Background: Patients afflicted with chronic kidney disease (CKD) typically suffer from cardiovascular disease (CVD) which is a leading cause of patient mortality. It has been demonstrated that two distinct physiological events contribute to this disease state. These include the abundance of abnormally high levels of protein-bound uraemic toxins as well as functionally aberrant endothelial progenitor cells (EPCs). Specifically, it has been demonstrated that the uraemic toxin p-cresol (pC; 4-methylphenol) inhibits EPC proliferation and tube formation in previous in vitro studies. More recently, however, it has been demonstrated that circulating pC is actually conjugated and that p-cresylsulphate (pCS) is its main metabolite. Therefore, within the context of this study, we examined the in vitro effects of pC and pCS treatment on cultured human EPCs.
Methods: Late-outgrowth EPCs were treated with physiological concentrations of pC or pCS (10, 40, 80, and 160 or 10, 40, 80, 160 and 320 µg/mL for up to 72 h, respectively) in the presence of 4% human serum albumin (HSA). Cell proliferation was determined using WST-1 assay, while migration and tube formation assays were used to evaluate EPC function in vitro. Cell cycle analyses were also performed to determine the effects of pC and pCS on cell cycle status.
Results: With regard to EPC proliferation, data demonstrate that pC in the presence or absence of HSA had an IC50 of 80.1 and 100.8 µg/mL 72 h post-treatment, respectively, while pCS-treated groups did not impair EPC proliferation. Similarly, pC-treated groups showed limited vessel formation and migration compared with controls and no detrimental effects were seen with pCS treatment. Lastly, pC treatment of EPCs caused cells to accumulate in the G2/M phase of the cell cycle with accompanied down-regulation of cyclin B1 and phosphorylated CDK1. pCS had no effect on cell cycle parameters.
Conclusions: Our data demonstrate that pC and pCS have different effects on EPC function. Since there is a dearth of data that have focused on the toxicity of pCS, further research should be performed to determine the exact biological toxicity of pCS on the cardiovascular system.