Uremic Patients with Increased Vascular Calcification Score Have Serum with High Calcific Potential: Role of Vascular Smooth Muscle Cell Osteoblastic Differentiation and Apoptosis

Background/Aims: Uremic patients experience premature vascular ageing that causes cardiovascular morbidity. In this study, we investigated the relationship between uremic serum calcific potential induced by high phosphate (Pi) and vascular calcification score (VCS). Methods: Vascular smooth muscle cells (VSMCs) were cultured with 3.5 mM Na3PO4 (Pi) with 10% uremic serum and calcium deposition, markers of osteoblastic transformation, and apoptosis were evaluated. Results: Culture with uremic serum and high-Pi significantly induced calcification (0.21 +/- 0.03 vs. 8.05 +/- 0.6; ctr vs. Pi; OD/mg protein; p < 0.01). We next stratified patients with respect of the degree of VCS in 2 groups: absence of vascular calcification (VC) no VC group and presence of VC VC group. We found that there was a significant correlation between VCS and uremic serum calcific potential induced by high Pi in vitro (p < 0.01). Interestingly, uremic sera of the VC group were more effective than sera from the no VC group, in downregulating alpha-actin and SM22 alpha, after treatment with high-Pi (41.3 +/- 4.7 vs. 23.3 +/- 2.9 and 25.6 +/- 6.8 vs. 8.14 +/- 2.3; VC vs. no VC group, alpha-actin and SM22 alpha respectively; Delta intensity area; p < 0.01). Similarly, sera from VC group were more effective than sera from no VC group in adjuvanting the high-Pi effect of increasing osteoblastic markers, such as bone morphogenic protein 2 (BMP2), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2; 39.1 +/- 11.3 vs. 5.0 +/- 2.6 BMP2; 12.2 +/- 4.2 vs. 1.7 +/- 0.3 OC; 2.9 +/- 0.4 vs. 1.2 +/- 0.2 RUNX2; VC vs. no VC group respectively; p < 0.05). We found a similar pattern with significantly higher apoptosis and necrosis induction by sera from the VC group compared to the no VC group (2.05 +/- 0.33 vs. 1.29 +/- 0.13 and 54.1 +/- 19.5 vs. 27.4 +/- 10.6; Pi; VC group vs. no VC group; enrichment factor of apoptotic or necrotic fragments, respectively; p < 0.05). Conclusions: We conclude that VCS of end-stage renal disease patients significantly correlates with serum-calcific potential induced by high Pi. In addition, uremic patients with higher VCS have sera with a higher potential to induce VSMC osteoblastic trans-differentiation, apoptosis, and necrosis.