Nephrol Dial Transplant (2003) 18: 1079-1085
© 2003 European Renal Association-European Dialysis and Transplant Association
Uraemic medium accelerates proliferation but does not induce apoptosis of endothelial cells in culture
1 Servicios de Hemoterapia-Hemostasia and 2 Nefrología, Hospital Clínic, IDIBAPS, Barcelona, Spain
Background. Chronic renal failure patients exhibit accelerated atherosclerosis, which is associated with a high incidence of cardiovascular death. We investigated the potential effect of uraemic medium on cell proliferation and apoptosis of endothelial cells in culture (ECs), two key processes in the development of atherosclerosis. Phosphorylation kinetics of the mitogen-activated protein kinase (MAPK) p42/44 and p38 were also evaluated.
Methods. ECs were cultured with growth media supplemented with pooled sera from healthy donors. Semiconfluent ECs were incubated for 24 h with media supplemented with pools of control or uraemic sera. Cell proliferation was assessed through morphometric analysis and by flow cytometry evaluation of cell cycle. To investigate if uraemic medium induces apoptosis in ECs, we used a combination of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay and activation of caspase-3 using flow cytometry. Changes in the phosphorylation levels of MAPK were evaluated in cell lysates by western blotting.
Results. Exposure to uraemic media caused an alteration in the morphology of ECs, showing irregular shape and size. The number of ECs at S+G2M phase in the cell cycle was found to be increased when exposed to uraemic media for 24 h (28.4±2.9 vs 20.2±2.6% in control ECs). There was a transient increase in levels of phosphorylation of MAPK in both cells, although these levels were significantly higher in ECs exposed to uraemic media, especially after 5 min. In contrast, no signs of apoptosis were observed in ECs incubated with uraemic medium at the conditions applied.
Conclusions. Under our experimental conditions, uraemic medium accelerates proliferation of ECs, but it does not seem to induce apoptosis. The increased proliferation observed could be related to a higher MAPK activity in these cells. Although the enhanced atherosclerosis cannot be explained on the basis of an apoptotic process, the proliferative status could contribute to intimal proliferation, which is considered to be an earlier step in the development of atherosclerosis.
Keywords: apoptosis; atherothrombosis; cell cycle; endothelial cells; MAPKs; uraemia
Correspondence and offprint requests to: Mireia Serradell Abel, PhD, Servicio de Hemoterapia y Hemostasia, Hospital Clínic, Villarroel 170, E-08036 Barcelona, Spain. Email: mpino{at}clinic.ub.es