Hyperphosphatemia Drives Procoagulant Microvesicle Generation in the Rat Partial Nephrectomy Model of CKD.

Nima Abbasian,Debbie Bursnall,Alan Bevington,Nigel J Brunskill,James O Burton,Alison H Goodall

doi:10.3390/jcm9113534

Abstract

Hyperphosphatemia has been proposed as a cardiovascular risk factor, contributing to long-term vascular calcification in hyperphosphatemic Chronic Kidney Disease (CKD) patients. However, more recent studies have also demonstrated acute effects of inorganic phosphate (Pi) on endothelial cells in vitro, especially generation of pro-coagulant endothelial microvesicles (MV). Hitherto, such direct effects of hyperphosphatemia have not been reported in vivo. Thirty-six male Sprague-Dawley rats were randomly allocated to three experimental groups: (1) CKD induced by partial nephrectomy receiving high (1.2%) dietary phosphorus; (2) CKD receiving low (0.2%) dietary phosphorus; and (3) sham-operated controls receiving 1.2% phosphorus. After 14 days the animals were sacrificed and plasma MVs counted by nanoparticle tracking analysis. MVs isolated by centrifugation were assayed for pro-coagulant activity by calibrated automated thrombography, and relative content of endothelium-derived MVs was assessed by anti-CD144 immunoblotting. When compared with sham controls, high phosphorus CKD rats were shown to be hyperphosphatemic (4.11 ± 0.23 versus 2.41 ± 0.22 mM Pi, p < 0.0001) with elevated total plasma MVs (2.24 ± 0.37 versus 1.31 ± 0.24 × 108 per ml, p < 0.01), showing increased CD144 expression (145 ± 25% of control value, p < 0.0001), and enhanced procoagulant activity (18.06 ± 1.75 versus 4.99 ± 1.77 nM peak thrombin, p < 0.0001). These effects were abolished in the low phosphorus CKD group. In this rat model, hyperphosphatemia (or a Pi-dependent hormonal response derived from it) is sufficient to induce a marked increase in circulating pro-coagulant MVs, demonstrating an important link between hyperphosphatemia and thrombotic risk in CKD.

Highlights

Renal function inversely correlates with cardiovascular mortality in humans [1]
We previously showed that applying elevated extracellular Pi concentration to cultured endothelial cell (EC) is sufficient to trigger their rapid release [9] through direct inhibition by Pi of the phosphoprotein phosphatase PP2A in ECs, culminating in cytoskeleton disruption and MV generation [10], an effect which may explain the pro-coagulant endothelial MVs previously reported in Chronic Kidney Disease (CKD) patients [14]
Animals in all three experimental groups were successfully matched for food consumption and showed no significant difference in final body weight (Figure 1i)

Summary

Introduction

Renal function inversely correlates with cardiovascular mortality in humans [1]. Elevation of plasma inorganic phosphate (Pi) (hyperphosphatemia) in Chronic Kidney Disease (CKD) is thought to be an important contributor to this, partly because of Pi’s effect on calcium deposition, resulting in vascular calcification [2,3,4,5,6,7]. Endothelial effects are of particular interest because CKD patients have been shown to have elevated circulating concentrations of pro-coagulant microvesicles (MVs) derived from endothelial cells, leading to a prothrombotic state, which may contribute to acute occlusive events [14,15,16]. MVs are submicron diameter vesicles shed from several cell types, notably platelets and vascular endothelial cells, following apoptosis or cellular activation [9,10,17,18]. They occur in plasma of healthy subjects, but their abundance, both from platelets and ECs, has been shown to increase in CKD patients [14]. A direct pro-coagulant effect of hyperphosphatemia has not been reported in vivo

Methods

Results

Discussion

Conclusion