Paola Ciceri1, Andrea Galassi2, Carlo Alfieri1, Piergiorgio Messa1, Mario Cozzolino3. 1. Renal Research Laboratory, Department of Nephrology, Dialysis and Renal Transplant, Fondazione Ca' Granda IRCCS, Ospedale Maggiore Policlinico, Milan, Italy. 2. Renal Division, ASST Santi Paolo e Carlo, Department of Health Sciences, University of Milan, Milan, Italy. 3. Renal Division, ASST Santi Paolo e Carlo, Department of Health Sciences, University of Milan, Milan, Italy, mario.cozzolino@unimi.it.
Abstract
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 α-actin and SM22α, 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, α-actin and SM22α respectively; Δ 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.
BACKGROUND/AIMS: Uremicpatients 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 α-actin and SM22α, 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, α-actin and SM22α respectively; Δ 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 diseasepatients significantly correlates with serum-calcific potential induced by high Pi. In addition, uremicpatients with higher VCS have sera with a higher potential to induce VSMC osteoblastic trans-differentiation, apoptosis, and necrosis.