Phosphate: a novel risk factor for cardiovascular disease and CKD progression

and Aims: Elevated serum phosphate is an independent risk factor for cardiovascular disease. Whether this is a direct effect of elevated phosphate or dependent on changes in intracellular calcium or calcium/phosphate product is unknown. We examined the direct effects of phosphate concentration in human resistance vessels and human umbilical vein endothelial cells (HUVECs). Methods: Surplus adipose tissue was removed from patients with chronic kidney disease (CKD) stage 5 undergoing live donor transplantation and their normal donors. Resistance vessels were dissected and incubated in a physiological saline solution (PSS) with normal (1.18mM) or high phosphate concentration (2.5mM) for 16 hours, then mounted on a myograph. Vasoconstrictor responses to phenylepherine (PE) and vasorelaxation responses to carbachol and sodium nitroprusside (SNP) were measured. Concentration-response curves were constructed for PE, carbachol and SNP. Area under the curve (AUC) was calculated and comparisons were made using either a t test or an ANOVA. HUVECs were grown in normal (0.5mM) and high (3mM) phosphate medium. eNOS and nitrotyrosine expression were measured by Western blot and intracellular calcium concentration measured by epifluorescence with FURA 2 AM. Gene expression was studied with PCR. Results: Vessels from patients with and without CKD incubated in high phosphate relax less well to carbachol (p<0.05). Vessels from patients without CKD relax less well to SNP (p<0.05); this difference is not seen in vessels from patients with CKD. Expression of total and phospho eNOS was reduced in HUVECs grown in high phosphate whilst nitrotyrosine expression was increased. Calcium concentration was not significantly different between HUVECs grown in high and normal phosphate. Genes involved in the cell cycle and growth were upregulated and expression of the phosphate transporters PiT1 and PiT2 was unchanged. HUVECs express Klotho and the FGF 1 receptor. Conclusions: Elevated phosphate decreases endothelium dependent vasodilatation in patients with and without CKD. This may be a marker of endothelial dysfunction, supported by the reduced eNOS protein expression and increased nitrotyrosine expression seen in HUVECs. Elevated phosphate also impairs endothelial independent relaxation in vessels from healthy patients without CKD. In vessels from healthy patients, elevated phosphate may alter cyclic GMP production and guanylate cyclase expression. These experiments indicate direct effects of elevated phosphate on the NO system, and on vascular function, and support the notion that phosphate has direct effects in uremia.