Endothelial progenitor cells, uraemic toxins, and the development of endothelial dysfunction in chronic kidney disease.

Abstract

Morbidity and mortality rates for cardiovascular disease (CVD) are increased among end stage kidney disease (ESKD) patients receiving dialysis treatment, and not corrected with kidney transplantation (KTx). Classic CVD risk factors do not fully predict the increased risk, with novel factors causing endothelial dysfunction (ED), leading to arteriosclerosis, congestive heart failure (CHF) and sudden death, key to disease pathogenesis. These novel factors include bone marrow (BM) derived endothelial progenitor cells (EPCs), which have key roles in maintenance, repair and growth of the endothelium. There is limited data about the role of EPCs and CVD in the ESKD population. This uraemic milieu includes p-cresol (sulfate, PC/S) and indoxyl sulfate (IS), toxins associated with CVD in ESKD. In this thesis, the relationship between CVD and ESKD, and the potential role of EPCs and uraemic toxins was examined from epidemiological, clinical and laboratory perspectives. Data was obtained for the period between 2002-2007 for all hospital separations in Australia. Analysis was performed based on ICD-9/10 coding. This showed (for the first time in an Australian population): (i) an increase in risk for CVD hospital separations among dialysis and KTx, with higher rates for CHF than acute cardiac events (ACE); (ii) an advantage for KTx recipients in regards to ACE, but not CHF hospital separations, over dialysis recipients, and (iii) for CHF, no increase in in-hospital mortality, or length of stay per separation for any ESKD group compared to controls. At a clinical level, in groups of haemodialysis (HDx), KTx patients and controls, low peripheral blood (PB) EPC numbers were correlated with surrogate markers of CVD and ED. No clear relationship of IS and PC/S with ED was seen (although study power was limited). For in vitro studies, techniques were developed for isolation (Flow sort and AutoMACS), enumeration (FACS) and culture expansion of EPCs from BM and umbilical cord blood samples. The effects of uraemic serum and toxins PC and IS on cultured endothelial cells (ECs) and EPCs in vitro was examined, as a model of vascular pathology in ESKD. Greater HUVEC VCAM-1 expression and reduced tube formation in Matrigel were observed in response to increasing PC concentration than IS. The effect of IS (but not PC) at higher concentration in Matrigel was reduced by the addition of EPCs. Akt/ERK expression by western blot, cell migration to VEGF, and supernatant investigation by FlowCytoMix for soluble cell surface markers, were also performed. Testing of HUVEC function post-exposure to sera from control, transplant and HDx recipients did not replicate the above results on the basis of sera PC and IS levels. In summary, this thesis has explored the increased burden of CVD in ESKD patients in Australia, the relationship of EPCs, both in vivo and in vitro, to vascular disease in this setting, and the role of uraemic toxins as agents for CVD. These results underline why certain therapies may not be effective in the ESKD population for CVD prevention, and suggest novel approaches are needed.