Deficient hemostasis coexists paradoxically with accelerated atherosclerosis in patients with cronic renal failure (CRF). The high incidence of aterothrombotic events in these patients has been associated with endothelial dysfunction. We have established an in vitro model of cell dysfunction by exposing endothelial cells (EC) to uremic media (

Serradell y cols, Thromb Haemost 86:1099,2001
). However, it is difficult to advance in this research using conventional protein identification techniques. We have applied a proteomic approach to exhaustively characterize the EC dysfunction in uremia. Extracts of confluent EC, grown in the presence of sera from control donors and from patients under hemodialysis, were collected and lysated. Proteins were resolved by molecular weigh (Mr range 10–50kDa) and isoelectric point (pI range 3–10) using two-dimensional gel electrophoresis (2-DE). Spots were visualized by silver staining and further identified by mass spectrometry (MALDI-TOF). A search for significant changes in protein phosphorylation and glycosilation, and overexpression or new presence of proteins was specifically performed in uremic EC. Identification of some of the most prominent proteins revealed molecules related to inflammation (HMGB1, 25148/5.8, Mr/pI; aldose reductase, 35854/6.5; a proteasome component, 29192/5.7) and oxidative stress (superoxide dismutase, 24772/8.3; gluthatione peroxidase, 21899/6.2), both processes associated with CRF. In addition, we observed a significant increase in the protein destrin (18506/8.1), an actin depolymerizing protein. This finding could be related to the previously reported increased detachment of uremic EC when exposed to flowing blood. Therefore, proteomics can be used to compare the proteome of control and uremic EC, helping to reveal information on the molecular basis of the disease. A more exhaustive analysis of 2-DE gels will provide answers and potential therapeutic targets in a short future.

Topics:
endothelial dysfunction, uremia, gel, aldehyde reductase, atherosclerosis, electrophoresis, gel, two-dimensional, hemodialysis, hemostasis procedures, hmgb1 protein, inflammation

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2005, The American Society of Hematology
2005
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