Effects of TNFα on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells

Abstract

In the pathogenesis of the hemolytic uremic syndrome (HUS), endothelial damage of glomeruli and arterioles of the kidney appears to play a central role. Previous studies have shown that verocytotoxin-1 (VT-1) cytotoxicity 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 microvascular endothelial cells (GMVEC) were examined. A reproducible method was developed for the isolation and purification of large numbers of highly purified GMVEC. The obtained GMVEC were over 99% pure; their endothelial origin was demonstrated by the expression of the endothelial antigens von Willebrand factor, EN-4, PECAM-1 and V,E-cadherin. Upon stimulation with TNF alpha the cells expressed the endothelial-specific adhesion molecule E-selectin. A limited number of fenestral structures was observed by scanning electron microscopy (SEM), suggesting glomerular 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 concentrations of VT-1. In non-stimulated GMVEC, cytotoxicity of VT-1 was inversely related to the degree and duration of confluence, subconfluent cells being the most sensitive. In highly confluent GMVEC, VT cytotoxicity required pre-exposure of the cells to the inflammatory mediator TNF alpha, which induced an increase in the number of VT receptors on GMVEC. Thin layer chromatography of extracted glycolipids from the GMVEC showed binding of VT-1 to globotriaosylceramide (Gb3), known to be the functional receptor for VT. There were no major differences in protein synthesis inhibition with equal concentrations VT-1 and VT-2. In conclusion, in this study we provide a reproducible method to isolate, purify and culture well characterized human GMVEC on a routine basis. In vitro studies with these GMVEC demonstrate that VT cytotoxicity depends on the degree of confluence and the additional preexposure to the inflammatory mediator TNF alpha. These observations provide further insight into the complex events that may occur in glomeruli in the pathogenesis of HUS.

Change in Conformation with Reduction of α-Helix Content Causes Loss of Neutrophil Binding Activity in Fully Cytotoxic Shiga Toxin 1

“…The binding experiments were performed in Eppendorf tubes precoated with PBS containing 1% BSA to avoid nonspecific loss of toxins (29). Freshly isolated endotoxin-free PMN (0.5 ϫ 10 6 ) were immediately incubated with native or fluorescent Stx1 (50 nM) in 250 l of PBS containing 1% BSA for 90 min at 37°C, with occasional stirring by gentle inversion of the tube.…”

Update on hemolytic uremic syndrome: Diagnostic and therapeutic recommendations

“…In humans this receptor is Gb3 (globotriosilceramyde) and is highly expressed on kidney tubular cells, and brain and gut endothelial cells. Moreover, in the kidney cytotoxicity is further amplified by tumor necrosis factor-α (TNF-α) [32,33] . After binding to cell surfaces, Shiga toxin is endocytosed and retrograde-transported to the Golgi apparatus and the endoplasmic reticulum; it is then translocated to the cytosol where it inactivates ribosomes and causes cell death [34] .…”

Role of RhoA and Rho Kinase in Lysophosphatidic Acid–Induced Endothelial Barrier Dysfunction

“…23,24 One hour before stimulation with LPA, confluent endothelial monolayers were deprived of serum and kept in medium 199 with 1% HSA. Unless indicated otherwise, ECs were stimulated with 13 mol/L LPA.…”