HIF-1α is a key molecule for the progression of EMT-mediated renal fibrosis

上皮-间充质转化(EMT)在介导肾小管间质纤维化中发挥重要作用。在人的活检标本中，纤维化区周围的肾小管上皮细胞(TEC)表达成纤维细胞特异性蛋白1(FSP1)，但α平滑肌肌动蛋白阳性的TEC非常罕见。因此，检测TECs中FSP1的表达对于检测肾间质纤维化中的EMT具有重要意义。以往的研究表明，慢性低氧是肾小管间质纤维化发生和发展的主要因素。在这项研究中，我们研究了体外和体内缺氧是否可能是介导EMT的一个因素。我们建立了用Cre-loxP系统标记的TECs原代培养的体外检测EMT的方法。低氧暴露后TECs开始伸长，5d后形态变为成纤维细胞样细胞。免疫细胞化学结果显示，在缺氧后5天，超过40%的TECs表达FSP1。缺氧反应的主要介质是缺氧诱导因子-1及其氧敏感成分缺氧诱导因子-1α，它被一种泛素连接酶迅速降解，即常氧条件下的von Hippel-Lindau肿瘤抑制因子。为了更直接地研究缺氧诱导因子-1α在体内是否在肾纤维化中发挥作用，我们利用Cre-loxP介导的重组技术建立了上皮靶向表达vhl基因的小鼠系(成分活性缺氧诱导因子-1)。单侧输尿管梗阻(UUO)诱导肾小管间质纤维化。UUO后第8天分别取梗阻和未梗阻肾脏进行免疫组织化学染色。与野生型相比，VHL突变小鼠未梗阻肾脏FSP1阳性细胞数显著增加。这些结果有力地表明，缺氧诱导因子-1α通过诱导上皮-间充质转化来激活肾纤维化中的组织成纤维细胞。

Epithelial-mesenchymal transition(EMT) plays a central role in mediating tubulointerstitial fibrosis. In human biopsy samples, tubular epithelial cells(TECs) surrounding the fibrotic area expressed fibroblast specific protein 1(FSP1), although TECs positive for α smooth muscle actin were very rare. Therefore, it is essential to examine FSP1 expression in TECs for the detection of EMT in renal fibrosis. Previous studies have implicated that chronic hypoxia is the main contributor to the development and progression of tubulointerstitial fibrosis. In this study, we examined whether hypoxia could be a factor mediating EMT in vitro and in vivo. We established the method to detect EMT in vitro, using primary culture of TECs genetically marked by Cre-loxP system. TECs started to elongate after hypoxic exposure and became fibroblast-like cells morphologically at 5 days after hypoxia. Immunocytochemistry revealed that more than 40% of TECs expressed FSP1 at 5 days after hypoxia. The chief mediator of hypoxic response is hypoxia-inducible factor 1(HIF-1), and its oxygen-sensitive component HIF-1α which is promptly degraded by a kind of ubiquitin ligase, the von Hippel-Lindau tumor suppressor (VHL) in normoxia. To study more directly whether HIF-1α functions in renal fibrosis in vivo, we generated a mouse line with epithelium-targeted expression of VHL gene (constitutively active HIF-1), using Cre-loxP-mediated recombination. Tubulointerstitial fibrosis was induced by unilateral ureteral obstruction(UUO). Both obstructed and unobstructed kidneys were harvested for immunohistochemistry 8 days after UUO. FSP1 positive cell numbers increased significantly in unobstructed kidneys of VHL mutant mice compared with wild type mice. These results strongly suggest that HIF-1α activates tissue fibroblasts in renal fibrosis via the induction of epithelial-mesenchymal transition(EMT).