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Aim: Activation of the complement system is a hallmark of renal ischemia/reperfusion injury (IRI). Recently, we demonstrated a novel pathogenic role for Mannan-binding lectin (MBL) in a rat model of IRI, independent of complement activation. In the current study we developed assays to monitor complement activity and complement activation products in rat serum and assessed the occurrence of complement activation over time and related this to renal function and local effects in the kidney.

Materials and Methods: Renal IRI was performed with or without prior MBL depletion in male Lewis rats (n=5-10) with 45 min ischemia. At given time points, blood samples were taken, rats were sacrificed and tissue was analysed for deposition of complement and presence of inflammatory mediators. SC5b-9 was measured in serum with an optimized sandwich ELISA capturing a C9 neo-epitope and detecting C6. Pathway-specific activities were determined from complement preserved serum with functional ELISAs; pathway specific activity was determined as amount of C5b-9 deposition on IgM-, Mannan- and LPS-coated, which quantify the activity of classical, lectin and alternative pathway (CP, LP and AP respectively). 

Results: Immediately after reperfusion, there are no signs of complement consumption in any of the tested pathways. In contrast MBL depletion resulted in transient and specific 70% inhibition of LP at 1h (P<0.05), which returned to baseline at 24h. Analysis of SC5b-9 showed gradual increase 48-72h post IRI (P<0.05). This increase was specific and not observed following MBL depletion or in sham operated rats. Further investigation showed that SC5b-9 increases up to day 7. However, renal C5b-9 deposition and decline in renal function peaks at 48-72h and is cleared by day 7. Histological analysis showed presence of OX42/CD11b positive macrophages in the kidney with correlation to SC5b-9 (r=0.64, p=0.007).

Conclusions: We have shown that in our rat model of renal IRI, complement activation is a relatively late event and can be monitored with SC5b-9. The relationship between circulating SC5b-9, local complement deposition and its functional role needs further evaluation, especially in the repair phase of IRI.