Pathogenic Role of Calprotectin on Vascular Calcification in CKD

Cardiovascular complications, particularly vascular calcification (VC), are major contributors to global mortality. In patients with chronic kidney disease (CKD), the prevalence of VC increases as renal function declines, a condition known as CKD–mineral and bone disorder (CKD-MBD), which markedly elevates cardiovascular risk and mortality. Calciprotein particles (CPPs), colloidal nanoparticles formed by calcium-phosphate complexes and serum proteins, play a crucial role in the initiation and progression of VC. We recently reported that serum calprotectin (CPT, also known as MRP8/14) may become a significant predictor of mortality in CKD patients with hyperphosphatemia. This study aims to elucidate the pathogenic role of CPT on cardiovascular calcification induced by CPPs in CKD-MBD, and also on CPPs formation.

CKD-MBD model was developed in myeloid lineage–specific MRP8 knockout (MyKO) mice, lacking circulating CPT, induced by adenine administration combined with a high-phosphate diet. Serum and kidney were collected to evaluate the severity of CKD-MBD. Aortic calcification was analyzed by q-PCR and Osteo680 fluoroscopic ex-vivo imaging. Secondary CPPs (CPP2) were generated by incubating calcium and phosphate-enriched culture medium (DMEM with 10% FBS) at 37°C. Vascular smooth muscle cells (VSMCs) were stimulated with CPP2 together with enriched CPT-containing cultured supernatant, collected from LPS-treated macrophages (RAW264.7). To investigate the specific role of calprotectin (CPT) in VSMC calcification, the experiments using siMRP8 and TAK242 (inhibitor of TLR4) were performed. The elemental composition of CPP2 prepared with or without CPT was analyzed by scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy (STEM-EDX). Calcification propensity (T50) was determined by measuring absorbance at 570 nm to assess the time course of CPP formation.

 In the CKD-MBD model, renal function was comparable between WT and MyKO mice. MyKO showed significantly attenuated aortic calcification and lower levels of serum CPP compared with WT. Ex-vivo imaging revealed the macrophage accumulation in the calcified lesion. In vitro, VSMC calcification and Ca²⁺ deposition induced by CPP2 were exacerbated by CPT-enriched cultured supernatant from LPS-stimulated macrophages. These effects, along with the upregulation of pro-osteogenic and pro-inflammatory genes, were effectively suppressed by CPT knockdown using siMRP8 or TAK242. Furthermore, CPT shortened the T50, indicating accelerated CPP2 formation, and STEM-EDX analysis revealed the higher calcium and phosphate content in CPP2 prepared with CPT.

Our findings indicate that inflammation-driven release of CPT from macrophages exacerbates CPP2-induced vascular calcification. Notably, we demonstrated for the first time that CPT promotes calcification by accelerating CPP2 formation. The pathogenic role of CPT in CPP2-mediated calcification aligns with our previous clinical observations in hemodialysis patients with hyperphosphatemia. These results suggest that CPT may serve as a novel therapeutic target for preventing vascular calcification in CKD–MBD.