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

Abstract

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 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 CH223191 prevented all. Roxadustat did not affect the above parameters. Reoxygenation activated AhR and increased CYP1A1, while CH223191 prevented both. Reoxygenation with or without 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.

Keywords: ischemia‑reperfusion injury; arylhydrocarbon receptor; cytochrome p450; nuclear factor erythroid2‑related factor 2; hypoxia‑inducible factor; ferroptosis.

Figure 1.

Reox activates AhR and induces CYP1A1 expression, while CH223191 inhibits both. RPTECs were cultured under ctrl conditions or subjected to Reox in the presence or absence of the AhR inhibitor CH223191. (A) Representative AhR and CYP1A1 western blot images. (B and C) Statistical analysis of the western blots. Reox decreases AhR levels, indicating its activation. CH223191 increases AhR levels both in ctrl cells and cells subjected to Reox. Reox enhanced CYP1A1 expression, whereas CH223191 reduced CYP1A1 under both conditions (C). Data are presented as the mean ± SEM of six independent experiments. *P<0.05 vs. ctrl; ^#P<0.05 vs. ctrl CH223191; ^{^}P<0.05 vs. Reox; ⁺P<0.05 vs. Reox CH223191. AhR, arylhydrocarbon receptor; CYP1A1, cytochrome P450 family 1 subfamily A member 1; RPTEC, renal proximal tubular epithelial cell; ctrl, control; Reox, reoxygenation.

Figure 2.

Inhibition of AhR prevents Reox-induced ROS production and attenuates lipid peroxidation and ferroptosis. RPTECs were cultured under ctrl conditions or subjected to Reox, with or without the AhR inhibitor CH223191 or the ferroptosis inhibitor tocophe. (A and B) Reox induced ROS production and lipid peroxidation, assessed by MDA levels. CH223191 prevented Reox-induced ROS production and attenuated lipid peroxidation. Reox caused cell necrosis. Necrosis was abrogated by α-tocopherol, demonstrating ferroptosis. *P<0.05 vs. ctrl; ^#P<0.05 vs. ctrl CH223191; ^{^}P<0.05 vs. Reox; ⁺P<0.05 vs. Reox CH223191. (C) CH223191 ameliorated Reox-induced cell necrosis. Data are presented as the mean ± SEM of six independent experiments. *P<0.05 vs. ctrl; ^#P<0.05 vs. ctrl tocophe; ^{^}P<0.05 vs. ctrl CH223191; ⁺P<0.05 vs. Reox, ^&P<0.05 vs. Reox tocophe; ^$P<0.05 vs. Reox CH223191. AhR, arylhydrocarbon receptor; RPTEC, renal proximal tubular epithelial cell; ROS, reactive oxygen species; MDA, malondialdehyde; ctrl, control; Reox, reoxygenation; tocophe; α-tocopherol.

Figure 3.

Inhibition of AhR protects from Reox-induced cell death. RPTECs were subjected to Reox with or without the AhR inhibitor CH223191. (A) Representative images of one of the experiments. Magnification, ×100. (B) Statistical analysis of all experiments. RPTECs died within 4 h of Reox, while CH223191 rescued the cells. Data are presented as the mean ± SEM of six independent experiments. *P<0.05 vs. Reox. AhR, arylhydrocarbon receptor; RPTEC, renal proximal tubular epithelial cell; Reox, reoxygenation.

Figure 4.

Apoptosis does not play a role in Reox-induced cell death and is not affected by inhibition of AhR. RPTECs were cultured under ctrl conditions or subjected to Reox in the presence or not of the AhR inhibitor CH223191. (A) Representative CC3 western blot images. (B) Statistical analysis of the western blots. Neither Reox nor CH223191 affects cell apoptosis. Data are presented as the mean ± SEM of six independent experiments. AhR, arylhydrocarbon receptor; RPTEC, renal proximal tubular epithelial cell; ctrl, control; Reox, reoxygenation; CC3, cleaved caspase-3.

