Inhibition of ferroptosis alleviates acute kidney injury caused by diquat in zebrafish

doi:10.12122/j.issn.1673-4254.2025.08.18

Abstract

Abstract:

Objective To investigate the role of ferroptosis in diquat-induced acute kidney injury (AKI) and its molecular mechanisms. Methods Transgenic zebrafish models with Tg (Eco.Tshb:EGFP) labeling of the renal tubules and Tg (lyz:dsRed2) labeling of the neutrophils were both divided into control group, gentamicin (positive control) group, diquat poisoning group, ferroptosis inhibitor group. The indicators of kidney injury, inflammatory response, and ferroptosis were examined in the zebrafish, and the changes in expressions of voltage-dependent anion-selective channel protein 1 (VDAC1) and mitochondrial ferritin (FTMT) were detected using Western blotting. Results AKI induced by diquat exhibited a significant dose-effect relationship, and the severity of injury was proportional to the exposure concentration. Diquat also caused marked oxidative stress and inflammatory responses in the zebrafish models. Rhodamine metabolism assay and HE staining revealed significantly declined glomerular filtration function of the zebrafish as diquat exposure concentration increased. Immunofluorescence staining highlighted significant changes in the expressions of ferroptosis markers GPX4 and FTH1 in zebrafish renal tissues following diquat exposure. In diquat-exposed zebrafish, treatment with ferrostatin-1, a ferroptosis inhibitor, obviously upregulated GPX4 and downregulated FTH1 expressions and improved the metabolic rate of glucan labeled with rhodamine B. Diquat exposure significantly upregulated the expression of VDAC1 and FTMT in zebrafish, and the application of ferrostatin-1 and VBIT-12 (a VDAC1 inhibitor) both caused pronounced downregulation of FTMT expression. Conclusion Ferroptosis is a critical mechanism underlying diquat-induced AKI, in which VDAC1 and FTMT play important regulatory roles, suggesting their potential as therapeutic target for AKI caused by diquat.

Key words: diquat, acute kidney injury, ferroptosis, voltage-dependent anion-selective channel protein 1, mitochondrial ferritin

Zejin OU, Ying LI, Shi CHEN, Ziyi WANG, Meiyi HE, Zhicheng CHEN, Shihao TANG, Xiaojing MENG, Zhi WANG. Inhibition of ferroptosis alleviates acute kidney injury caused by diquat in zebrafish[J]. Journal of Southern Medical University, 2025, 45(8): 1743-1750.

Figures/Tables 7

Fig.1 Effect of diquat on kidney development in zebrafish (Original magnification: ×40).Three replicates are shown in each group.

Fig.2 Inflammatory response induced by diquat in zebrafish (×40).

Fig.3 Fluorescence quantitative analysis of the effect of diquat on ROS level in live-stained zebrafish (×40). A: Control group. B: Gentamicin-positive group. C: Diquat 10 μmol/L group. D: Diquat 20 μmol/L group. E: Diquat 40 μmol/L group. F: Results of quantitative analysis.

Fig 4 Effect of ferroptosis inhibition on expressions of GPX4 and FTH1 in the kidneys of diquat-exposed zebrafish (×400). A, B: Immunofluorescence and quantitative analysis of GPX4. C,D: Immunofluorescence and quantitative analysis of FTH1. The white arrows indicated the zebrafish kidney tissue. *P<0.05.

Fig.5 Effect of diquat exposure on glomerular filtration rate in zebrafish (×40). The metabolism of rhodamine B-labeled dextran was detected using fluorescence quantification. A: Control group. B: Gentamicin-positive group. C: Diquat 10 μmol/L group. D: Diquat 20 μmol/L group. E: Diquat 40 μmol/L group. F: Diquat 40 μmol/L group+Fer-1 intervention group. G: The results of quantitative analysis. *P<0.05, ***P<0.001.

Fig.6 Pathological analysis of the effect of diquat exposure on renal injury in zebrafish (HE staining, ×400). A: Control group. B: Gentamicin-positive group. C: Diquat exposure 40 μmol/L group. D: Diquat 40 μmol/L group+Fer-1 intervention group.

Fig.7 Effect of diquat exposure and drug interventions on protein expressions of VDAC1 and FTMT. A: Protein expressions of VDAC1 and FTMT detected using Western blotting. B: Quantitative analysis of the protein expressions (n=20). *P<0.05, **P<0.01, ***P<0.001. Each experimental repetition involved 20 larvae.

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