Figure 7. Combination of excess iron and protein overload exacerbates ferroptosis and cell death in FtH deficient human proximal renal tubular cells.

2X10⁵ HK-2 cells (human proximal renal tubular cells) were treated with scramble siRNA or siRNA to H-ferritin which resulted in ~ 83% knockdown of H-ferritin gene (FtH1) (A). The ferritin light chain (FtL) was not affected (B). Following knockdown (83% after 52 h), cells were treated with vehicle, Ferrous sulphate (200 μg/mL), or human albumin (20 mg/mL) and the supernatants were analyzed for LDH levels after 12 h. Vehicle and Fe treated medium had comparable level of LDH in scramble or FtH1 knock down (FtH1 KD) groups (C). Albumin significantly but comparable induced cell death in both groups. However, concomitant treatment with Fe and albumin significantly increased in cell death in FtH1 KD HK-2 cells (C). The involvement of ferroptosis in these conditions was evaluated by measuring the protein expression of ACSL4 and GPX4. Addition of iron did not induce ferroptosis in FtH1 sufficient or knockdown cells (D). Albumin comparably reduced the expression of GPX4 (E) in scramble and FtH1 KD cells, but increased the expression of ACSL4, which tended to be higher in the FtH1 knockdown cells (F-H). Concomitant addition of Fe and albumin significantly increased ACSL4 expression in FtH1 sufficient cells, which was further elevated by FtH1 deficiency (G-H). Data was analyzed using 1-way and 2-way ANOVA with Holm-Šídák’s multiple comparisons test and represented as mean ± SEM. **P < 0.001, ***P < 0.0001.