Functional assessment of mouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury.
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.
Leiden University Medical Center (LUMC), Leiden, The Netherlands. Electronic address: Kooten@lumc.nl.
Juha P
JP
Kotimaa
The University Medical Center Groningen, The Netherlands.
Maaike B
MB
van Werkhoven
Leiden University Medical Center (LUMC), Leiden, The Netherlands.
Joseph
J
O'Flynn
Leiden University Medical Center (LUMC), Leiden, The Netherlands.
Ngaisah
N
Klar-Mohamad
Hycult Biotech, Uden, The Netherlands.
Jan
J
van Groningen
Hycult Biotech, Uden, The Netherlands.
Geurt
G
Schilders
Hycult Biotech, Uden, The Netherlands.
Helma
H
Rutjes
Leiden University Medical Center (LUMC), Leiden, The Netherlands; The University Medical Center Groningen, The Netherlands.
Mohamed R
MR
Daha
The University Medical Center Groningen, The Netherlands.
Marc A
MA
Seelen
Leiden University Medical Center (LUMC), Leiden, The Netherlands.
Cees
C
van Kooten
D000818:Animals; D003167:Complement Activation / Q000235:genetics / Q000276:immunology*; D003179:Complement C3b / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015932:Complement C3c / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D003186:Complement C9 / Q000276:immunology / Q000378:metabolism; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005240:Feasibility Studies; D007668:Kidney / Q000098:blood supply / Q000276:immunology* / Q000378:metabolism; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D015427:Reperfusion Injury / Q000276:immunology*; D015203:Reproducibility of Results; D013997:Time Factors
D003186:Complement C9; D003179:Complement C3b; D015932:Complement C3c
Alternative pathway; Classical pathway; Lectin pathway; Mouse; Renal ischaemia/reperfusion injury; The complement system