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.