[The role of ferroptosis in renal injury induced by diquat].

Abstract

Objective: To investigate the role of ferroptosis in renal cell injury induced by diquat (DQ) . Methods: From January to October 2022, human renal tubular epithelial (HK-2) cells were treated with DQ for 48 h, and different doses of ferroptosis inhibitors [deferoxamine (DFO), Fer-1] were added, and cells were harvested 24 h later. The experiment was divided into 6 groups (n=6) : control group, DQ group (60 μmol/L), 20 μmol/L DFO (DFO-H) group, 10 μmol/L DFO (DFO-L) group, 5 μmol/L Fer-1 (Fer-1-H) group, 0.5 μmol/L Fer-1 (Fer-1-L) group. From December 2022 to June 2023, male C57bl/6 mice were selected to establish the animal model, and the experimental group was divided into 4 groups (n=6) : control group, DQ group (25 mg/kg), DFO group (100 mg/kg) and Fer-1 group (2.5 μmol/kg). The changes of renal tissue were detected by HE staining. The fluorescence probe of ferrous ions was used to detect the change of iron ions in cells, and the colorimetric determination of total iron and ferrous ions in mouse kidney tissues was performed. Reverse transcription real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to detect mRNA and protein expression changes related to ferroptosis signaling pathway. TUNEL staining was used to detect apoptosis. Enzyme-linked immunosorbent assay (ELISA) was used to detect the changes of antioxidant-related proteins and oxidative stress-related products. Differences among groups were analyzed by one-way analysis of variance. Results: In vitro test, compared with the control group, the iron ion level of HK-2 cells in DQ group was increased, the mRNA and protein expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and ferritin heavy chain (FTH) were decreased, the mRNA and protein expression levels of transferrin receptor 1 (TFR1) and divalent metal transporter 1 (DMT1) were increased, and the apoptosis level was significantly increased (P<0.05). The expression levels of glutathione (GSH) and super oxide dismutase (SOD) in HK-2 cells in DQ group were significantly lower than those in control group (P<0.05), and the expression levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were significantly higher than those in control group (P<0.05). Compared with DQ group, iron ion levels in HK-2 cells in the intervention groups of DFO and Fer-1 at different doses were decreased (P<0.001), and GPX4, SLC7A11 and FTH mRNA and protein expression levels were increased, and the mRNA and protein expression levels of TFR1 and DMT1 were decreased in DFO-H and DFO-L groups (P<0.05). The apoptosis levels in the intervention groups of DFO and Fer-1 at different doses were decreased compared with DQ group (P<0.001), the expression levels of GSH and SOD were higher than those in DQ group (P<0.05), and the expression levels of ROS were lower than those in DQ group (P<0.05). In vivo, HE staining showed that the renal tissue of DQ group mice had obvious renal tubular epithelial cell injury with inflammatory cell infiltration. Compared with DQ group, DFO group and Fer-1 group had less damage of renal tubular epithelial cells and less inflammatory cell infiltration. Compared with the control group, the total iron content and ferrous iron content in kidney tissue of mice in DQ group were increased, the mRNA and protein expression levels of GPX4, SLC7A11 and FTH were decreased, the mRNA and protein expression levels of TFR1 and DMT1 were increased, and the apoptosis level was increased in DQ group (P<0.05). The levels of GSH and SOD in DQ group were lower than those in control group, while the levels of MDA and ROS in DQ group were higher than those in control group (P<0.05). Compared with DQ group, the total iron content and ferrous iron content in DFO group, and ferrous iron content in Fer-1 group were decreased (P<0.001), the mRNA and protein expression levels of GPX4, SLC7A11 and FTH in kidney tissues of mice in DFO group and Fer-1 group were increased (P<0.05), and the mRNA and protein expression levels of TFR1 and DMT1 were decreased (P<0.05). The level of apoptosis in DFO group and Fer-1 group was lower than that in DQ group (P<0.001). Compared with DQ group, the expression levels of GSH in kidney tissues, and the expression levels of SOD in serum and kidney tissues in DFO group were increased (P<0.05), and the expression levels of GSH and SOD in serum and kidney tissues in Fer-1 group were increased (P<0.05). The expression levels of MDA and ROS in serum and kidney tissues of DFO group and Fer-1 group were lower than those of DQ group (P<0.05) . Conclusion: Ferroptosis may be involved in renal cell injury induced by DQ poisoning, and ferroptosis inhibitor may alleviate DQ-induced renal injury by inhibiting ferroptosis.