[Protective effects of SESN2 on Erastin-induced ferroptosis in renal tubular cells and acute kidney injury].

OBJECTIVES: Prevention and treatment of perioperative acute kidney injury (AKI) remain a major focus in clinical practice. This study aims to investigate the role and underlying mechanism of sestrin2 (SESN2) in Erastin-induced ferroptosis of renal tubular cells and AKI in ICR mice.

METHODS: For in vitro experiments, human renal proximal tubular epithelial cells (HK-2) stably transfected with lentivirus were divided into three groups: control group, Erastin model group, and SESN2 intervention group. The control group was transfected with empty vector lentivirus (LV-Con). The Erastin model group was transfected with LV-Con and then treated with 10 μmol/L Erastin for 24 h. The SESN2 intervention group was transfected with SESN2-overexpressing lentivirus (LV-SESN2) followed by Erastin treatment. For in vivo experiments, after confirming successful establishment of the Erastin-induced AKI mouse model by measuring serum creatinine (SCr) and blood urea nitrogen (BUN), 18 healthy adult male ICR mice were randomly divided into three groups (n=6 per group): control group, Erastin model group, and SESN2 intervention group. The control group received tail vein injection of control virus (AAV9-Con). The Erastin model group received AAV9-Con followed by intraperitoneal injection of Erastin (30 mg/kg) after 4 weeks. The SESN2 intervention group received tail vein injection of SESN2-overexpressing virus (AAV9-Sesn2), followed by Erastin injection at the same dose after 4 weeks. Serum and renal tissues were collected 24 h after modeling. Enzyme-linked immunosorbent assay (ELISA) was used to measure lactate dehydrogenase (LDH), interleukin (IL)-6, and tumor necrosis factor (TNF)-α levels in cell and tissue supernatants. Reactive oxygen species (ROS) levels were assessed using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) fluorescence staining. Malondialdehyde (MDA), glutathione (GSH), and ferrous ion (Fe2+) levels were determined by colorimetric assays. Cell apoptosis was calculated using Annexin V-FITC/propidium iodide (PI), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and flow cytometry. The mRNA and protein expression levels of SESN2, nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPX4), and ferroptosis suppressor protein 1 (FSP1) were detected by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) and Western blotting (WB).

RESULTS: Compared with the in vitro/in vivo control groups, the Erastin model groups and SESN2 intervention groups showed significantly increased levels of LDH, IL-6, TNF-α, MDA, ROS, and Fe2+, along with significantly decreased GSH levels and increased apoptosis rates (all P<0.001). The mRNA and protein expression levels of GPX4 and FSP1 were significantly decreased, whereas those of SESN2 and Nrf2 were significantly increased (all P<0.001). Compared with the Erastin model groups, the SESN2 intervention groups exhibited significantly reduced levels of LDH, IL-6, TNF-α, MDA, ROS, and Fe2+, increased GSH levels, and decreased apoptosis rates (all P<0.001). In addition, the mRNA and protein expression levels of SESN2, Nrf2, GPX4, and FSP1 were significantly increased (all P<0.001).

CONCLUSIONS: SESN2 exerts protective effects against Erastin-induced ferroptosis in renal tubular cells and ferroptosis-related AKI by activating the Nrf2 signaling pathway, upregulating ferroptosis-related molecules GPX4 and FSP1, and inhibiting oxidative stress, lipid peroxidation, and inflammatory responses.