The SOX4/EZH2/SLC7A11 signaling axis mediates ferroptosis in calcium oxalate crystal deposition-induced kidney injury

Abstract

Epigenetic regulation is reported to play a significant role in the pathogenesis of various kidney diseases, including renal cell carcinoma, acute kidney injury, renal fibrosis, diabetic nephropathy, and lupus nephritis. However, the role of epigenetic regulation in calcium oxalate (CaOx) crystal deposition-induced kidney injury remains unclear. Our study demonstrated that the upregulation of enhancer of zeste homolog 2 (EZH2)-mediated ferroptosis facilitates CaOx-induced kidney injury. CaOx crystal deposition promoted ferroptosis in vivo and in vitro. Usage of liproxstatin-1 (Lip-1), a ferroptosis inhibitor, mitigated CaOx-induced kidney damage. Single-nucleus RNA-sequencing, RNA-sequencing, immunohistochemical and western blotting analyses revealed that EZH2 was upregulated in kidney stone patients, kidney stone mice, and oxalate-stimulated HK-2 cells. Experiments involving in vivo EZH2 knockout, in vitro EZH2 knockdown, and in vivo GSK-126 (an EZH2 inhibitor) treatment confirmed the protective effects of EZH2 inhibition on kidney injury and ferroptosis. Mechanistically, the results of RNA-sequencing and chromatin immunoprecipitation assays demonstrated that EZH2 regulates ferroptosis by suppressing solute carrier family 7, member 11 (SLC7A11) expression through trimethylation of histone H3 lysine 27 (H3K27me3) modification. Additionally, SOX4 regulated ferroptosis by directly modulating EZH2 expression. Thus, this study demonstrated that SOX4 facilitates ferroptosis in CaOx-induced kidney injury through EZH2/H3K27me3-mediated suppression of SLC7A11.

Keywords: EZH2; Ferroptosis; Kidney injury; Kidney stones; SOX4.

Fig. 1

Ferroptosis is stimulated in kidney stone mice, and ferroptosis inhibition mitigates Gly-induced kidney injury. A Diagram of mouse moulding. B Effects of Lip-1 on bodyweight (n = 6). C H&E, Von-Kossa, and TUNEL staining (n = 6). D MDA, GSH, and Fe2 + levels (n = 6). E Serum BUN and Cr levels (n = 6). F GO and KEGG enrichment analyses of DEGs between the Gly and control groups (n = 3). G Immunofluorescence of SLC7A11, GPX4, and ACSL4 in vivo (n = 6). H Western blotting analysis of SLC7A11, GPX4, ACSL4 and PTGS2. Scale bar = 50 µm. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group; ^#P < 0.05 and ^##P < 0.01 compared with the Gly group

Fig. 2

EZH2 is upregulated in kidney stone patients, kidney stone mice, and oxalate-stimulated HK-2 cells. A Single-nucleus RNA sequencing dataset analysis of CKD patients and healthy controls. B Volcano map of the DEGs between the Gly and control groups. C Western blotting quantitative analysis of EZH2 in vivo and in vitro. D Expression levels of EZH2 in kidney stone patients, kidney stone mice, and the control groups were examined using immunohistochemical analysis. E Immunofluorescence analysis of EZH2. Scale bar = 50 µm. *P < 0.05 and **P < 0.01 compared with the control group

Fig. 3

EZH2 knockout alleviates CaOx-induced kidney injury and ferroptosis. A Diagram of mouse moulding. B Effect of EZH2 knockout on bodyweight (n = 6). C H&E and Von-Kossa staining (n = 6). D Serum BUN and Cr levels in different groups (n = 6). E Immunofluorescence analysis of SLC7A11, GPX4, and ACSL4 in kidney tissues (n = 6). F Representative images were acquired using a TEM. G Western blotting analysis of EZH2, SLC7A11, GPX4, ACSL4 and PTGS2. H Relative MDA, GSH, and Fe2 + levels (n = 6). I EZH2, SLC7A11, GPX4, and ACSL4 mRNA levels were testing using qRT-PCR. Scale bar = 50 µm. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the EZH2^fl/fl group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.01 compared with the Gly + EZH2^fl/fl group

Fig. 4

EZH2 knockdown mitigates oxalate-induced ferroptosis in HK-2 cells. A The effect of EZH2 knockdown on lipid peroxidation in vitro. B Effects of EZH2 knockdown on lipid ROS levels were analyzed using the C11-BODIPY fluorescence probe. C Immunofluorescence analysis of SLC7A11, GPX4, and ACSL4. D Relative lipid peroxidation, MDA, GSH, and Fe2 + levels in HK-2 cells. E Western blotting analysis of EZH2, SLC7A11, GPX4, ACSL4 and PTGS2. F The mRNA levels of EZH2, SLC7A11, GPX4, ACSL4 and PTGS2. Scale bar = 50 µm. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the shNC group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 compared with the Ox + shNC group

Fig. 5

EZH2 regulates ferroptosis by directly suppressing SLC7A11 expression. A Heat map showed the genes significantly upregulated in the shEZH2 group. Red and blue colors denote ferroptosis-related upregulated and downregulated genes, respectively. B Western blotting analysis of H3K27me3. C Genome browser view of the H3K27me3 density at the SLC7A11 promoter region. D Analysis of EZH2-SLC7A11 promoter interaction using the CHIP assay. E SLC7A11 inhibition reverses the effects of the EZH2 knockdown on ferroptosis. The lipid ROS levels were analyzed using the C11-BODIPY probe. F Western blotting analysis of EZH2, SLC7A11, and GPX4. **P < 0.01, ***P < 0.001 compared with the shNC group; ^###P < 0.001 compared with the Ox + shNC group in B. *P < 0.05 and **P < 0.01 compared with the Ox group, ^#P < 0.05 and ^##P < 0.01 compared with the Ox + shEZH2 group in F

Fig. 6

EZH2 is a direct transcriptional target of the transcription factor SOX4. A Sequence logo of SOX4. B Potential binding sites of SOX4 at the promoter of EZH2. C Correlation between SOX4 and EZH2 expression levels. The mRNA levels of SOX4 were correlated with EZH2 (r = 0.53; P = 0.0026). D Relative enrichment of SOX4 on the promoter region of EZH2 gene was evaluated by CHIP-qPCR in HK-2 cell. E The effect of SOX4 knockdown on lipid peroxidation. F Relative lipid peroxidation, MDA, GSH, and Fe2 + levels. G Western blotting analysis of SOX4, EZH2, SLC7A11, and GPX4. Scale bar = 50 µm. *P < 0.05 and **P < 0.01 compared with the siNC group; ^#P < 0.05 and ^##P < 0.01 compared with the Ox + siNC group

Fig. 7

GSK-126 suppresses Gly-induced ferroptosis. A Tubular injury and CaOx crystal deposition were evaluated using H&E and Von-Kossa staining (n = 6). B Serum levels of BUN and Cr in different groups (n = 6). C Immunofluorescence analysis of SLC7A11, GPX4, and ACSL4 in kidney tissues (n = 6). D Western blotting analysis of SLC7A11, GPX4, ACSL4 and PTGS2 in kidney tissues. Scale bar = 50 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group; ^#P < 0.05 and ^##P < 0.01 compared with the Gly group