The hemolytic uremic syndrome (HUS) causes acute renal failure in children and leads to substantial morbidity, and mortality. The HUS follows verotoxin producing Escherichia coli colitis implicating verotoxin in the endothelial cell injury characteristic of HUS. The investigators propose that verotoxin induces sublethal endothelial cell injury and leads to activation of PMNs and induction of NOS leading to generation of reactive oxygen and nitrogen molecules which amplifies endothelial cell injury.
The specific aims are 1) To determine mechanisms of verotoxin (VT-1 and VT-2) mediated glomerular endothelial cell (GEC) injury and the role of PMNs in GEC injury. The cellular targets of verotoxin (with and without PMNs) will be investigated in GEC by determining ATP depletion, protein oxidation, lipid peroxidation, DNA damage and the mechanisms of DNA damage, and antioxidant defense mechanisms. 2) To determine VT-1 & VT-2 induced alterations in production of nitric oxide (NO). Preliminary data suggest that VT-1 increases NO production in GEC. Both VT-1 & VT-2 induced alterations in NO production and enzyme activity, VT-1 and VT-2 induced alterations in cNOS and iNOS mRNA and protein expression and peroxynitrite induced cell injury will be determined in GEC. 3) To determine VT-1 & VT-2 induced PMN activation and induction of cytokines that activate PMNs. Both VT-1 & VT-2 induced activation of PMNs to release oxidants and protease enzymes, VT-1 & VT-2 induced IL-8 and IL-8 mRNA in cells, and the interaction of VT-1, VT-2 and IL-8 in activating PMNs will be investigated. 4) To determine the role of reactive oxygen and nitrogen molecules in mediating GEC injury. Both VT-1 and VT 2 induced GEC injury (with and without PMNs ) will be investigated with scavengers of reactive oxygen molecules with mutant PMNs lacking NADPH oxidase and the ability to generate oxidants, with antibodies to leukocyte adhesion molecules and with inhibitors of cNOS and iNOS. Once the mechanisms of verotoxin mediated cell injury are understood, strategies to prevent or limit cell injury can be formulated.
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