Ferroptosis-related changes in the kidney are observed in diabetic mice.

(A) Immunohistochemical staining results and semiquantification analyses of GPX4, SLC7A11, FTH-1 and COX2 in the kidneys of nondiabetic, diabetic control and diabetic treated with Fer-1 mice. (B) Western blot analysis and quantification of GPX4, SLC7A11, FTH-1, TFR-1 and COX2 in the kidneys of nondiabetic, diabetic and diabetic mice treated with Fer-1. (C-G) The mRNA expression levels of GPX4 (C), SLC7A11 (D), FTH-1 (E), TFR-1 (F) and PTGS2 (G) in kidney tissue in mice. (H-K) GSH (H), MDA (I), 4-HNE (J) and iron concentrations (K) were detected in the kidney tissue of mice. Data are the mean ± SEM. **p vs. NC group; ***p vs. DM group.</p

High glucose-induced ferroptosis was alleviated by treatment with aspirin.

(A) DCHF-DA staining revealed ROS (green) generation in high glucose-cultured HK-2 cells compared to control cells, which was partially alleviated by AS treatment. (B) Western blot analysis of GPX4, SLC7A11, FTH-1, TFR-1 and COX2 protein expression in cells in the Ctrl, HG, Fer-1, DMSO and AS groups. (C-G) GPX4 (C), SLC7A11 (D), FTH-1 (E), TFR-1 (F) and PTGS2 (G) mRNA expression in cells in the Ctrl, HG and AS groups. (H-K) GSH (H), MDA (I),4-HNE(J) and iron concentrations (K) of cells in the Ctrl, HG and AS groups. (L-M) The KIM-1 (L) and NGAL (M) increased mRNA levels induced by high glucose were relieved by treatment with AS. Data are the mean ± SD. **p vs. Ctrl group; ***p vs. HG group.</p

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Ferroptosis is a critical component of high glucose-cultured HK-2 cells.

(A) CCK8 was used to detect the viability of HK-2 cells cultured in different concentrations of glucose medium for 48 h. (B) DCHF-DA staining revealed the generation of ROS (green) in high glucose-cultured HK-2 cells compared to the control cells, which was partially reduced by Fer-1 treatment for 48 h. (C) Western blot analysis of GPX4, SLC7A11, FTH-1, COX2 and TFR-1 protein expression in cells in the Ctrl and HG groups. (D-G) GPX4 (D), SLC7A11 (E), FTH-1 (F) and TFR-1 (G) mRNA levels in cells in the Ctrl, HG, Fer-1, DMSO and mannitol groups.(H-K) GSH (H), MDA (I),4-HNE(J) and iron concentrations (K) of cells in the Ctrl, HG, Fer-1, DMSO and mannitol groups. Data are expressed as the mean ± SD. **p vs. Ctrl group; ***p vs. HG group.</p

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Aspirin ameliorates ferroptosis indices in the diabeitc kidney.

(A) GPX4, SLC7A11, FTH-1 and COX2 of the kidneys in nondiabetic, diabetic control and diabetic treated with AS mice were assessed based on immunohistochemical staining results and semiquantification analyses. (B) Western blot analysis of GPX4, SLC7A11, FTH-1, TFR-1 and COX2 in the kidneys of nondiabetic, diabetic control and diabetic mice treated with AS. (C-G) GPX4 (C), SLC7A11 (D), FTH-1 (E), TFR-1 (F) and PTGS2 (G) mRNA expression in the kidney tissue of mice were measured. (H-K) The GSH (G), MDA (H), 4-HNE (I) and iron concentrations (J) in the kidney tissue were analyzed. (L) The mitochondrial morphology of cells was detected by transmission electron microscopy in the Ctrl, HG and AS groups. (M) Schematic model: COX2/GPX4-mediated ferroptosis in the process of diabetic kidney. Data are the mean ± SEM. **p vs. NC group; ***p vs. DM group.</p

Cellular ferroptosis-induced RSL-3 was inhibited by transfection with COX2-siRNA.

