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. 2018 May 1;314(5):F702-F714.
doi: 10.1152/ajprenal.00044.2017. Epub 2017 May 17.

Heme oxygenase-1 Mitigates Ferroptosis in Renal Proximal Tubule Cells

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Free PMC article

Heme oxygenase-1 Mitigates Ferroptosis in Renal Proximal Tubule Cells

Oreoluwa Adedoyin et al. Am J Physiol Renal Physiol. .
Free PMC article

Abstract

Ferroptosis is an iron-dependent form of regulated nonapoptotic cell death, which contributes to damage in models of acute kidney injury (AKI). Heme oxygenase-1 (HO-1) is a cytoprotective enzyme induced in response to cellular stress, and is protective against AKI because of its antiapoptotic and anti-inflammatory properties. However, the role of HO-1 in regulating ferroptosis is unclear. The purpose of this study was to elucidate the role of HO-1 in regulating ferroptotic cell death in renal proximal tubule cells (PTCs). Immortalized PTCs obtained from HO-1+/+ and HO-1-/- mice were treated with erastin or RSL3, ferroptosis inducers, in the presence or absence of antioxidants, an iron source, or an iron chelator. Cells were assessed for changes in morphology and metabolic activity as an indicator of cell viability. Treatment of HO-1+/+ PTCs with erastin resulted in a time- and dose-dependent increase in HO-1 gene expression and protein levels compared with vehicle-treated controls. HO-1-/- cells showed increased dose-dependent erastin- or RSL3-induced cell death in comparison to HO-1+/+ PTCs. Iron supplementation with ferric ammonium citrate in erastin-treated cells decreased cell viability further in HO-1-/- PTCs compared with HO-1+/+ cells. Cotreatment with ferrostatin-1 (ferroptosis inhibitor), deferoxamine (iron chelator), or N-acetyl-l-cysteine (glutathione replenisher) significantly increased cell viability and attenuated erastin-induced ferroptosis in both HO-1+/+ and HO-1-/- PTCs. These results demonstrate an important antiferroptotic role of HO-1 in renal epithelial cells.

Keywords: acute kidney injury; erastin; ferrostatin-1; iron; proximal tubule.

