Role of tissue kallikrein in alternative complement pathway activation in STEC-HUS

Project Summary/Abstract Shiga toxin-associated hemolytic uremic syndrome (STEC-HUS) is a type of HUS caused by infection with Shiga toxin and lipopolysaccharide (LPS)-producing enterohemorrhagic Escherichia coli (STEC), a foodborne pathogen, resulting in bloody diarrhea. HUS follows the diarrheal prodrome, with the highest incidence in young children. Pediatric mortality from STEC-HUS has improved in the last 20 years, but children still have significant morbidity secondary to chronic or end-stage kidney disease. Adult mortality is higher, especially in the elderly. The pathophysiology of STEC-HUS is hallmarked by endothelial injury secondary to Shiga and LPS toxemia, and there is increasing evidence to suggest that the alternative complement pathway (AP) is overactivated in STEC-HUS. The AP is part of the innate immune system, responsible for ridding the body of foreign invaders. In the "tick over" hypothesis of AP activation, a low level of the hydrolyzed form of complement factor C3 (C3H2O) is continuously formed. Upon forming a complex with C3H2O, complement factor B (FB) is cleaved by complement factor D (FD) to yield the initial C3 convertase, C3H2O-Bb, which proteolyzes C3 into C3a and C3b. AP activation continues with heterodimerization between C3b and FB and subsequent cleavage of FB by FD, yielding the C3 convertase, C3bBb, that amplifies C3 proteolysis into C3a and C3b. Rapid inactivation by complement factors H and I limits AP amplification. However, activation of C3 by non-complement serine proteases brings the paradigm of the "tick over" hypothesis into question. Expression of the serine protease tissue kallikrein 1 (KLK1) is elevated in models of endotoxemia. The role(s) for this inflammatory serine protease in mediating AP overactivation in the setting of STEC-HUS has not been explored. The investigators have demonstrated that KLK1 cleaves factor C3 of the AP in the fluid phase. This application proposes biochemical studies using cellular and animal models to test the hypothesis that the non-complement serine protease KLK1 significantly contributes to the AP overactivation that is associated with STEC-HUS. These studies will determine the impact of non-canonical mechanisms of AP activation and open a new area of research on complement biology in microangiopathic and infectious diseases.