(A) Immunofluorescence analysis with GPX4 antibody (red) and nuclear staining with DAPI (blue) of HK-2 cells in the Si-Ctrl, Si-COX2, RSL-3+Si-Ctrl, RSL-3+Si-COX2 and DMSO+Si-Ctrl groups. (B) The cell viability measurements in HK-2 cells treated with RSL-3 100 nm for 48 h were partially improved by transfection of COX2-siRNA. (C) Western blotting results of COX2, GPX4, SLC7A11, FTH-1 and TFR-1 in cells in each group. (D-H) The mRNA expression of GPX4 (D), SLC7A11 (E), FTH-1 (F), TFR-1 (G) and PTGS2 (H) in each group of cells. (I-L) GSH (I), MDA (J), 4-HNE(K) and iron concentrations (L) of HK-2 cells in the Si-Ctrl, Si-COX2, RSL-3+Si-Ctrl and RSL-3+Si-COX2 groups. (M-N) KIM-1 (M) and NGAL (N) increased mRNA expression induced by RSL-3 and were relieved by transfection of COX2-siRNA. Data are the mean ± SEM. **p vs. Si-Ctrl group; ***p vs. Si-Ctrl+ RSL-3 group.</p

Knockdown of COX2 ameliorated the ferroptosis sensitivity of cells cultured in high glucose.

(A) Immunofluorescence analysis with GPX4 antibody (red) and nuclear staining with DAPI (blue) in the HK-2 cells of the Si-Ctrl group, Si-COX2 group, HG+Si-Ctrl group and HG+Si-COX2 group. (B) Western blotting showed the protein expression of COX2, GPX4, SLC7A11, FTH-1 and TFR-1 in the cells of each group. G) GPX4 (C), SLC7A11 (D), FTH-1 (E), TFR-1 (F) and PTGS2 (G) mRNA expression in cells in each group. (H-J) GSH (H), MDA (I), 4-HNE(J)and iron concentrations (K) of cells in each group. (K-L) KIM-1 (L) and NGAL (M) increased mRNA expression was alleviated by transfection of COX2-siRNA in high glucose. Data are expressed as the mean ± SD. **p vs. Si-Ctrl group; ***p vs. HG+Si-Ctrl group.</p

Inhibition of ferroptosis by Fer-1 ameliorated the pathological changes of kidney fibrosis in diabetic kidney disease.

(A) Schematic diagram. Five doses of STZ (40 mg/kg/day IP) were injected to show the induction of diabetes in DM and Fer-1 mice. (B-G) Body weight (B), blood glucose (C), 24-h urine volume (D), urinary albuminuria (E), albumin-to-creatinine ratio ACR (F) and kidney weight/body weight (G) were monitored in the NC, Fer-1, DM and vehicle-P groups. (H) HE staining, PAS staining and Masson staining of kidneys in nondiabetic and diabetic control Fer-1 mice. The relative area of fibrosis (%) and relative collagen (%) were assessed by the ImageJ program. (I-J) The increased KIM-1 and NGAL mRNA expression was relieved by Fer-1 in diabetic mice. Data are the mean ± SEM. **p vs. NC group; ***p vs. DM group.</p

Cellular ferroptosis induced by RSL-3 was alleviated by Fer-1 and aspirin.

(A)DCHF-DA staining revealed the generation of ROS (green) in the HK-2 cells cultured in 100 nmol/L RSL-3 medium compared to the control cells, and it was partially reduced by aspirin. (B) Cell viability was measured in HK-2 cells cultured with different concentrations of RSL-3 for 48 h, and it was partially improved by 400 nm Fer-1 or 400 μm aspirin treatment. (C) Cell viability measurements in HK-2 cells treated with RSL-3 100 nm for 48 h were partially improved by treatment with different concentrations of Fer-1 or AS. (D) Western blot analysis of SLC7A11, GPX4, FTH-1, TFR-1 and COX2 protein expression of cells in Ctrl, DMSO, RSL-3 100 nm, RSL-3 100 nm+Fer-1 400 nm, RSL-3 100 nm+AS400 μm and AS 400 μm groups. (E-I) GPX4 (E), SLC7A11 (F), FTH-1 (G), TFR-1 (H) and PTGS2 (I) mRNA expression in cells of each group. (J-M) GSH (J), MDA (K), 4-HNE(L)and iron concentrations (M) in the cells of each group. (N-O) The increased mRNA expression of KIM-1 (N) and NGAL (O) induced by RSL-3 was relieved by treatment with Fer-1 or aspirin. Data are expressed as the mean ± SD. **p vs. Ctrl group; ***p vs. RSL-3 group.</p