Inhibition of HIF-la activity is a novel strategy for the treatment of tubulointerstitial fibrosis

慢性缺氧是肾小管间质纤维化进展的主要因素。在本研究中，我们利用选择性缺失肾近端上皮细胞HIF-1α表达的小鼠模型，在体内和体外研究了低氧诱导因子-1α(HIF-1α)在多大程度上介导上皮-间充质转化，并评价了HIF-1α在肾间质纤维化中的作用。此外，我们还建立了一种体外检测肾小管上皮细胞的方法，使用Cre-重组酶激活的β-半乳糖苷酶(LacZ)来鉴定和追踪原代肾小管上皮细胞。为确定HIF-1a是否参与体内肾小管间质纤维化的发生发展，采用单侧尿路梗阻(UUO)方法建立HIF-1a突变小鼠和野生型小鼠肾间质纤维化模型。UUO后第8天取肾脏进行免疫组织化学染色，用改良的ABC-过氧化物酶方法进行FSP1(成纤维细胞特异性蛋白1，EMT的标志)染色。…的FSP1+细胞数与野生型小鼠相比，HIF-1α突变型小鼠梗阻肾脏的ERE显著降低。此外，低氧(1%O_2)而不是常氧(20.9%O_2)培养5天后，LacZ标记的培养的TEC呈成纤维细胞样形态。LacZ和FSP1双重染色显示，8.2%的TECs在常氧条件下接受EMT(LacZ+/FSP1+)，28.3%的TECs在低氧条件下接受EMT。我们还以YC-1(3-(5‘-羟甲基-2’-呋喃)-1-苯基吲唑)作为抗HIF-1α的试剂，发现阻断HIF-1α活性可通过抑制上皮细胞间充质转化而成为治疗肾小管间质纤维化的新途径。综上所述，我们的结果提示，缺氧通过激活HIF-1在诱导肾小管间质纤维化和体内、外肾间质纤维化中起关键作用，抗HIF-1α可通过抑制上皮细胞间充质转化来治疗肾间质纤维化。较少

Chronic hypoxia is a main contributor to the progression of tubulointerstitial fibrosis. In this study, we investigated the extent to which hypoxia-inducible factor 1α (HIF-1α) mediates epithelial-mesenchymal-transition (EMT) in vivo and in vitro using a mouse model in which renal proximal epithelial HIF-1α expression was selectively deleted and assessed the role of HIF-1α in EMT-mediated renal fibrosis. In addition, we developed a method with which to detect EMT in vitro, using Cre-recombinase activated β-galactosidase (LacZ) to identify and track primary tubular epithelial cells (TECs). To determine whether HIF-la is involved in the development of tubulointerstitial fibrosis in vivo, renal fibrosis was induced by unilateral urethral obstruction (UUO) in HIF-la-mutant and wild-type mice. Kidneys were harvested for immunohistochemistry 8 days after UUO and stained for FSP1 (fibroblast-specific protein 1, a marker for EMT) using a modified ABC-peroxidase method. Numbers of FSP1+ cells w … More ere significantly lower in obstructed kidneys from HIF-1α-mutant than wild type mice. Moreover, after exposure to hypoxia (1% O_2), but not normoxia (20.9% O_2), for 5 days cultured TECs permanently marked with LacZ assumed a fibroblast-like morphology. And double staining for LacZ and FSP1 revealed that whereas 8.2% of TECs underwent EMT (LacZ+/FSP1+) under normoxic conditions, 28.3% underwent EMT under hypoxic conditions. We also determined that blockade of HIF-1α activity can be a novel approach for the treatment of tubulointerstitial fibrosis through the inhibition of epithelial-mesenchymal-transition (EMT) in vitro and in vivo, using YC-1(3-(5'-hydroxymethy1-2'-fury1)-1-benzyl indazole) as an anti-HIF-1α agent.Taken together, our results suggest that, through activation of HIF-1, hypoxia plays a key role in the induction of EMT and in EMT-mediated renal fibrosis in vitro and in vivo and anti-HIF-1α agents may be utilized for the treatment of tubulointerstitial fibrosis through the inhibition of EMT. Less