Inhibiting the Complement System Does Not Reduce Injury in Renal Ischemia Reperfusion PIERCE PARK,* MARK HAAS, ² PATRICK N. CUNNINGHAM,* JESSY J. ALEXANDER,* LIHUA BAO,* JOEL M. GUTHRIDGE, ‡ DAMIAN M. KRAUS, ‡ V. MICHAEL HOLERS, ‡ and RICHARD J. QUIGG* *Department of Medicine, Section of Nephrology, The University of Chicago, Chicago, Illinois; ² Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and ‡ Department of Medicine, Division of Rheumatology, University of Colorado Health Sciences Center, Denver, Colorado. Abstract. The complex pathogenesis of ischemia reperfusion injury (IRI) includes endothelial expression of adhesion mol- ecules, leukocyte recruitment and activation, reactive oxygen species production, and apoptotic and necrotic cell death. A role for complement in IRI of different organs, including kidney, has been proposed on the basis of results of experi- ments that used pharmacologic inhibitors as well as animals that were deficient in individual complement proteins. Here, renal IRI in mice was examined. Animals that were deficient in C3 had partial protection from IRI induced by 27.5 min of bilateral renal ischemia, followed by 20 h of reperfusion (blood urea nitrogen [BUN] values, 46.6  6.9 and 68.4  7.9 mg/dl in C3 -/- and C3 +/+ mice; n = 7 and 8, respectively; P = 0.033). Given the reduction in IRI in C3 -/- mice, it was investigated, by use of the rodent C3 convertase inhibitor CR1-related gene/protein y-Ig (Crry-Ig), whether exogenous administration of a complement inhibitor could lessen renal injury. Despite the use of Crry-Ig in high doses, there was no significant reduction of injury induced by 20 to 30 min of ischemia followed by up to 30 h of reperfusion. Histologic examination revealed acute tubular necrosis and neutrophilic infiltration, both of which correlated significantly with BUN values (P  0.001). Of interest, C3 deposition around renal tubules was significantly less in animals with IRI, compared with that in unmanipulated controls (P  0.001). In Crry-Ig– treated animals, C3 deposition was inversely proportional to BUN values (r =-0.63; P  0.001), which presumably indicates that severe vascular IRI allowed access of the 160 kD Crry-Ig to the interstitium. Thus, renal IRI in mice may have a partial complement dependence, yet pharmacologic inhibition of the complement system does not seem to be effective, likely because of the presence of other mediator systems that operate in parallel. A number of complex events occur during tissue ischemia and reperfusion, including cell surface expression of intracellular P-selectin and other normally hidden antigens, as well as upregulated expression of a number of proteins, such as E- selectin and intercellular adhesion molecule-1 (1,2). Once reperfusion occurs, the access of inflammatory mediators, in- cluding antibody, complement, and blood cells, sets off an inflammatory reaction in which neutrophils and molecular oxygen are key mediators (3,4). Not surprising, given its direct exposure to blood-borne inflammatory elements, the endothe- lial surface seems to be the most important cell in ischemia reperfusion injury (IRI), because it produces multiple factors such as adhesion molecules, cytokines, leukotrienes, endothe- lin, and platelet-activating factor, contributing to the inflam- mation (1,5). Human complement receptor 1 (CR1) is a potent comple- ment inhibitor that is active toward C3 and C5 convertases of both the alternative and classical pathways of complement. Given this, CR1 has been produced as a soluble recombinant protein (sCR1). The first use of sCR1 came in a rat model of IRI in the heart (6). In this setting, infusion of sCR1 at the time of arterial ligation protected against the resultant myocardial infarction. Inhibition of complement protects against the neu- trophilic influx that occurs (6). Subsequently, the use of sCR1 has been applied successfully in IRI models in intestine, skel- etal muscle, and liver (7–10). An elegant series of experiments performed by Carroll et al. (11,12) defined events in skeletal muscle and intestinal IRI. Mice that were deficient in complement components C3 and C4, as well as in Ig, were protected from IRI. Reconstitution of IgM in Ig-deficient mice restored IRI. Taken together, these data indicate that IgM natural antibody-mediated activation of the classical pathway on endothelium is a proximate event in IRI in these two organs. In renal IRI, tubular cells also are prominently injured, which leads to the pathologic picture of acute tubular necrosis. The events that occur are complex but involve complement activation on endothelial cells, endothelial cell P- and E-selec- tin (but not L-selectin) engagement of neutrophils, and an Received October 25, 2000. Accepted January 8, 2001. Correspondence to Dr. Richard J. Quigg, University of Chicago, Section of Nephrology, AMB, S-508, MC 5100, 5841 S. Maryland, Chicago, IL 60637. Phone: 773-702-0757; Fax: 773-702-4816; E-mail: rquigg@medicine. bsd.uchicago.edu 1046-6673/1207-1383 Journal of the American Society of Nephrology Copyright © 2001 by the American Society of Nephrology J Am Soc Nephrol 12: 1383–1390, 2001