Figure 5.

AhR activation status does not affect Nrf2 activation or transcriptional activity. RPTECs were cultured under ctrl conditions or subjected to Reox with or without the AhR inhibitor CH223191. (A) Representative western blots of Nrf2 levels (corresponding to its activation status) and the expression of the Nrf2 transcriptional targets xCT (SLC7A11) and SOD-3. (B-D) Statistical analysis of the western blots. Neither Reox nor CH223191 affects Nrf2 activity, or the expression of xCT and SOD-3. Data are presented as the mean ± SEM of six independent experiments. AhR, arylhydrocarbon receptor; Nrf2, nuclear factor erythroid 2-related factor 2; xCT, cystine-glutamate antiporter; SOD-3, superoxide dismutase; RPTEC, renal proximal tubular epithelial cell; ctrl, control; Reox, reoxygenation.

Figure 6.

AhR activation status does not affect HIF-1α levels or its transcriptional activity. RPTECs were cultured under ctrl conditions or subjected to Reox with or without the AhR inhibitor CH223191. (A) Representative western blot images of HIF-1α levels (corresponding to its activation status) and its transcriptional target LDH-A. (B and C) Statistical analysis of the western blots. Neither Reox nor CH223191 affected HIF-1α levels. LDH-A levels increased under Reox, yet CH223191 did not affect LDH-A expression in both ctrl cells and cells subjected to Reox. Data are presented as the mean ± SEM. *P<0.05 vs. ctrl; ^#P<0.05 vs. ctrl CH223191; ^{^}P<0.05 vs. Reox; ⁺P<0.05 vs. Reox CH223191. AhR, arylhydrocarbon receptor; RPTEC, renal proximal tubular epithelial cell; HIF-1α, hypoxia-inducible factor 1α; LDH-A, lactate dehydrogenase-A; ctrl, control; Reox, reoxygenation.

Figure 7.

Roxadustat increases HIF-1α level and transcriptional activity but does not affect ROS production, lipid peroxidation, or cell necrosis. RPTECs were cultured under ctrl conditions or subjected to Reox with or without the HIF-1α activator roxadustat. (A) Representative western blot images of HIF-1α levels (corresponding to its activation status) and its transcriptional target LDH-A. (B and C) Statistical analysis of the western blots. Roxadustat enhanced HIF-1α levels in both ctrl cells and cells subjected to Reox. Roxadustat also upregulated LDH-A expression in both ctrl cells and cells subjected to Reox. Compared to ctrl cells, LDH-A levels increased in cells subjected to Reox. (D) ROS production, (E) lipid peroxidation and (F) cell necrosis were also assessed. Roxadustat did not affect Reox-induced ROS production, lipid peroxidation, and cell necrosis. Data are presented as the mean ± SEM. *P<0.05 vs. ctrl; ^#P<0.05 vs. ctrl roxadustat; ^{^}P<0.05 vs. Reox; ⁺P<0.05 vs. Reox roxadustat. RPTEC, renal proximal tubular epithelial cell; HIF-1α, hypoxia-inducible factor 1α; LDH-A, lactate dehydrogenase-A; ROS, reactive oxygen species; MDA, malondialdehyde; ctrl, control; Reox, reoxygenation.

Figure 8.

Activation of HIF-1α protects from Reox-induced cell death. RPTECs were subjected to Reox with or without the HIF-1α activator roxadustat. (A) Representative images of one of the experiments. Magnification, ×100. (B) Statistical analysis of all experiments. Roxadustat did not alter RPTEC resistance to Reox-induced cell death, since both roxadustat-treated and untreated cells died within 4 h of observation. Data are presented as the mean ± SEM of six independent experiments. RPTEC, renal proximal tubular epithelial cell; HIF-1α, hypoxia-inducible factor 1α; Reox, reoxygenation.