Qiao Rongfang, Cui Xiaohui, Hu Yitong, Wei Haoqing, Xu Hu, Zhang Cong, Du Chunxiu, Chang Jiazhen, Li Yaqing, Ming Wenhua, Qi Yinghui, Guan Youfei, Zhang Xiaoyan
Advanced Institute for Medical Sciences, Dalian Medical University, Dalian, China.
Kidney Health Institute, Health Science Center, East China Normal University, Shanghai, China.
Kidney Dis (Basel). 2024 Oct 22;10(6):504-518. doi: 10.1159/000542087. eCollection 2024 Dec.
Patients with acute mountain sickness (AMS) due to hypoxia at high altitudes often exhibit abnormal water metabolism. Hypoxia-inducible factors (HIFs) are major regulators of adaptive responses to hypoxia. As transcription factors, HIFs are involved in the regulation of erythropoiesis, iron metabolism, angiogenesis, energy metabolism, and cell survival by promoting the transcriptional expression of hundreds of target genes. Roxadustat, a novel drug for the treatment of anemia associated with chronic kidney disease (CKD), acts by inhibiting the degradation of HIFs to increase their protein levels. However, the clinical use of roxadustat is frequently associated with peripheral edema, suggesting the involvement of HIFs in regulating the body's water balance possibly by modulating water reabsorption in the kidney.
We first evaluated the effect of hypoxia (8% O) on mouse urine output. We then performed in vitro experiments using hypoxia (1% O) and roxadustat on mouse primary proximal tubular cells (mPTCs). The quantitative polymerase chain reaction, Western blot, and immunofluorescence were used to assess AQP1 mRNA and protein expression levels. Luciferase, Chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) were used to investigate the transcriptional regulation of AQP1 by HIF1α.
We found that mice exposed to hypoxia (8% O) had significantly reduced urine volume compared to mice exposed to normoxia (21% O). Hypoxia significantly elevated AQP1 expression at both mRNA and protein levels. In vitro experiments using mouse primary cultured proximal tubular cells (mPTCs) revealed that both hypoxia and roxadustat increased AQP1 expression. Mechanistically, overexpression of HIF1α, but not HIF2α, markedly increased AQP1 protein expression. Furthermore, the upregulation of AQP1 by hypoxia and roxadustat can be blocked by the HIF1α inhibitor PX-478 in mPTCs. Finally, we found that the AQP1 gene promoter contains a putative hypoxia response element and confirmed that AQP1 is a target gene of HIF1α using Luciferase reporter, ChIP, and EMSA assays.
This study demonstrates that hypoxia can reduce the urine volume of mice via upregulating AQP1 expression by HIF1α in the proximal tubular epithelial cells. Our findings also suggest a potential mechanism involved in water metabolism disorders in patients with AMS and in patients with CKD receiving roxadustat treatment.
高海拔地区因缺氧导致急性高原病（AMS）的患者常出现水代谢异常。缺氧诱导因子（HIFs）是对缺氧适应性反应的主要调节因子。作为转录因子，HIFs通过促进数百个靶基因的转录表达，参与红细胞生成、铁代谢、血管生成、能量代谢和细胞存活的调节。罗沙司他是一种用于治疗慢性肾脏病（CKD）相关贫血的新型药物，其作用机制是抑制HIFs的降解以提高其蛋白水平。然而，罗沙司他的临床应用常伴有外周水肿，提示HIFs可能通过调节肾脏对水的重吸收参与调节机体水平衡。
我们首先评估了缺氧（8%氧气）对小鼠尿量的影响。然后使用缺氧（1%氧气）和罗沙司他对小鼠原代近端肾小管细胞（mPTCs）进行体外实验。采用定量聚合酶链反应、蛋白质免疫印迹和免疫荧光法评估水通道蛋白1（AQP1）mRNA和蛋白表达水平。利用荧光素酶报告基因检测、染色质免疫沉淀（ChIP）和电泳迁移率变动分析（EMSA）研究HIF1α对AQP1的转录调控。
我们发现，与暴露于常氧（21%氧气）的小鼠相比，暴露于缺氧（8%氧气）的小鼠尿量显著减少。缺氧显著提高了AQP1在mRNA和蛋白水平的表达。使用小鼠原代培养近端肾小管细胞（mPTCs）进行的体外实验表明，缺氧和罗沙司他均增加AQP1的表达。机制上，HIF1α而非HIF2α的过表达显著增加AQP1蛋白表达。此外，在mPTCs中，缺氧和罗沙司他对AQP1的上调作用可被HIF1α抑制剂PX - 478阻断。最后，我们发现AQP1基因启动子含有一个假定的缺氧反应元件，并通过荧光素酶报告基因检测、ChIP和EMSA实验证实AQP1是HIF1α的靶基因。
本研究表明，缺氧可通过上调近端肾小管上皮细胞中HIF1α介导的AQP1表达来减少小鼠尿量。我们的研究结果还提示了AMS患者以及接受罗沙司他治疗的CKD患者水代谢紊乱的潜在机制。
