Functionalassessmentofmouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury☆

Thecomplementsystemisanessentialcomponent ofourinnateimmunity,bothfortheprotectionagainstinfectionsandforproperhandlingofdyi ngcells.However,thecomplementsystemcanalsocontribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities,thereisanincreasinginterestinmeasurementofthecomplementsystemactivationinthesystemiccompartment, both in the clinical setting as well as in experimental models. Here we describe inparallel a sensitive andspecific sandwich ELISAdetecting mouse C3 activation fragments C3b/C3c/iC3b,aswellasfunctionalcomplementELISAsdetec tingspecificactivitiesofthethreecomplementpathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation ofC3b/C3c/iC3bfragmentsat24hfollowingreperfusion,whichreturnedtobase-lineat3and7dayspostreperfusion.WhenthepathwayspecificcomplementactivitiesweremeasuredatthelevelofC3 activation, we found no significant reduction in any of the pathways. However, the functionalcomplement activity of all three pathways was significantly reduced when measuredatthe levelof C9, with the strongest reduction being observed in the alternative pathway. For all threepathways there was a strong correlation between the amount of C3 fragments and the reductionin functional complement activity. Moreover, at 24 h both C3 fragments and the functionalcomplement activities showed a correlation with the rise in serum creatinine. Together ourresults show that determinationofthesystemicpathwayspecificcomplementactivity isfeasibleinexperimentalmouse models and that theyare usefulinunderstandingcomplement activationand inhibition in vivo.© 2015 Elsevier B.V. All rights reserved.