Ewha Medical Research Center, Ewha Womans University College of Medicine, Seoul 07804, Korea.
Department of Biomedical Laboratory Science, Jungwon University, Goesan 28024, Korea.
BMB Rep. 2023 Mar;56(2):196-201. doi: 10.5483/BMBRep.2022-0145.
Renal fibrosis is the final manifestation of chronic kidney disease (CKD) regardless of etiology. Hypoxia-inducible factor-2 alpha (HIF-2α) is an important regulator of chronic hypoxia, and the late-stage renal tubular HIF-2α activation exerts protective effects against renal fibrosis. However, its specific role in progressive renal fibrosis remains unclear. Here, we investigated the effects of the long-term tubular activation of HIF-2α on renal function and fibrosis, using in vivo and in vitro models of renal fibrosis. Progressive renal fibrosis was induced in renal tubular epithelial cells (TECs) of tetracycline-controlled HIF-2α transgenic (Tg) mice and wild-type (WT) controls through a 6-week adenine diet. Tg mice were maintained on doxycycline (DOX) for the diet period to induce Tg HIF-2α expression. Primary TECs isolated from Tg mice were treated with DOX (5 μg/ml), transforming growth factor-β1 (TGF-β1) (10 ng/ml), and a combination of both for 24, 48, and 72 hr. Blood was collected to analyze creatinine (Cr) and blood urea nitrogen (BUN) levels. Pathological changes in the kidney tissues were observed using hematoxylin and eosin, Masson's trichrome, and Sirius Red staining. Meanwhile, the expression of fibronectin, E-cadherin and α-smooth muscle actin (α-SMA) and the phosphorylation of p38 mitogenactivated protein kinase (MAPK) was observed using western blotting. Our data showed that serum Cr and BUN levels were significantly lower in Tg mice than in WT mice following the adenine diet. Moreover, the protein levels of fibronectin and E-cadherin and the phosphorylation of p38 MAPK were markedly reduced in the kidneys of adenine-fed Tg mice. These results were accompanied by attenuated fibrosis in Tg mice following adenine administration. Consistent with these findings, HIF-2α overexpression significantly decreased the expression of fibronectin in TECs, whereas an increase in α-SMA protein levels was observed after TGF-β1 stimulation for 72 hr. Taken together, these results indicate that long-term HIF-2α activation in CKD may inhibit the progression of renal fibrosis and improve renal function, suggesting that long-term renal HIF-2α activation may be used as a novel therapeutic strategy for the treatment of CKD. [BMB Reports 2023; 56(3): 196-201].
肾纤维化是慢性肾脏病（CKD）的终末表现，无论病因如何。缺氧诱导因子-2 ɑ（HIF-2ɑ）是慢性缺氧的重要调节因子，晚期肾小管 HIF-2ɑ的激活对肾纤维化具有保护作用。然而，其在进行性肾纤维化中的具体作用尚不清楚。本研究通过体内和体外肾纤维化模型，研究了长期肾小管 HIF-2ɑ激活对肾功能和纤维化的影响。在四环素控制的 HIF-2ɑ转基因（Tg）小鼠和野生型（WT）对照的肾小管上皮细胞（TECs）中通过 6 周腺嘌呤饮食诱导进行性肾纤维化。在饮食期间，Tg 小鼠用强力霉素（DOX）喂养以诱导 Tg HIF-2ɑ表达。从 Tg 小鼠分离的原代 TECs 用 DOX（5 μg/ml）、转化生长因子-β1（TGF-β1）（10 ng/ml）和两者的组合处理 24、48 和 72 小时。收集血液以分析肌酐（Cr）和血尿素氮（BUN）水平。通过苏木精和伊红、马松三色和天狼星红染色观察肾脏组织的病理变化。同时，通过 Western blot 观察纤维连接蛋白、E-钙粘蛋白和α-平滑肌肌动蛋白（α-SMA）的表达和 p38 丝裂原活化蛋白激酶（MAPK）的磷酸化。我们的数据显示，在给予腺嘌呤饮食后，血清 Cr 和 BUN 水平在 Tg 小鼠中明显低于 WT 小鼠。此外，在腺嘌呤喂养的 Tg 小鼠肾脏中，纤维连接蛋白和 E-钙粘蛋白的蛋白水平以及 p38 MAPK 的磷酸化明显降低。这些结果伴随着腺嘌呤给药后 Tg 小鼠纤维化的减轻。与这些发现一致的是，HIF-2ɑ过表达显著降低了 TECs 中纤维连接蛋白的表达，而在 TGF-β1 刺激 72 小时后观察到 α-SMA 蛋白水平增加。总之，这些结果表明，CKD 中 HIF-2ɑ 的长期激活可能抑制肾纤维化的进展并改善肾功能，表明长期肾脏 HIF-2ɑ 的激活可能作为治疗 CKD 的一种新的治疗策略。[BMB 报告 2023;56(3):196-201]。
