Calcification potential of plasma from patients with chronic kidney disease

Ashish Patidar1, Dhruv Singh3, Peter Winocour2, Ken Farrington3, Anwar Baydoun1. 1University of Hertfordshire, Hatfield, United Kingdom, 2QEII Hospital, Welwyn Garden City, United Kingdom, 3Lister Hospital, Stevenage, United Kingdom.

Vascular calcification (VC) is a complex but regulated process of bio mineralization that resembles the differentiation of vascular smooth muscle cells to osteoblast like cells. This condition is normally associated with renal and/or cardiovascular disease where calcification leads to organ failure with detrimental consequences. Several different, non-mutually exclusive mechanisms including loss of inhibitors of tissue mineralization or the presence of osteogenic processes have been implicated in the calcification process. Which of these mechanisms predominate in various disease states remains unclear. We have therefore initiated studies to address these issues and have started by examining, in an in vitro vascular smooth muscle cell model, the calcification potential of plasma from patients in different stages of CKD. Ethics approval for the use of the latter was granted by the Hertfordshire Research Ethics Committee.

In our studies, vascular calcification was induced in rat cultured aortic smooth muscle cells (RASMCs) using a buffer consisting of calcium chloride (7 mM) and β-glycerophosphate (7 mM). Controls were incubated in culture medium alone. To determine the optimum calcification period, cells were incubated with the calcification buffer (CB) for various time points up to 7 days. Plasma samples were obtained from 6 predialysis patients with stage 4 and 5 CKD (Group I) and 6 patients requiring haemodialysis (Group II). The plasma was added to the culture medium at a concentration of 10 %. Calcification was determined using the calcium DICA-500 assay kit (Bioassay, Cambridge, UK) and confirmed microscopically for deposition of octacalcium phosphate. Statistical analysis was performed where relevant using one way ANOVA followed by a post-hoc Tukey test.

The time course studies indicate that optimum calcification occurred after 5 days of incubation of cells in CB, declining thereafter as a result of increased cell death. Calcification was evident as elevated calcium levels (1.3 Ca+2μg μg Protein-1) in cell lysates at day 5 and as deposition of octacalcium phosphate crystals in the cell monolayers. Inclusion of 10 % plasma sample from CKD subjects caused an increase in calcification. This was more pronounced with plasma from Group II patients when compared to those in Group I. In the former, calcium levels were in the range of 0.38 Ca+2μg μg Protein-1 compared to 0.2 Ca+2μg μg Protein-1 in Group I. These preliminary findings show clearly, and for the first time, that plasma from patients on haemodialysis has significantly higher calcification potential when compared to those in the predialysis phase. Moreover, the data suggest the presence of factors in the circulation of CKD patients which can induce calcification even in the absence of the pathological milieu. Further studies with bigger patient groups are required to confirm our findings and elucidate the underlying mechanisms associated with the calcification potential in patients with CKD.