Post-diarrheal hemolytic uremic syndrome (HUS) is the leading cause of acute renal failure in children. The HUS is characterized by acute renal injury, microangiopathic hemolytic anemia, and thrombocytopenia. Renal damage predominantly involves glomerular endothelial cell swelling and detachment, fibrin accumulation, and thrombosis. Marked decreases in glomerular filtration rate (GFR) can occur without obvious histologic changes, suggesting augmented vasoconstrictor influence. The HUS is typically associated with enteric infection by shiga-like toxin (SLT) producing Escherichia coli. The toxin binds to a cell surface glycosphingolipid, GB3, is internalized, and inhibits protein synthesis. The above observations, taken together with the finding that SLT is toxic for endothelial cells, have let to the belief that SLT damage to human glomerular endothelial cells (HGEN) is central to the pathogenesis of HUS renal disease. However, little is known about how SLT interacts with glomerular endothelial cells, particularly in humans. Further, little is known about why HGEN appears to be a major target of SLT, or other factors, in HUS. We have developed a new method for studying HGEN. The current application will take advantage of this technique in order to address the above issues. Finally, very little is know about the molecular biologic events that control cell sensitivity to SLT. The current project includes studies that provide crucial information about this process. Accordingly, the specific aims are: 1) development of a human glomerular endothelial cell model to study the biologic actions of SLT; 2) determination of HGEN susceptibility to SLT toxicity including measurement of baseline GB3 expression by HGEN, identification of inflammatory factors regulating HGEN SLT sensitivity, and elucidation of paracrine and autocrine regulation of HGEN SLT sensitivity; 3) determination of the biologic effects of SLT and inflammatory factors on HGEN including evaluation of factors mediating SLT-induced HGEN detachment, examination of SLT modulation of HGEN-regulated fibrin accumulation, and analysis of inflammatory factor and SLT effects on HGEN vasoactive mediator production; and 4) cloning of the gene encoding human UDP-galactose: lactosylceramide alpha 1-4-galactosyl-transferase, the rate-limiting enzyme in GB3 formation. These studies provide essential information on how and why HGEN are damaged in HUS.
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