Renal Ischemia Induces Epigenetic Changes in Apoptotic, Proteolytic, and Mitochondrial Genes in Swine Scattered Tubular-like Cells

Figures

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  Figure 1. Characterization of scattered tubular-like cells (STCs) isolated from pig kidneys. (A): Flow cytometry analysis of isolated STCs co-expressing CD133 and CD24 (97%) and representative images of CD133+ (pink)/CD24+ (green) cells. (B): Immunofluorescence staining (original magnification: ×40) of swine STCs with antibodies against the surface markers CD24 (green), CD133 (red), vimentin (pink), and kidney injury molecule (KIM)-1 (green). Cells processed without primary antibodies served as negative controls.

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  Figure 2. Renal artery stenosis (RAS) induces significant changes in 5-methylcytosine (5mC) levels in swine STCs. (A): Heat map of genes with significant changes in 5mC levels between Normal- and RAS-STCs. (B): Volcano plot of dysregulated 5mC genes. The vertical axis (y-axis) corresponds to the -log 2 (p-value) and the horizontal axis (x-axis) displays the log 2-fold change (RAS-STCs/Normal-STCs) value. Genes with higher (n = 239) and lower (n = 236) 5mC levels in RAS- versus Normal-STCs are shown with red and blue dots, respectively, while non-significant genes are shown as grey dots. A p-value ≤ 0.05 and fold changes ≥1.4 or ≤0.7 are indicated by black dashed lines. (C): Representative reads for ODF3B and TP23 genes in Normal- and RAS-STCs were visualized using Integrative Genomics Viewer. Gray rectangles indicate regions (peaks) with higher and lower 5mC levels in RAS- versus Normal-STCs. (D–F): Gene ontology (GO) analysis of cellular component (D), molecular function (E), and biological process (F) of genes with significant changes in 5mC levels between Normal- and RAS-STCs.

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  Figure 3. RAS induces significant changes in 5-hydroxymethylcytosine (5hmC) levels in swine STCs. (A): Heat map of genes with significant changes in 5hmC levels between Normal- and RAS-STCs. (B): Volcano plot of dysregulated 5hmC genes. Genes with higher (n = 275) and lower (n = 315) 5hmC levels in RAS- versus Normal-STCs are shown with red and blue dots, respectively. (C): Representative reads for CLDN11 and ZAR1 genes in Normal- and RAS-STCs. D–F: Gene ontology (GO) analysis of cellular component (D), molecular function (E), and biological process (F) of genes with significant changes in 5hmC levels between Normal- and RAS-STCs.

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  Figure 4. RAS-induced 5mC and 5hmC marks correlate with changes in gene expression in swine STCs. (A) Scatter plots of gene sets with significant overlap (5mC levels and mRNA expression) between Normal- and RAS-STCs, containing apoptotic, proteolytic, and mitochondrial genes. (B) Scatter plot of gene sets with significant overlap (5hmC levels and mRNA expression) between Normal- and RAS-STCs, containing apoptotic, proteolytic, and mitochondrial genes. (C) Expression (qPCR) of randomly selected genes dysregulated in RAS- versus Normal-STCs followed the same patterns as the mRNA-seq findings. * p < 0.05 vs. Normal-STCs.

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  Figure 5. RAS alters apoptosis, proteolysis, and mitochondrial function in swine STCs. (A) Representative images of TUNEL staining and its quantification, as well as quantification of BCL-2 expression (qPCR) in Normal- and RAS-STCs. (B) Quantification of amino acid metabolites (LC-MS) in Normal- and RAS-STCs. (C) Protein expression of ubiquitin (Western blotting) in Normal- and RAS-STCs (Figure S1). (D) Representative transmission electron microscopy images and quantification of mitochondrial density, area, and matrix density in Normal- and RAS-STCs. (E) Mito-SOX (red) and tetramethylrhodamine ethyl ester (TMRE, green), and mitochondrial ATP generation (ATP/ADP ratio) in Normal- and RAS-STCs. * p < 0.05 vs. Normal-STCs.