Mitochondrial DNA methylation is involved in contrast-induced renal tubular epithelial cell injury

Abstract

Mitochondrial DNA (mtDNA) methylation may be associated with mitochondrial damage; this study investigates their relationship in contrast-induced renal tubular epithelial cell (RTEC) injury. We stimulated HK-2 cells with iohexol to establish an in vitro model and analyzed the methylation level of mtDNA by bisulfite amplicon sequencing. The mitochondrial membrane potential, mitochondrial reactive oxygen species (mtROS), intracellular ROS, and changes in mitochondrial ultrastructure were evaluated as indicators of mitochondrial damage. Iohexol significantly inhibited cell viability and induced cell apoptosis, increasing both mtROS and intracellular ROS levels. Additionally, the methylation levels of mtDNA-encoded genes cytochrome c oxidase subunit I (COX I) (3.09%, *p < 0.05), cytochrome c oxidase subunit II (COX II) (4.51%, **p < 0.01), cytochrome c oxidase subunit III (COX III) (3.50%, **p < 0.01) and cytochrome B (CYTB)(4.66%, *p < 0.05) were increased, accompanied by enhanced transcription of both COX I and COX III. 5-Aza-dC, as a DNA methylation inhibitor, was dissolved in dimethyl sulfoxide (DMSO) vehicle to explore the role and mechanism of inhibiting mtDNA methylation in contrast-induced RTEC injury. HK-2 cells were further divided into four groups: vehicle control (DMSO alone), vehicle pretreated contrast - induced group (CI) (DMSO-CI), inhibitor control (5-Aza-dC), and inhibitor pretreated CI (5-Aza-dC-CI). Intriguingly, administration of 5-Aza-dC effectively attenuated mtDNA methylation, leading to improvements in these parameters and restoration of cell viability while reducing apoptosis. In conclusion, mtDNA methylation is involved in the mechanism of contrast-induced RTEC injury, potentially mediated by over-transcription of COX I and III, abnormal mtROS production, and subsequent mitochondrial damage and dysfunction. Inhibiting mtDNA methylation can provide protective effects against contrast - induced RTEC injury by reducing ROS (mtROS) production.

Keywords: CI-AKI; Mitochondrial DNA methylation; ROS; apoptosis; mitochondrial damage.

Graphical abstract

Figure 1.

Iohexol injured cell viability and induced apoptosis in renal tubular epithelial cells (RTECs). (a) Cell viability was determined by CCK-8 assay (n ≥ 3); (b, c) Flow cytometry analysis of the cell apoptotic rate and quantitation analysis (n ≥ 3); Representative images. Data were expressed as the means ± SD. ***p < 0.001.

Figure 2.

Iohexol induced mtDNA methylation and promoted partly mtDNA transcription in renal tubular (RTECs). (a) The mtDNA methylation level was analyzed by BSAS (n ≥ 5); (b) The mRNA expression levels of mtCOX I-III and mtCYTB were measured by qPCR (n ≥ 3). Data were expressed as the means ± SD. *p < 0.05; **p < 0.01.

Figure 3.

Iohexol induced accumulation of ROS, mitochondrial damage, and morphological abnormalities in renal tubular epithelial cells (RTECs). (a, b) The intracellular reactive oxygen species (ROS) level in cells was detected by DCFH-DA fluorescence (n ≥ 3). Representative images. Scale bars:100µm. Magnification: ×100; (c, d) The mitochondrial ROS level in cells was detected by MitoSOX Red (n ≥ 3). Representative images. Scale bars: 50 µm; (e, f) The mitochondrial membrane potential of cells was observed via JC-1 staining (n ≥ 3). Representative images. Red: aggregated JC-1; Green: monomeric JC-1; Scale bars:100 μm. Magnification: ×200; (g) The mitochondrial morphology of cells was assessed by transmission electron microscopy (TEM) (n ≥ 3). Representative images. Blue arrows: normal mitochondria, red arrows: damaged mitochondria. Scale bars: 500 nm. Original magnification: ×2.5 × 10⁴. Data were expressed as the means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. 5-Aza-dC: 5-Aza-2’-deoxycytidine.

Figure 4.

Inhibiting iohexol induced-mtDNA methylation alleviated cell damage and apoptosis in renal tubular epithelial cells (RTECs). (a) The mtDNA methylation level of mtCOX I-III and CYTB was analyzed by BSAS (n ≥ 5); (b) Cell viability was determined by CCK-8 assay (n ≥ 3). (c, d) Flow cytometry analysis of the cell apoptotic rate and quantitation analysis (n ≥ 3); Representative images. Data were expressed as the means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. 5-Aza-dC:5-Aza-2’-deoxycytidine.

Figure 5.

Inhibiting iohexol-induced mtDNA methylation reversed excessive transcription of certain mtDNA, reduced ROS accumulation, repaired mitochondrial damage, and morphological abnormalities. (a) The mRNA expression levels of mtCOX I-III and mtCYTB were detected by qPCR (n ≥ 3). (b, c) The mitochondrial membrane potential of cells was observed via JC-1 staining (n ≥ 3). Representative images. Red: aggregated JC-1; Green: monomeric JC-1; Scale bars:100 μm. Magnification: ×200; (d, e) The intracellular reactive oxygen species (ROS) level in cells was detected by DCFH-DA fluorescence (n ≥ 3). Representative images. Scale bars:100μm. Magnification: ×100; (f, g) The mitochondrial ROS level in cells was detected by MitoSOX Red (n ≥ 3). Representative images. Scale bars:50µm. (h) The mitochondrial morphology of cells was assessed by transmission electron microscopy (TEM) (n ≥ 3). Representative images. Blue arrows: normal mitochondria, red arrows: damaged mitochondria. Scale bars: 500 nm. Original magnification: ×2.5 × 10⁴. Data were expressed as the means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. 5-Aza-dC:5-Aza-2’-deoxycytidine.