Analysis of the Hypoxic Response in a Mouse Cortical Collecting Duct-Derived Cell Line Suggests That Esrra Is Partially Involved in Hif1α-Mediated Hypoxia-Inducible Gene Expression in mCCDcl1 Cells

The kidney is strongly dependent on a continuous oxygen supply, and is conversely highly sensitive to hypoxia. Controlled oxygen gradients are essential for renal control of solutes and urine-concentrating mechanisms, which also depend on various hormones including aldosterone. The cortical collecting duct (CCD) is part of the aldosterone-sensitive distal nephron and possesses a key function in fine-tuned distal salt handling. It is well known that aldosterone is consistently decreased upon hypoxia. Furthermore, a recent study reported a hypoxia-dependent down-regulation of sodium currents within CCD cells. We thus investigated the possibility that cells from the cortical collecting duct are responsive to hypoxia, using the mouse cortical collecting duct cell line mCCD(cl1) as a model. By analyzing the hypoxia-dependent transcriptome of mCCD(cl1) cells, we found a large number of differentially-expressed genes (3086 in total logFC< -1 or >1) following 24 h of hypoxic conditions (0.2% O-2). A gene ontology analysis of the differentially-regulated pathways revealed a strong decrease in oxygen-linked processes such as ATP metabolic functions, oxidative phosphorylation, and cellular and aerobic respiration, while pathways associated with hypoxic responses were robustly increased. The most pronounced regulated genes were confirmed by RT-qPCR. The low expression levels of Epas1 under both normoxic and hypoxic conditions suggest that Hif-1 alpha, rather than Hif-2 alpha, mediates the hypoxic response in mCCD(cl1) cells. Accordingly, we generated shRNA-mediated Hif-1 alpha knockdown cells and found Hif-1 alpha to be responsible for the hypoxic induction of established hypoxically-induced genes. Interestingly, we could show that following shRNA-mediated knockdown of Esrra, Hif-1 alpha protein levels were unaffected, but the gene expression levels of Egln3 and Serpine1 were significantly reduced, indicating that Esrra might contribute to the hypoxia-mediated expression of these and possibly other genes. Collectively, mCCD(cl1) cells display a broad response to hypoxia and represent an adequate cellular model to study additional factors regulating the response to hypoxia.

Automated summary

The kidney is highly dependent on a continuous oxygen supply and is sensitive to hypoxia. In this study, the authors investigated the response of cortical collecting duct cells to hypoxia using a mouse cell line. They found that hypoxia led to significant changes in gene expression, with decreased oxygen-linked processes and increased pathways associated with hypoxic responses. Knockdown experiments revealed the involvement of certain genes in the hypoxic induction of other genes. Overall, the study suggests that the mCCD(cl1) cell line is a suitable model for studying the cellular response to hypoxia.