Sirt1 +/+ mice,while Sirt1 expression intensity was significantly reduced in Sirt1 +/-mice.Compared with the WT-Sham group,WT-Nx group had increased proteinuria,BUN,Scr,glomernlar sclerosis index and tubulointerstitial fibrosis index at 12 weeks after operation (all P ＜ 0.01),and KO-Nx group had exacerbated the above up-regulations (all P ＜ 0.01).Compared with those in WT-Sham group,the expressions of fibronectin,collagen Ⅰ and TGF-β were up-regulated in WT-Nx group (all P ＜ 0.01),and were significantly augmented in KO-Nx group (all P ＜ 0.01).Compared with those in WT-Sham group,renal mRNA and protein expressions of VEGF and Flk-1 were decreased in WT-Nx group,and KO-Nx group aggravated their down-regulation (all P ＜ 0.01).Conclusions Sirt1 gene knockout can increase proteinuria and Scr,and aggravate renal pathology and renal fibrosis in 5/6 nephrectomized mice,which is associated with the inhibition of VEGF/Flk-1 signaling pathway.It is suggested that Sirt1 may be a potential therapeutic target of chronic kidney disease.
ABSTRACT
OBJECTIVE: To investigate the effect of resveratrol (RSV) on angiotensin II (Ang II) induced cardiomyocytes hypertrophy and FoxO1/MnSOD signaling pathway. METHODS: The cardiomyocytes isolated from neonatal Wistar rats were cultured with pancreatin and preplating technique and divided into five groups: control (CON), Ang II (1 x 10(-6) mol/L, Ang II), Ang II + RSV (10 µmol/L), Ang II + RSV (25 µmol/L), and Ang II + RSV (50 µmol/L). 3H-Leucine incorporation method were used to determine the cardiomyocyte protein synthesis rate. Cell size was measured by phase contrast microscope. The gene expression of A type natriuretic factor (ANF) was detected by real-time PCR. Western blot was used to measure the expression of MnSOD, FoxO1 and acety-FoxO1. Immunoprecipitation was used to detect the interaction between Sirt1 and FoxO1. RESULTS: Cardiomyocyte protein synthesis rate in Ang II group was significantly higher in Ang II group than in the control group ((1,971 ± 175) cpm vs. (1,216 ± 136) cpm, P < 0.05), which could be significantly attenuated by RSV (10 µmol/L, 25 µmol/L, 50 µmol/L, all P < 0.05), especially in Ang II + RSV (25 µmol/L) group( (1,374 ± 143) cpm). Cardiomyocytes size in Ang II group was significantly larger than control group ((29.3 ± 3.2) µm vs. (19.4 ± 1.8) µm, P < 0.05), which could be significantly reduced by cotreatment with RSV (10 µmol/L, 25 µmol/L, 50 µmol/L, all P < 0.05), especially in Ang I + RSV (25 µmol/L) group ((20.8 ± 2.1) µm). Ang II also significantly upregulated ANP mRNA expression of cardiomyocytes (4.4 ± 0.4 vs. 1.0 ± 0.1 in control group, P < 0.05), which could be significantly inhibited by cotreatment with RSV, especially in Ang II + RSV (25 µmol/L) group (2.2 ± 0.2). Ang II significantly decreased MnSOD expression of cardiomyocytes compared with control group (P < 0.05), which was reversed by RSV (25 µmol/L). The binding level of Sirt1 and FoxO1 was significantly lower (1.00 ± 0.11 vs. 1.63 ± 0.16, P < 0.05), and the expression of acetylation of FoxO1 was significantly higher in Ang II group than in control group (1.48 ± 0.16 vs. 1.00 ± 0.13, P < 0.05), which was significantly reversed by cotreatment with RSV (25 µmol/L). CONCLUSIONS: Resveratrol treatment can inhibit Ang II induced cardiomyocyte hypertrophy. This protective effect is associated with reduced FoxO1 acetylation and activation of Sirt1, suggesting that Sirt1 may serve as a potential therapeutic target of cardiomyocyte hypertrophy.
