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

Abstract

The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways 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 of C3b/C3c/iC3b fragments at 24 h following reperfusion, which returned to base-line at 3 and 7 days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24 h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo.

Deficiency of the complement regulatory protein CD59 accelerates the development of diabetes-induced atherosclerosis in mice

“…This interpretation was investigated measuring levels of iC3b in serum of STZ-induced diabetic mice. Serum levels of iC3b, a product of the proteolytic cleavage of C3b, increase as a consequence of complement activation and is an accepted parameter to assess complement activation in experimental animals (Kotimaa et al, 2015). Supplemental figure 3 shows that serum levels of iC3b were increased by 40% (p < 0.05) even in mCD59ab +/+ /ApoE −/− mice as early as 2 weeks after STZ treatment as compared with their non-diabetic counterparts.…”

“…Briefly, blood was collected from the tail tip of ApoE −/− mice at various times after STZ injection and allowed to clot at 4°C for 1 hour (Kotimaa et al, 2015). Serum was separated from blood cells by centrifugation at 1000 × g for 15 min at 4°C and aliquots were stored at −80°C until analysis.…”

Classical Complement Pathway Activation in the Kidneys of Women With Preeclampsia

“…25 Zymosan-activated CD1 mouse serum (IMSCD1-COMPL, Innovative Research) was used as a standard and set to 100 AU/ml as described previously. 26 …”

Properdin binds independent of complement activation in an in vivo model of anti–glomerular basement membrane disease

“…All blood samples were placed directly on ice and prepared as plasma or serum as described previously. 45 The impact of C3 and properdin deficiency was investigated by injecting either anti-GBM or control IgG (0.5 mg) in WT, C3 KO, and properdin KO mice. The severity of renal injury was evaluated by 24-hour urine collection and proteinuria analysis from anti-GBM antibody-injected WT (n ¼ 10), properdin-KO (n ¼ 8), and C3 KO (n ¼ 9) mice and WT mice injected with control antibody (n ¼ 5).…”