Smad6和Smad7基因治疗对肾小管间质纤维化进程的影响

Effects of Smad 6 and Smad 7 gene therapy on renal tubulointerstitial fibrosis of unilateral ureteral obstruction

目的观察腺相关病毒(rAAV)介导的Smad6和Smad7基因治疗对单侧输尿管梗阻性(UUO)肾小管间质纤维化进程的影响。方法构建产生表达Smad6和Smad7基因的无辅毒腺相关病毒载体,再将此病毒颗粒通过肾动脉途径转移到UUO模型。30只Wistar大鼠随机分为假手术组、梗阻组、LacZAAV转染组、Smad6AAV转染组和Smad7AAV转染组(n=6),于术后第3周处死大鼠。采用β-半乳糖苷酶染色和免疫组化观察外源基因的定位,Western印迹法观察外源基因、胞内磷酸化Smad2、PAI-1和α-SMA的表达;底物酶谱法检测MMP-2和MMP-9活性,分光光度法测定肾组织羟脯氨酸含量。结果Smad6和Smad7成功地转染到外髓的肾小管间质区,定位在肾小管和集合管细胞,而血管细胞、肾小球或间质细胞均未见有AAV转移基因的表达。Smad7基因治疗可显著降低肾组织PAI-1、α-SMA的表达和肾组织羟脯氨酸含量,增加MMP-2和MMP-9活性,这些作用与Smad7阻断Smad2磷酸化有关。相反,Smad6没有这样的治疗作用。结论Smad7基因转移能有效缓解单侧输尿管梗阻肾小管间质纤维化,AAV介导的Smad7基因转移有望成为治疗肾小管间质纤维化的方法之一。

Objective To investigate the effects of recombinant adeno associated viral (rAAV) vector mediated Smad 6 and Smad 7 gene transfer on renal tubulointerstitial fibrosis of unilateral ureteral obstruction(UUO). Methods Two recombinant adeno associated viruses(AAV)expressing Smad 6 and Smad 7 gene were produced without helper virus and then were delivered into the rat kidney of UUO through the renal artery. Thirty rats were divided into sham operated group,UUO group,LacZ AAV transfection group, Smad6 AAV transfection group and Smad 7 AAV transfection group(n=6).The rats were sacrificed at postoperative week 3. The localizations of LacZ, Smad 6/Flag and Smad 7/Flag genes in the rat kidney were demonstrated by β galactosidase staining and immunocytochemical staining. The levels of phospho Smad 2, PAI 1, and α smooth muscle actin(α SMA) preteins were measured by Western blot analysis. MMP 2 and MMP 9 activities were detected by substrate zymography. The content of hydroxyproline in renal tissue was determinated by spectrophotometric method.Results The expression of transgene was restricted to the outer medullary region of the kidney and was localized to tubule epithelial cells and collecting duct cells. No transduction was observed in blood vessels, glomeruli, or the interstitium. Compared to LacZ AAV controls, expression of Smad 7 transgene resulted in marked decrease of PAI 1, α SMA protein expression and hydroxyproline content in renal tissue,and increase of MMP 2 and MMP 9 activities. These were closely associated with inhibition of TGF induced Smad 2 activation. In contrast, Smad 6 did not possess such effects on TGF beta mediated tubulointerstitial fibrosis in UUO model. Conclusion Gene transfer of Smad 7 but not Smad 6 can efficiently inhibit TGF beta mediated tubulointerstitial fibrosis in UUO model, and the rAAV delivering the Smad 7 gene may provide a new therapeutic potential for renal tubulointerstitial fibrosis.