Conclusion
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.
Methods
We first evaluated the effect of hypoxia (8% O2) on mouse urine output. We then performed in vitro experiments using hypoxia (1% O2) 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.
Results
We found that mice exposed to hypoxia (8% O2) had significantly reduced urine volume compared to mice exposed to normoxia (21% O2). 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.