Reoxygenation induces reactive oxygen species production and ferroptosis in renal tubular epithelial cells by activating aryl hydrocarbon receptor

During the reperfusion phase of ischemia-reperfusion injury, reactive oxygen species (ROS) production aggravates the course of many diseases, including acute kidney injury. Among the various enzymes implicated in ROS production are the enzymes of the cytochromes P450 superfamily (CYPs). Since arylhydrocarbon receptor (AhR) controls the expression of certain CYPs, the involvement of this pathway was evaluated in reperfusion injury. Because AhR may interact with the nuclear factor erythroid 2-related factor 2 (Nrf2) and the hypoxia-inducible factor-1α (HIF-1α), whether such an interaction takes place and affects reperfusion injury was also assessed. Proximal renal proximal tubular epithelial cells were subjected to anoxia and subsequent reoxygenation. At the onset of reoxygenation, the AhR inhibitor {"type":"entrez-nucleotide","attrs":{"text":"CH223191","term_id":"44935898","term_text":"CH223191"}}CH223191, the HIF-1α activator roxadustat, or the ferroptosis inhibitor α-tocopherol were used. The activity of AhR, Nrf2, HIF-1α, and their transcriptional targets were assessed with western blotting. ROS production, lipid peroxidation and cell death were measured with colorimetric assays or cell imaging. Reoxygenation induced ROS production, lipid peroxidation and cell ferroptosis, whereas {"type":"entrez-nucleotide","attrs":{"text":"CH223191","term_id":"44935898","term_text":"CH223191"}}CH223191 prevented all. Roxadustat did not affect the above parameters. Reoxygenation activated AhR and increased CYP1A1, while {"type":"entrez-nucleotide","attrs":{"text":"CH223191","term_id":"44935898","term_text":"CH223191"}}CH223191 prevented both. Reoxygenation with or without {"type":"entrez-nucleotide","attrs":{"text":"CH223191","term_id":"44935898","term_text":"CH223191"}}CH223191 did not alter Nrf2 or HIF-1α activity. Thus, AhR is activated during reoxygenation and induces ROS production, lipid peroxidation and ferroptotic cell death. These detrimental effects may be mediated by AhR-induced CYP overexpression, while the Nrf2 or the HIF-1α pathways remain unaffected. Accordingly, the AhR pathway may represent a promising therapeutic target for the prevention of reperfusion injury.