摘要目的 探讨5/6肾切除诱导的肾脏纤维化小鼠中沉默信息调节因子2同源体1(Sirt1)基因敲除的影响及其对血管内皮生长因子(VEGF)/胎肝激酶1(Flk-1)信号通路的作用.方法 雄性8周龄Sirt1+/+野生型小鼠及Sirt1+/+杂合子小鼠按随机数字表分为4组:Sirt1+/+假手术组(WT-Sham组,n=6)、Sirt1+/-假手术组(KO-Sham组,n=6)、Sirt1+/+5/6肾切除组(WT-Nx组,n=6)、Sirt1+/-5/6肾切除组(KO-Nx组,n=6).代谢笼收集24 h尿液检测尿蛋白含量,收集血和肾组织标本检测血肌酐、尿素氮以及肾脏组织病理改变.免疫组化检测各组小鼠肾组织Sirt1、Ⅰ型胶原(CollagenⅠ)、转化生长因子β(TGF-β)的表达;实时定量PCR和Western印迹分别检测肾组织Sirt1、纤连蛋白(Fibronectin)、Collagen Ⅰ、VEGF、Flk-1 mRNA和蛋白表达.结果 Sirt1+/+小鼠中Sirt1在肾小球内皮细胞、足细胞、系膜细胞及肾小管上皮细胞均有表达,而Sirt1+/-小鼠Sirt1表达强度降低.WT-Nx组小鼠术后12周24 h尿蛋白量、血尿素氮、血肌酐、肾小球硬化指数和肾小管间质纤维化指数均高于WT-Sham组(均P＜0.01),而KO-Nx组小鼠术后12周上述指标比WT-Nx组更高(均P＜0.01).与WT-Sham组比较,WT-Nx组肾组织Fibronectin、Collagen Ⅰ和TGF-β表达均上调(均P＜0.01);而KO-Nx组比WT-Nx组更高(均P＜0.01).WT-Nx组肾组织VEGF、Flk-1 mRNA和蛋白表达均低于WT-Sham组(均P＜0.01);而KO-Nx组VEGF、Flk-1表达比WT-Nx组更低(均P＜0.05). 结论 Sirt1基因敲除可进一步增加5/6肾切除小鼠术后的尿蛋白量和血肌酐,加重肾脏病理和肾脏纤维化,其作用机制可能与抑制VEGF/Flk-1信号通路有关,提示Sirt1可能是慢性肾脏病潜在的干预靶点.

abstractsObjective 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.