Deletion of soluble epoxide hydrolase suppressed chronic kidney disease-related vascular calcification by restoring Sirtuin 3 expression

Abstract

Vascular calcification is common in chronic kidney disease (CKD) and contributes to cardiovascular disease (CVD) without any effective therapies available up to date. The expression of soluble epoxide hydrolase (sEH) is different in patients with and without vascular calcification. The present study investigates the role of sEH as a potential mediator of vascular calcification in CKD. Both Ephx2^-/- and wild-type (WT) mice fed with high adenine and phosphate (AP) diet were used to explore the vascular calcification in CKD. Compared with WT, deletion of sEH inhibited vascular calcification induced by AP. sEH deletion also abolished high phosphorus (Pi)-induced phenotypic transition of vascular smooth muscle cells (VSMCs) independent of its epoxyeicosatrienoic acids (EETs) hydrolysis. Further gene expression analysis identified the potential role of Sirtuin 3 (Sirt3) in the sEH-regulated VSMC calcification. Under high Pi treatment, sEH interacted with Sirt3, which might destabilize Sirt3 and accelerate the degradation of Sirt3. Deletion of sEH may preserve the expression of Sirt3, and thus maintain the mitochondrial adenosine triphosphate (ATP) synthesis and morphology, significantly suppressing VSMC calcification. Our data supported that sEH deletion inhibited vascular calcification and indicated a promising target of sEH inhibition in vascular calcification prevention.

Fig. 1. Deletion of soluble epoxide hydrolase (sEH) inhibited high adenine and phosphate (AP)-induced mouse vascular calcification.

Ephx2^−/− and wild-type (WT) mice were fed with AP (N = 15 per AP group) or normal diet (ND) (N = 6 per ND group) for 16 weeks. At the end of the experiment, the numbers of living mice were 10 in the Ephx2^−/− AP and 6 in WT AP group while no death occurred in two ND groups. A Scheme of AP-induced chronic kidney disease (CKD) vascular calcification model. B Survival curves. C Body weights. D Serum urea. E Serum creatinine. F Serum calcium. G Serum phosphorus. H Serum alkaline phosphatase (ALP). I Alizarin Red S staining of whole mouse aortas. J Von Kossa staining and immunohistochemistry of bone morphogenetic protein-2 (BMP2) and smooth muscle 22 alpha (SM22α). K–M Representative western blot bands (K) and quantitative analysis of BMP2 (L) and SM22α (M) in VSMC. Data are presented as mean ± SD vs. WT ND: *P < 0.05; ^#P < 0.01; ^$P < 0.001; ^&P < 0.0001. B, C: Ephx2^−/− AP: N = 15; WT AP: N = 15; Ephx2^−/− ND: N = 6; WT ND: N = 6. D–H: Ephx2^−/− AP: N = 10; WT AP: N = 6; Ephx2^−/− ND: N = 6; WT ND: N = 6.

Fig. 2. Deletion of soluble epoxide hydrolase (sEH) ameliorated high phosphate (Pi)-induced vascular smooth muscle cell (VSMC) calcium deposition and phenotypic transition.

A Alizarin Red S staining of VSMCs. B The related OD value (562 nm) of Alizarin Red S staining. C Quantification of calcium contents of VSMCs. The calcium contents of each group were normalized to the related protein concentrations. Data are presented as mean ± SD. *P < 0.05 vs. wild-type (WT) 0d; ^#P < 0.01 vs. WT 0 d; ^&P < 0.0001 vs. WT 0 d. D–H Representative western blot bands (D), and the quantitative analysis of bone morphogenetic protein-2 (BMP2) (E), runt-related transcription factor 2 (Runx2) (F), alpha smooth muscle actin (α-SMA) (G), and smooth muscle 22 alpha (SM22α) (H) in VSMCs. Data are presented as mean ± SD. *P < 0.05; ^#P < 0.01; ^$P < 0.001; ^&P < 0.0001.

Fig. 3. Sirtuin 3 (Sirt3) was required in the inhibitory effect of soluble epoxide hydrolase (sEH) deletion on calcium deposition in vascular smooth muscle cells (VSMCs).

