Silencing FABP4 alleviated ferroptosis of renal tubular epithelial cells induced by hypoxia/reoxygenation through regulating Nrf2/GPX4 axis

Objective  To investigate the effects and mechanisms of fatty acid binding protein 4 (FABP4) on ferroptosis in human renal tubular epithelial cells (HK-2) treated with hypoxia/reoxygenation (H/R).

Methods  HK-2 cells were cultured in vitro and subjected to hypoxia for 24 hours followed by reoxygenation for different durations (1, 3, 6 hours). The messenger RNA (mRNA) and protein levels of FABP4 in HK-2 cells were detected at each time point using real-time fluorescent quantitative polymerase chain reaction and Western blotting. Small interfering RNA (siRNA) technology was used to silence the expression of FABP4 gene in HK-2 cells, which were then treated with H/R (24 hours of hypoxia and 6 hours of reoxygenation) or treated with the Nrf2 inhibitor ML385. Cell proliferation activity was assessed using cell counting kit-8 (CCK-8). Lactate dehydrogenase (LDH) levels were measured by enzyme-linked immune absorbent assay (ELISA). Malondialdehyde (MDA), glutathione (GSH) and ferrous ion (Fe²⁺) levels were determined by biochemical technology. Reactive oxygen species (ROS) levels were detected using the 2',7'-dichlorodihydrofluorescein diacetate fluorescence probe. Protein expression levels of FABP4, nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) were measured by Western blotting.

Results  The mRNA and protein levels of FABP4 in HK-2 cells increased with prolonged reoxygenation time (all P<0.05). H/R treatment reduced cell proliferation activity, increased LDH levels in the cell supernatant, and elevated MDA, Fe²⁺ and ROS levels in HK-2 cells while decreasing GSH levels and the protein levels of Nrf2, HO-1, GPX4 and SLC7A11 (all P<0.05). Silencing FABP4 enhanced the proliferation activity of H/R-treated HK-2 cells (P<0.05), reduced MDA, Fe²⁺ and ROS levels, increased GSH levels, and elevated the protein levels of Nrf2, HO-1, GPX4 and SLC7A11 (all P<0.05). However, these beneficial effects of FABP4 silencing on H/R-induced ferroptosis in HK-2 cells were reversed by co-treatment with ML385.

Conclusions  Silencing FABP4 alleviated H/R-induced ferroptosis in HK-2 cells, possibly by activating the Nrf2/GPX4 axis.