Oxidative phosphorylation promotes vascular calcification in chronic kidney disease

Abstract

Metabolism has been reported to associate with the progression of vascular diseases. However, how vascular calcification in chronic kidney disease (CKD) is regulated by metabolic status remains poorly understood. Using a model of 5/6 nephrectomy, we demonstrated that the aortic tissues of CKD mice had a preference for using oxidative phosphorylation (OXPHOS). Both high phosphate and human uremic serum-stimulated vascular smooth muscle cells (VSMCs) had enhanced mitochondrial respiration capacity, while the glycolysis level was not significantly different. Besides, 2-deoxy-d-glucose (2-DG) exacerbated vascular calcification by upregulating OXPHOS. The activity of cytochrome c oxidase (COX) was higher in the aortic tissue of CKD mice than those of sham-operated mice. Moreover, the expression levels of COX15 were higher in CKD patients with aortic arch calcification (AAC) than those without AAC, and the AAC scores were correlated with the expression level of COX15. Suppressing COX sufficiently attenuated vascular calcification. Our findings verify the relationship between OXPHOS and calcification, and may provide potential therapeutic approaches for vascular calcification in CKD.

Fig. 1. Glucose metabolism was enhanced in calcified VSMCs and aortas.

A, B Transcriptome analysis of control or high phosphate-treated MOVAS were performed. A Volcano plot of gene expression changes. B The top ten enriched KEGG pathways in the transcripts. C ATP concentration of HASMC (unpaired t test; control n = 4, high phosphate n = 5). D ATP concentration of HASMC treated with high phosphate (one-way ANOVA; n = 4). E Relative glucose consumption of HASMC (unpaired t test; n = 5). F Calcium contents in HASMC (one-way ANOVA; n = 5). G Extracellular lactic acid concentration of HASMC (unpaired t test; n = 6). H Scheme. Mice underwent a two-step, 5/6 nephrectomy, followed by high phosphate diet for 3 months. I ATP concentration of aortas of mice (unpaired t test; sham n = 5, 5/6 nephrectomy n = 6). J Lactic acid concentration of abdominal aortas of mice (unpaired t test; sham n = 5, 5/6 nephrectomy n = 6). ***P < 0.001, ****P < 0.0001, n.s. not significant.

Fig. 2. OXPHOS was increased in high phosphate-induced calcification.

A GSEA plots of KEGG pathways showed that OXPHOS was enriched in the high phosphate group. B Heatmap of selected enriched terms from GSEA plots. C OCRs were measured in control or high phosphate-treated HASMC (n = 3). D Basal respiration, maximum respiration, proton leak, and ATP-linked respiration in OCRs were analyzed (unpaired t test; n = 3). E Relative mRNA expression of OXPHOS-related and glycolysis-related genes in HASMC as detected by RT-qPCR (unpaired t test; n = 5–6). F Relative mRNA expression of genes related to the influx of pyruvate into mitochondria (unpaired t test; n = 5–6). G Relative mtDNA content (unpaired t test; n = 5). H Measurement of the mitochondrial mass (unpaired t test; n = 4). I Measurement of the ROS level (unpaired t test; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant.

Fig. 3. OXPHOS was enhanced in uremia-related calcification.

HASMC were cultured in medium treated with serum from healthy control or uremic patients. A Total calcium content in HASMC (unpaired t test; n = 4). B Relative mRNA expression of Runx2, Sox9, ALPL in HASMC as detected by RT-qPCR (unpaired t test; n = 6). C Relative glucose consumption of HASMCs (unpaired t test; n = 5). D ATP concentration of HASMC (unpaired t test; n = 5). E Extracellular lactic acid concentration of HASMC (unpaired t test; n = 6). F OCRs were measured in HASMC (n = 3). G Basal respiration, maximum respiration, proton leak, and ATP-linked respiration in OCRs were analyzed (unpaired t test; n = 3). H Relative mRNA expression of genes related to the influx of pyruvate into mitochondria (unpaired t test; n = 5–6). I Relative mRNA expression of OXPHOS-related and glycolysis-related genes in HASMCs as detected by RT-qPCR (unpaired t test; n = 6). J Relative mtDNA content (unpaired t test; n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. not significant.

Fig. 4. 2-DG accelerated high phosphate-induced calcification by upregulating OXPHOS.

A Relative mRNA expression of OXPHOS-related genes in HASMC as detected by RT-qPCR (one-way ANOVA; n = 4–6). B Extracellular lactic acid concentration of HASMC (one-way ANOVA; n = 6). C Total calcium content in HASMC (one-way ANOVA; n = 5). D Representative images and quantification of Alizarin red staining of HASMC (one-way ANOVA; n = 4). E Relative mRNA expression of Runx2, Sox9, ALPL in HASMC as detected by RT-qPCR (one-way ANOVA; n = 4–6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5. Cytochrome c oxidase is involved in enhanced OXPHOS of calcified aortas and VSMCs.

A GSEA plots of GO pathways showed that COX was enriched in the high phosphate group. B The top ten enriched organelles of GO cellular component terms in the transcripts. C The top ten enriched GO molecular function terms in the transcripts. D COX activities measured in mice (unpaired t test; n = 5). E Immunohistochemistry of aortic COX15 expression in sham and 5/6 nephrectomy mice. F COX activities measured in HASMC treated with serum from healthy control or uremic patients (unpaired t test; n = 3). G Western blot and its quantification analysis in HASMC treated with serum from healthy control or uremic patients (unpaired t test; n = 3). H COX activities measured in HASMC treated with control or high phosphate (unpaired t test; n = 3). I Western blot and its quantification analysis in HASMC treated with control or high phosphate (unpaired t test; n = 3). *P < 0.05, **P < 0.01. Scale bar = 20 µm.

Fig. 6. Inhibition of cytochrome c oxidase attenuated high phosphate-induced calcification.

A, B Aortic rings were cultured in control or high phosphate medium with or without ADDA 5 hydrochloride supplementation. A Representative images of Alizarin red staining of aortic rings from mice. B Total calcium content in aortic rings (one-way ANOVA; n = 3). C Representative images and quantification of Alizarin red staining of HASMCs (one-way ANOVA; n = 3–5). D Total calcium content in HASMC (one-way ANOVA; n = 4). E Relative mRNA expression of Runx2, Sox9, ALPL in HASMC as detected by RT-qPCR (one-way ANOVA; n = 4). F Relative mtDNA content (unpaired t test; n = 4). G Measurement of the mitochondrial mass (one-way ANOVA; n = 4–5). H Measurement of the ROS level (one-way ANOVA; n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar = 20 µm.

Fig. 7. The expression of Cox15 is enhanced in the artery of patients with CKD.

A Representative images and quantification of immunohistochemistry staining of COX15 in arteries of patients with or without CKD (unpaired t test; Control n = 5, CKD n = 34). B Western blot and its quantification analysis in arteries of patients with or without CKD (unpaired t test; n = 4). C Comparison of the intensity of COX15 expression in CKD patients with aortic arch calcification and those without aortic arch calcification (unpaired t test; no calcification n = 6, calcification n = 23). *P < 0.05. Scale bar = 50 µm.