A Heatmap of all differential expression genes (DEGs) between Ephx2^−/− (EP) and wild-type (WP) VSMCs with high phosphate (Pi) treatment. B Volcano plots displaying the fold change (log2) of all genes analyzed. C Enrichment plot of Central Carbon Metabolism in Cancer gene set in gene set enrichment analysis (GSEA). D Venn diagram displaying DEGs in the GSEA gene sets. E, F Western blot analysis (A) and its quantitative analysis of Sirt3 (B) between Ephx2^−/− and wild-type (WT) VSMCs treated with high phosphate (Pi). G, H Alizarin S red staining (G) of VSMCs with scramble small interfering RNA transfection (siRNA) or Sirt3 siRNA treatment. The related staining OD values (562 nm) were shown (H). I Quantification of calcium contents of VSMCs. The calcium contents of each group were normalized to the related protein concentrations. J–L Western blot analysis and its quantitative analysis of alpha smooth muscle actin (α-SMA) (K) and bone morphogenetic protein-2 (BMP2) (L) protein expression levels in VSMCs with scramble siRNA or Sirt3 siRNA treatment, respectively, under high Pi stimulation. Data are presented as mean ± SD. *P < 0.05; ^#P < 0.01; ^$P < 0.001; ^&P < 0.0001.

Fig. 4. Soluble epoxide hydrolase (sEH) affected the degradation of Sirtuin 3 (Sirt3).

A, B High phosphate (Pi) triggered the combination of sEH and Sirt3. Cellular lysates were immunoprecipitated (IP) with anti-sEH antibody and then immunoblotted (IB) with anti-Sirt3 (A), or IP with anti-Sirt3 antibody and then IB with anti-sEH (B). C, D High Pi treatment markedly accelerated Sirt3 degradation in wild-type (WT) compared with Ephx2^−/− VSMCs. CHX indicates cycloheximide. Data are presented as mean ± SD. *P < 0.05 vs. WT 6 h. Data are presented as mean ± SD. *P < 0.05.

Fig. 5. Soluble epoxide hydrolase (sEH) deletion increased Sirtuin 3 (Sirt3)-related mitochondrial adenosine triphosphate (ATP) production and improved mitochondrial morphology.

A–C Western blot (A) and its quantitative analysis (B, C) of acetylated and total peroxisome proliferator-activated receptor-γ co-activator-1 alpha (PGC-1α) between Ephx2^−/− and wild-type (WT) VSMCs treated with high phosphate (Pi). D ATP detection in vascular smooth muscle cells (VSMCs) from Ephx2^−/− and WT with scramble small interfering RNA transfection (siRNA) or Sirt3 siRNA treatment, respectively, under high Pi stimulation. E The transmission electron microscopy (TEM) showing the fragmented mitochondrial morphology in VSMCs with scramble siRNA or Sirt3 siRNA treatment. Scale bar: 500 nm. Data are presented as mean ± SD. *P < 0.05; ^#P < 0.01; ^&P < 0.0001.

Fig. 6. Schematic cartoon showing the mechanism of soluble epoxide hydrolase (sEH)-related vascular calcification.

Under chronic kidney disease (CKD) condition, sEH interacted with Sirtuin 3 (Sirt3), leading to Sirt3 instable and degradation. This relative Sirt3 downregulation induced the increased acetylation of peroxisome proliferator-activated receptor γ co-activator-1 alpha (PGC-1α). Acetylated PGC-1α could not exert its function on gene transcription. Furthermore, the mitochondrial adenosine triphosphate (ATP) production and morphology were also impaired. All these changes triggered vascular smooth muscle cell (VSMC) phenotypic transition and calcium deposition. When sEH deletion, the sEH–Sirt3 complex could not be created and Sirt3 turned to be more stable. The sustained level of Sirt3 was able to increase the function of PGC-1α and ameliorate the mitochondrial dysfunction. As a result, VSMC phenotypic transition and calcium deposition were reduced, and vascular calcification was inhibited. TF transcription factor.