Phosphate as a cardiovascular risk factor: effects on vascular and endothelial function

Stevens, Kathryn K. (2014) Phosphate as a cardiovascular risk factor: effects on vascular and endothelial function. PhD thesis, University of Glasgow.

Abstract

Chronic kidney disease (CKD) is prevalent affecting 8.5% of the population in the UK and it is associated with premature cardiovascular disease (CVD) and death. The association between CKD and accelerated CVD arises as a consequence of traditional and non-traditional cardiovascular (CV) risk factors, including serum phosphate. Currently, there is no therapeutic intervention which has been shown to effectively reverse the increased CV risk in CKD. Phosphate metabolism is disordered in CKD particularly in the advanced stages (CKD 4 and 5). Even at the upper limit of the normal reference range, serum phosphate has been shown to be associated with CV mortality and morbidity including left ventricular hypertrophy, vascular calcification (VC) and endothelial dysfunction (ED). These associations also extend to populations without CKD. Serum phosphate is an appealing CV risk factor because it can be modified by dietary and pharmacological therapies. However, there has been no study linking lowered phosphate with improved outcomes and whilst several small mechanistic studies have suggested a role of phosphate in VC, oxidative stress and more recently ED, the precise mechanism of action of phosphate as a CV risk factor remains elusive. Our lack of understanding of the mechanism of action of phosphate makes it difficult to ascertain how to best manage phosphate.

The hypothesis of this thesis is that long term exposure to elevated phosphate is associated with endothelial and vascular dysfunction and it is this which contributes to the elevated CV risk seen in CKD. Endothelial and vascular dysfunction will be evident in individual cell lines and in blood vessels as well as in humans, who have been exposed to sustained oral phosphate. This hypothesis has been explored in a translational manner in three ways:

1. The function of resistance vessels from rats and from humans, with and without CKD, has been studied. Experiments were performed looking at endothelium dependent and independent relaxation of vessels exposed to either normal or high phosphate concentration medium. The anti-oxidant, ascorbic acid and zaprinast, a phosphodiesterase 5 inhibitor were studied in the rat vessels to assess if these additions altered the vessels’ relaxation response.

2. Cells were cultured in normal and high phosphate concentration medium from the outset to mimic the chronicity of the uraemic environment. The nitric oxide (NO) pathway was studied considering eNOS expression, VEGF and cGMP production, intracellular calcium concentration and NO production. Proliferation and cell growth pathways have also been studied.

3. A cross-over clinical trial was performed in 19 healthy volunteers, without CKD. Volunteers attended for three visits. Prior to each visit, they fasted for 12 hours and performed a 24 hour urine collection. Bloods were drawn and endothelial function was measured with flow mediated dilatation and vascular stiffness with pulse wave velocity and analysis. There was a baseline visit and then two further visits. Prior to visit two, volunteers were randomised to take phosphate supplementation or phosphate binding medication for two weeks, followed by a wash out period and then volunteers took the other tablet for two weeks, before attending for a final visit.

In rat vessels, there was impaired endothelium dependent and independent relaxation in vessels exposed to high phosphate concentration medium. Vessels in high phosphate produced less basal NO and less cGMP. The impaired relaxation could be ameliorated with the addition of a phosphodiesterase 5 inhibitor. This suggests reversibility of the detrimental effects of phosphate. In human vessels from patients without CKD, there was similarly attenuated endothelium dependent and independent relaxation. In vessels from patients with CKD, there was impaired endothelium dependent relaxation but independent relaxation was preserved. The CKD vessels exposed to normal phosphate medium relaxed to the same degree as their counterparts from patients without CKD, again suggesting that the effects of phosphate may be reversible. These effects are independent of intracellular calcium concentration which was found to be similar in cells cultured in normal or high phosphate medium. There was evidence of disruption to the NO pathway with reduced eNOS expression in human and rat endothelial cells and reduced protein kinase G expression in vascular smooth muscle cells. NO measured by the Griess reaction was lower in cells cultured in high phosphate medium. NO has an inhibitory effect on growth and cells cultured in high phosphate proliferated more (measured with the MTT assay) and were bigger than cells cultured in normal phosphate medium. Gene expression studies showed alterations in growth genes and cell cycle regulators.

ED was demonstrated in healthy volunteers exposed to sustained oral phosphate loading. This was independent of serum phosphate level which was unchanged. Urinary phosphate and fibroblast growth factor 23 level independently predicted ED and suggest that whilst the normal homeostatic mechanisms maintain serum phosphate within the normal reference range, total body phosphate was elevated and urinary phosphate excretion was a surrogate for this. These relationships are novel and have not been demonstrated previously.

It has previously been difficult to separate the effects of phosphate from other effects of the uraemic environment, including acidosis. The studies in this thesis have achieved this and the results provide strong evidence of an association between phosphate and ED. There is also evidence that these effects may be reversible. In contrast to conventional thinking that the effects of phosphate, like VC, are largely irreversible, these studies suggest that there may be dynamic effects of phosphate. This may be explained by alterations in intracellular phosphate. These findings have important implications for patients with CKD because they provide a sound explanation for the increased CV risk seen with phosphate and advocate further study of phosphate lowering (and outcome) as a therapeutic strategy to reverse this elevated CV risk.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Phosphate, chronic kidney disease, cardiovascular risk, endothelium, endothelial dysfunction.
• Subjects: R Medicine > R Medicine (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Supervisor's Name: Jardine, Professor Alan G. and Delles, Dr. Christian
• Date of Award: 2014
• Depositing User: Dr Kathryn Stevens
• Unique ID: glathesis:2014-5301
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 Jun 2014 14:14
• Last Modified: 29 Jun 2017 15:38
• URI: https://theses.gla.ac.uk/id/eprint/5301