Calprotectin Plays an Important Role in the Pathogenesis of Vascular Calcification in CKD

Background: Cardiovascular complications are a major contributor to global mortality rates, with atherosclerosis and vascular calcification (VC) being significant factors. In particular, patients with CKD experience a higher prevalence of VC as their renal function deteriorates, well-known as CKD-MBD, leading to increased mortality rates. Calciprotein particles (CPPs) are colloidal nanoparticles that play a crucial role in the initiation and progression of VC, partly through TLR4 signaling. We recently reported that serum calprotectin (CPT, also known as MRP8/14 or S100A8/A9) levels, which amplify TLR4 signaling, have been found to have a predictive role in mortality among hemodialysis patients with high phosphatemia. The aim of this study is to clarify the pathogenic role of CPT on the cardiovascular calcification involving CPPs in CKD patients. Methods: Secondary CPP (CPP2) was generated using a phosphate-enriched culture medium (DMEM/10% FBS) incubated at 37°C. Vascular smooth muscle cells (VSMCs) were stimulated by CPP2 concomitant with recombinant CPT or cultured supernatant of macrophages (RAW264.7) treated with LPS. To characterize the significant role of CPT in the calcification of VSMCs, RNA interference experiments using siMRP8 were performed. In in vivo experiment, the CKD-MBD model was developed in myeloid-lineage cell-specific MRP8 KO mice (MyMRP8KO) using adenine combined with a high-phosphate diet. Results: In VSMCs, the addition of cultured medium from LPS-stimulated RAW264.7, which contains markedly increased CPT, aggravated calcification and Ca2+ deposition induced by CPP2. This phenotype was associated with the amplified expressions of pro-osteogenic and -inflammatory genes, which were effectively suppressed by CPT knockdown with siMRP8. In the CKD-MBD model, the aortic calcification was ameliorated in MyMRP8KO compared to WT. Conclusion: Our study suggested that the inflammation can enhance the calcification through CPT. Inflammation-mediated release of CPT from macrophages exacerbates CPP2-induced VC. The pathogenic role of CPT in CPP2-induced calcification might support our previous cohort study in hemodialysis patients with high phosphatemia. CPT might become a novel therapeutic target to protect against VC in patients with CKD-MBD.