Calprotectin Accelerates Pathogenic Calciprotein Particle Maturation to Promote Vascular Calcification in Chronic Kidney Disease

BACKGROUND: Vascular calcification is a hallmark of chronic kidney disease (CKD)-mineral and bone disorder and a major determinant of cardiovascular mortality. Secondary calciprotein particles (CPP2) are key mediators of vascular calcification; however, the mechanisms regulating their maturation and pathogenic properties remain incompletely understood. CPT (Calprotectin, S100A8/A9), a myeloid-derived inflammatory mediator, is elevated in CKD and predicts cardiovascular events and mortality, particularly under high-phosphate conditions, but the underlying mechanisms remain unclear. METHODS: We investigated the role of CPT in vascular calcification using CKD-mineral and bone disorder mice with myeloid-specific CPT deletion. Aortic calcification was assessed by ex vivo imaging, histology, and calcium quantification. Serum calciprotein particle (CPP) fractions were determined by flow cytometry. Mechanistic studies were performed in vascular smooth muscle cells exposed to CPP2 in the presence or absence of macrophage-derived CPT. Calcification propensity was evaluated using the T50 assay, and CPP2 elemental composition was analyzed by high-angle annular dark-field scanning transmission electron microscopy-energy-dispersive X-ray spectroscopy. RESULTS: Myeloid-specific CPT deletion significantly attenuated aortic calcification and osteogenic gene expression in CKD-mineral and bone disorder mice without altering renal function or systemic mineral parameters. These changes were accompanied by an increased circulating immature CPP fraction. Macrophage-derived CPT markedly enhanced CPP2-induced vascular smooth muscle cell calcification by accelerating CPP maturation, reflected by a shortened T50, and by altering CPP2 composition to be enriched in calcium and phosphate. Although no direct interaction between CPT and CPPs was detected, CPT modified the physicochemical milieu, including ionized calcium and pH. Consistently, CPP2 from CPT-deficient serum exhibited substantially reduced calcification potency, indicating that CPT shapes CPP pathogenicity by modulating particle quality rather than quantity. Importantly, CPT-mediated CPP maturation occurred in a cell-free milieu, indicating a TLR4-independent mechanism distinct from its known receptor-mediated actions. CONCLUSIONS: CPT is a previously unrecognized regulator of pathogenic CPP maturation and composition, thereby promoting vascular calcification in CKD-mineral and bone disorder. Targeting CPT may represent a novel therapeutic strategy to attenuate vascular calcification in CKD.