Epidemic hemolytic uremic syndrome (eHUS) is a leading cause of acute renal injury and failure in children (65% of cases of eHUS), who may also suffer seizures, neurocognitive deficits from cerebral ischemic events, and other organ system failure. This project will examine the mechanisms whereby Shiga toxin (Stx)-induced complement activation and Stx interaction with the endothelium generate procoagulant tissue factor (TF) activity and platelet adhesion molecules in this thrombotic microangiopathy. We hypothesize that Stx activates the mannose binding lectin-2 (MBL-2) pathway of complement, leading to the binding of the complement effectors C3b, C5, C5b-9, and MBL2 on or near endothelial cell (EC) Gb3, the inducible receptor on ECs for Stx, and on or near toll-like receptor 4 (TLR4). Activation of endothelium via cytokines and Stx follows, with subsequent upregulation of platelet adhesion molecules and expression of EC pro-coagulant tissue factor (TF) activity, leading to the development of eHUS and further thrombin and factor Xa-mediated complement activation.
Specific Aims /Hypotheses: 1: To demonstrate that complement activation by Stx leads to C3d, C5a, and MBL-2 binding to ECs and the appearance of membrane-bound C5b-9, with expression of procoagulant TF activity and platelet adhesion molecules (P-selectin, high MW von Willebrand Factor (vWF) multimers). To show that complement is activated via the MBL-2 lectin pathway, and not the alternative pathway. 2: To demonstrate that Stx, complement activation, and upregulated procoagulant TF and platelet adhesion molecules contribute to platelet thrombus formation during flow using an in vitro flow model of eHUS employing monolayers of HUVECs or HGECs. The studies incorporate the effects of shear rate (convection of complement components, clotting factors) characteristic of glomerular arterioles, and shear stress, which are in vivo conditions. 3: To identify the molecular mechanisms of complement activation in a novel humanized MBL-2 mouse model of Stx-2-induced renal injury. This proposal will demonstrate that Stx leads to MBL-2 lectin pathway complement activation, procoagulant EC TF activity, and platelet-fibrin thrombus formation in flowing blood, in vitro and in vivo, thereby providing novel therapeutic targets (i.e., MBL-2 and/or TF) for a devastating disease that presently lacks a definitive therapy. 1
The childhood epidemic hemolytic uremic syndrome (eHUS), caused by the bacterial toxin Shiga toxin, is a poorly understood disease in which blood clotting at the level of the smallest blood vessels causes kidney failure in 65% of affected children, brain impairment in up to 30%, and death in up to 5%. We will clarify important and interactive roles for misdirection of an important part of the immune system, on the one hand, and blood clotting, on the other. Using models for eHUS based on a) growing human umbilical vein or renal glomerular endothelial cells in the laboratory and exposing them to human plasma or flowing donor blood to which has been added Shiga toxin, and b) injecting mice in a special mouse model which replicates important parts of the human immune system, we will identify future novel treatments, including blocking the clotting and complement pathways with unique antibodies, for a devastating disease for which there is at present only supportive care and no specific treatment.