转录因子FOXK1在肾脏缺血再灌注后纤维化中的作用及其机制探讨

Role of Transcription Factor FOXK1 on the Progression of Ischemia-reperfusion Induced Renal Fibrosis and Its Underlying Mechanism

目的:探讨转录因子FOXK1在肾脏缺血再灌注(I/R)诱导肾间质纤维化中的作用及其机制。方法:包括动物实验和细胞实验。(1)动物实验采用C57BL/6小鼠,随机分为四组:FOXK1^(+/+)假手术组(FOXK1^(+/+)sham组)、FOXK1^(-/-)假手术组(FOXK1^(-/-)sham组)、FOXK1^(+/+)+I/R组、FOXK1^(-/-)+I/R组。肾动脉钳制法建立I/R模型;HE、Masson染色观察各组肾组织病理变化,免疫组化法和Western印记法分别检测肾上皮细胞标志物闭锁小带蛋白(ZO-1)、E-钙粘蛋白(E-cadherin)、肌成纤维细胞标志物α-平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)和Rho/ROCK信号通路蛋白RhoA、ROCK1、p-Cofilin的表达。(2)细胞实验采用人肾小管上皮细胞(HK-2),用慢病毒转染,分别设置对照shRNA组(Ctrl shRNA组)、FOXK-1 shRNA组、Ctrl shRNA+转化生长因子-β1组(Ctrl shRNA+TGF-β1组)和FOXK-1 shRNA+TGF-β1组,用10ng/ml TGF-β1刺激48h。Western印记法检测ZO-1、E-cadherin、α-SMA、Vimentin、RhoA、ROCK1、p-Cofilin及ROCK1作用底物p-LIMK的表达;划痕实验观察各组HK-2细胞迁移能力。结果:与两sham组比较,FOXK1^(+/+)+I/R组肾小管间质损伤明显,胶原纤维沉积伴大量炎性细胞浸润,上皮ZO-1、E-cadherin表达下调,α-SMA、Vimentin、RhoA、ROCK1、p-Confilin表达增加(均P<0.05);与FOXK1^(+/+)+I/R组相比,FOXK1^(-/-)+I/R组肾小管间质损伤减轻,胶原纤维沉积减少,ZO-1、E-cadherin表达增加,α-SMA、Vimentin、RhoA、ROCK1、p-Cofilin表达降低(均P<0.05)。细胞实验结果显示,与Ctrl shRNA组和FOXK1 shRNA组比较,Ctrl shRNA+TGF-β1组HK-2细胞α-SMA、Vimentin、RhoA、ROCK1、p-LIMK蛋白表达增加(均P<0.05),细胞迁移速度增加;FOXK1 shRNA+TGF-β1组较Ctrl shRNA+TGF-β1组上述纤维化相关蛋白和Rho/ROCK信号通路蛋白表达明显降低(均P<0.05),细胞迁移明显减少。结论:转录因子FOXK1通过激活Rho/ROCK信号通路在肾脏I/R引起的肾间质纤维化过程中起重要调节作用,抑制FOXK1激活可能是防治肾脏纤维化进展的新策略。

Objective:To investigate the role and molecular mechanism of transcription factor FOXK1 in ischemia reperfusion(I/R)-induced renal interstitial fibrosis and its mechanism.Method:Animal and cellular experiments were included.(1)C57BL/6 mice were used for animal experiments and randomly divided into four groups:FOXK1^(+/+)sham-operated group(FOXK1^(+/+)sham group),FOXK1^(-/-)sham-operated group(FOXK1^(-/-)sham group),FOXK1^(+/+)+I/R group,FOXK1^(-/-)+I/R group.The I/R model was established by renal artery clipping method;Hematoxylin-eosin and Masson staining was performed to observe the histopathological changes.The expression of renal epithelial cell markers zonula occludens-1(ZO-1),E-cadherin,myofibroblast markerα-smooth muscle actin(α-SMA),Vimentin and Rho/ROCK signalling pathway proteins RhoA,ROCK1,p-Cofilin in kidney tissues were detected by immunohistochemistry and Western blotting.(2)Cell experiments were performed by using human renal tubular epithelial cells(HK-2).HK-2 cells were transfected with lentivirus and divided into four groups:control shRNA(Ctrl shRNA)group,FOXK-1 shRNA group,Ctrl shRNA+transforming growth factor-β1(TGF-β1)group and FOXK-1 shRNA+TGF-β1 group respectively.After stimulation with 10 ng/ml TGF-β1for 48 h,the expression of ZO-1,E-cadherin,α-SMA,Vimentin,RhoA,ROCK1,p-Cofilin and ROCK1-acting substrates p-LIMK were detected by Western blotting;the migration ability of HK-2 cells in each group was observed by wound healing assay.Results:Compared with the two sham groups,the FOXK1^(+/+)+I/R group showed significant tubular interstitial injury,collagen fiber deposition with massive inflammatory cell infiltration,down-regulated expression of epithelial ZO-1 and E-cadherin,and increased expression ofα-SMA,Vimentin,RhoA,ROCK1and p-Confilin(all P0.05);Compared with the FOXK1^(+/+)+I/R group,the FOXK1^(-/-)+I/R group showed attenuated tubular interstitial damage,reduced collagen deposition,increased expression of ZO-1 and E-cadherin(all P0.05),and decreased expression ofα-SMA,Vimentin,RhoA,ROCK1 and p-Cofilin(all P0.05).The results of cellular assays showed that compared with the control shRNA group and FOXK1 shRNA group,the expression ofα-SMA,Vimentin,RhoA,ROCK1,p-LIMK protein were increased in the Ctrl shRNA+TGF-β1 group(all P<0.05)and the cell migration rate were increased;the FOXK1 shRNA+TGF-β1 group showed significantly reduced the expression of fibrosis-related proteins and Rho/ROCK signaling pathway proteins(all P0.05)and significantly reduced the migration ability compared with the Ctrl shRNA+TGF-β1 group.Conclusion:The transcription factor FOXK1 plays a critical role in renal I/R-induced interstitial fibrosis regulation through activation of the Rho/ROCK signaling pathway.Inhibition of FOXK1 activation may be a new strategy to prevent and treat the progression of renal interstitial fibrosis.