Abstract

Background:As a predominant complication of chronic kidney disease (CKD), vascular calcification (VC) warrants investigation into its mechanisms. The epigenetic modulator Enhancer of Zeste Homologue 2 (EZH2) is implicated in various kidney disorders. This investigation aims to examine its function in the development of VC related to CKD.Methods:Radial arteries collected from individuals with kidney failure (with or without calcification) following arterial venous fistula surgery were processed for Von Kossa and immunohistochemical (IHC) staining. A VC- associated CKD model was established by administering a diet containing adenine and phosphate (1.2%) and 3-deazaneplanocin A (3-DZNeP) was used to block EZH2 activity. To induce calcification, thoracic aortas isolated from SD rats were cultured ex vivo in a medium containing 2.6 mmol/L Pi. In parallel, an in vitro model of calcification was established by treating A7r5, a rat vascular smooth muscle cell line (VSMC), with a calcifying medium, and 3-DZNeP was used to inhibit EZH2. Adenovirus was used to overexpress EZH2 and siRNA was used to silence bone morphogenetic protein (BMP) 2.Results:EZH2 protein expression was higher in calcified arteries than in non-calcified arteries from individuals after the surgical creation of an arteriovenous fistula. In the mouse model, the upregulation of EZH2 and Runx2 at both the transcriptional and translational levels was demonstrated in calcified aortas. 3-DZNeP treatment decreased VC and transcriptional levels of EZH2. The ex vivo experiments revealed that high Pi stimulation significantly enhanced EZH2 expression and its histone mark, H3K27me3 (methylation of Histone H3 at lysine 27). This epigenetic alteration was accompanied by an elevation in osteogenic markers and a reduction in the expression of VSMC differentiation markers. In vitro, EZH2 and H3K27me3 were noticeably elevated in calcifying VSMCs. Mechanistically, overexpression of EZH2 aggravated the VSMCs’ calcification