Figures

Fig. 1.
Fig. 1.
Erastin increases heme oxygenase-1 (HO-1) expression in renal proximal tubular epithelial cells (PTCs). A: HO-1 mRNA expression in cultured immortalized HO-1+/+ PTCs after 0.1 or 1 μM erastin treatment at the indicated time points. Representative Western blot (B) and densitometry showing protein levels (C) of HO-1 in HO-1+/+ PTCs treated with erastin over time. Representative Western blot (D) and densitometry showing protein levels (E) of HO-1 in HO-1+/+ PTCs treated with RSL3 over time. Data shown represent the means ± SE of three independent experiments performed each time in triplicate; *P < 0.05 and **P < 0.01 compared with lower concentration at each time point.
Fig. 2.
Fig. 2.
Erastin treatment reduces cell viability in a time- and dose-dependent manner. Cell viability in HO-1+/+ PTCs after treatment with either 0.1 or 1 μM erastin for 8 h (A) or 16 h (B). C: representative Western blot showing absence of cleaved caspase 3 in HO-1+/+ PTCs treated with the indicated doses of erastin for 16 h. HO-1−/− PTCs treated with 50 μM cisplatin (a known inducer of apoptosis) for 16 h were used as a positive control. All lanes are from the same blot. “Control” label indicates where the blot was cropped to include relevant lanes. Data are represented as means ± SE from three independent experiments with seven or eight replicates in each experiment; **P < 0.01 compared with vehicle-treated controls.
Fig. 3.
Fig. 3.
Lack of HO-1 increases PTC sensitivity to erastin-induced ferroptosis. A: phase contrast microscopy images of HO-1+/+and HO-1−/− PTCs treated with either 0.1 or 1 μM erastin for 16 h. B: cell viability after treatment of PTCs with 0.1 or 1 μM erastin or 1; *P < 0.05 compared with 0.1 μM erastin-treated HO-1+/+ PTCs; **P < 0.01 compared with 0.1 μM erastin-treated HO-1−/− PTCs; #P < 0.01 compared with erastin-treated HO-1+/+ PTCs. C: cell viability after treatment of PTCs with 10 μM RSL3 for 16 h; *P < 0.01 compared with 1 μM RSL3 treatment; #P < 0.05 compared with RSL3-treated HO-1+/+ PTCs. Data shown represent means ± SE of three independent experiments with four to six replicates in each experiment.
Fig. 4.
Fig. 4.
Iron chelation attenuates erastin-induced ferroptosis. Representative phase contrast microscopy images of HO-1+/+ (A) and HO-1−/− (B) PTCs treated with either 0.1 or 1 μM erastin with or without 0.1 mM deferroxamine mesylate (DFO, iron chelator) for 16 h.
Fig. 5.
Fig. 5.
Iron chelation increases cell viability in erastin-treated cells. Cell viability in HO-1+/+ (A) and HO-1−/− (B) PTCs treated with either 0.1 or 1 μM erastin with or without 0.1 mM DFO for 16 h. C: representative Western blot showing HO-1 protein levels in HO-1+/+ PTCs treated with erastin in the presence or absence of DFO for 16 h. Data represented as means ± SE of three independent experiments with six to eight replicates in each experiment; **P < 0.01 compared with vehicle-treated cells; #P < 0.01 compared with 1 μM erastin-treated cells (HO-1+/+ or HO-1−/−).
Fig. 6.
Fig. 6.
Iron supplementation exacerbates erastin-induced ferroptosis. Representative phase contrast microscopy images of HO-1+/+ (A) and HO-1−/− (B) PTCs treated with either 0.1 or 1 μM erastin with or without 10 μg/ml ferric ammonium citrate (FAC, iron source) for 16 h.
Fig. 7.
Fig. 7.
Iron supplementation and/or HO-1 deficiency potentiate erastin-induced ferroptosis. Cell viability in HO-1+/+ (A) and HO-1−/− (B) PTCs treated with either 0.1 or 1 μM erastin with or without 10 μg/ml FAC (ferric ammonium citrate) for 16 h compared with vehicle-treated cells. Data represented as means ± SE of four to six independent experiments with three to eight replicates in each experiment; **P < 0.01 compared with vehicle-treated PTCs; #P < 0.01 compared with 0.1 or 1 μM erastin-treated cells.
Fig. 8.
Fig. 8.
N-acetyl-l-cysteine (NAC) cotreatment rescues cells from erastin-induced ferroptosis. Representative phase contrast microscopy images of HO-1+/+ (A) and HO-1−/− (B) PTCs treated with either 0.1 or 1 μM erastin with or without 0.1 mM NAC (glutathione reducing agent) for 16 h.
Fig. 9.
Fig. 9.
Glutathione supplementation rescues renal epithelial cells from erastin-induced ferroptosis. Cell viability of HO-1+/+ (A) and HO-1−/− (B) PTCs treated with either 0.1 or 1 μM erastin with or without 0.5 or 1 mM NAC for 16 h (magnification ×10). C: representative Western blot showing protein levels of HO-1+/+ PTCs treated with either 0.1 or 1 μM erastin with or without NAC cotreatment. Data represented as means ± SE of three or four independent experiments with four to eight replicates in each experiment; *P < 0.05 and **P < 0.01 compared with vehicle-treated cells; #P < 0.01 compared with erastin-only treatments.
Fig. 10.
Fig. 10.
Ferrostatin-1, a ferroptosis inhibitor, attenuates erastin-induced ferroptosis. Representative phase contrast microscopy images of HO-1+/+ (A) and HO-1−/− (B) PTCs treated with either 0.1 or 1 μM erastin with or without 0.1 or 1 μM ferrostatin for 16 h.
Fig. 11.
Fig. 11.
Ferrostatin-1 (Fer-1) attenuates erastin-induced ferroptosis in HO-1+/+ and HO-1−/− PTCs. Cell viability in HO-1+/+ (A) and HO-1−/− (B) PTCs treated with either 0.1 or 1 μM erastin with or without Fer-1 (0.1 or 1 μM) for 16 h. C: representative Western blot showing cells treated with erastin in the presence or absence of Fer-1. Data represented as means ± SE of four to six independent experiments with four to eight replicates in each experiment; *P < 0.05 and **P < 0.01 compared with vehicle-treated cells; #P < 0.01 compared with erastin-only treatments.

Comment in

  • Heavy metal suicide.
    Linkermann A, Stockwell B, Vanden Berghe T. Linkermann A, et al. Am J Physiol Renal Physiol. 2017 Oct 1;313(4):F959-F960. doi: 10.1152/ajprenal.00273.2017. Epub 2017 May 31. Am J Physiol Renal Physiol. 2017. PMID: 28566503 No abstract available.

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