Vitamin D alleviates indoxyl sulfate-induced inflammatory and cholesterol dysregulation in macrophages: Implications for vascular health in patients receiving hemodialysis

Indoxyl sulfate (IS) is a protein-bound uremic toxin that accumulates in patients with chronic kidney disease (CKD) and promotes oxidative stress, endothelial dysfunction, vascular smooth muscle cell proliferation, and fibrosis, thereby contributing to vascular stenosis in patients receiving hemodialysis. CKD is also commonly associated with vitamin D deficiency, which is linked to vascular calcification, immune dysregulation, and inflammation. This study aims to investigate the preventive effects of 1,25(OH)2D3 (active vitamin D) against IS-induced macrophage inflammatory activation and cholesterol dysregulation. Macrophages were pretreated with 30 nM 1,25(OH)2D3 for 12 and 24 h, followed by exposure to IS at concentrations of 125 and 250 µg/mL for 24 h. Morphological changes were observed under a microscope. To assess macrophage phenotype-associated changes, qPCR was performed to analyze the expression of M1-like/pro-inflammatory markers (TNF-α and IL-1β) and M2-like/anti-inflammatory phenotype-associated markers (CD163 TGF-β and IL-10). Cholesterol metabolism was assessed using a cholesterol efflux assay, qPCR analysis of ABCA1 and ABCG1 and Oil Red O staining for intracellular lipid accumulation. Protein expression of inflammatory mediators, TGF-β1, and cholesterol efflux transporters was further evaluated by western blotting. 1,25(OH)2D3 pretreatment modulated IS-associated inflammatory responses, as reflected by changes in selected M1-like/pro-inflammatory mediators, including iNOS, IL-6 and IL-1β. Conversely, 1,25(OH)2D3 increased selected M2-like/anti-inflammatory phenotype-associated markers, including CD163 and IL-10. Furthermore, 1,25(OH)2D3 pretreatment preserved cholesterol efflux capacity and modulated ABCA1 and ABCG1 expression in a time- and transporter-dependent manner, accompanied by reduced intracellular lipid accumulation as shown by Oil red O staining. These findings suggest that 1,25(OH)2D3 may protect macrophages against IS-induced inflammatory activation and cholesterol dysregulation, highlighting its potential as a preventive or modulatory approach for macrophage-mediated vascular dysfunction in CKD.