EFFECTS OF TNF-ALPHA ON VEROCYTOTOXIN CYTOTOXICITY IN PURIFIED HUMAN GLOMERULAR MICROVASCULAR ENDOTHELIAL-CELLS

In the pathogenesis of the hemolytic uremic syndrome (HUS), endothelia l damage of glomeruli and arterioles of the kidney appears to play a c entral role. Previous studies have shown that verocytotoxin-1 (VT-1) c ytotoxicity on human vein endothelial cells require additional stimuli , in particular the inflammatory mediator tumor necrosis factor alpha (TNF alpha). In this study the effects of VT on human glomerular micro vascular endothelial cells (GMVEC) were examined. A reproducible metho d was developed for the isolation and purification of large numbers of highly purified GMVEC. The obtained GMVEC were over 99% pure; their e ndothelial origin was demonstrated by the expression of the endothelia l antigens von Willebrand factor, EN-4, PECAM-1 and V,E-cadherin. Upon stimulation with TNF alpha the cells expressed the endothelial-specif ic adhesion molecule E-selectin. A limited number of fenestral structu res was observed by scanning electron microscopy (SEM), suggesting glo merular origin of the endothelial cells. Cytotoxicity of VT-1 to GMVEC was evaluated by determination of the number of viable adherent cells and by assay of overall protein synthesis after exposure to varying c oncentrations of VT-1. In non-stimulated GMVEC, cytotoxicity of VT-1 w as inversely related to the degree and duration of confluence, subconf luent cells being the most sensitive. In highly confluent GMVEC, VT cy totoxicity required pre-exposure of the cells to the inflammatory medi ator TNF alpha, which induced an increase in the number of VT receptor s on GMVEC. Thin layer chromatography of extracted glycolipids from th e GMVEC showed binding of VT-1 to globotriaosylceramide (Gb3), known t o be the functional receptor for VT. There were no major differences i n protein synthesis inhibition with equal concentrations VT-1 and VT-2 . In conclusion, in this study we provide a reproducible method to iso late, purify and culture well characterized human GMVEC on a routine b asis. In vitro studies with these GMVEC demonstrate that VT cytotoxici ty depends on the degree of confluence and the additional preexposure to the inflammatory mediator TNF alpha. These observations provide fur ther insight into the complex events that may occur in glomeruli in th e pathogenesis of HUS.