Up-regulation of GCLM alleviates ferroptosis in renal tubular epithelial cells induced by hypoxia reoxygenation injury

Abstract

Objective To explore the role and mechanism of glutamate-cysteine ligase modifier subunit (GCLM) in alleviation of hypoxia reoxygenation (HR) injury in renal tubular epithelial cells. Methods Human proximal tubular epithelial HK2 cells were cultured and divided into control group (Ctrl group), HR group, and HR+GCLM overexpression group (HR+GCLM OE group). Cell model of HR was established by hypoxia (94% N2+1% O2+5% CO2, glucose and serum-free DMEM/F12 medium) for 24 h, and then followed by of reoxygenation (95% air +5% CO2, DMEM/F12 medium with 10% FBS) for 6 h. The cells of the HR+GCLM OE group was transfected with REST overexpression plasmid for 24 h and then exposed to HR injury. The protein levels of GCLM and glutathione peroxidase 4(GPX4) were detected with Western blotting, the contents of glutathione (GSH), malonaldehyde (MDA) and reactive oxygens (ROS) were measured, and mitochondrial morphology was observed with transmission electron microscopy (TEM). Results Under HR condition, the HK2 cells showed significantly accumulated MDA (P < 0.01) and ROS (P < 0.01) contents, obviously reduced GSH level (P < 0.01), typical ferroptosis changes under TEM, and decreased expression of GCLM and GPX4 (P < 0.01). However, overexpression of GCLM inhibited MDA and ROS production (P < 0.01) while increased GSH generation and GPX4 expression (P < 0.01), and alleviated HR injury. Conclusion Up-regulation of GCLM protects renal tubular epithelial cells against HR injury probably by suppressing ferroptosis, indicating a potential therapeutic effect of GCLM in acute kidney injury.

Keywords

• ferroptosis
• hypoxia reoxygenation injury
• acute kidney injury
• glutamate-cysteine ligase modifier subunit