C3(H2O) prevents rescue of complement-mediated C3 glomerulopathy in Cfh–/– Cfd–/– mice
Yuzhou Zhang, Adam Keenan, Dao-Fu Dai, Kristofer S. May, Emily E. Anderson, Margaret A. Lindorfer, John B. Henrich, Gabriella R. Pitcher, Ronald P. Taylor, Richard J.H. Smith
Yuzhou Zhang, Adam Keenan, Dao-Fu Dai, Kristofer S. May, Emily E. Anderson, Margaret A. Lindorfer, John B. Henrich, Gabriella R. Pitcher, Ronald P. Taylor, Richard J.H. Smith
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Research Article Nephrology

C3(H2O) prevents rescue of complement-mediated C3 glomerulopathy in Cfh–/– Cfd–/– mice

Abstract

Therapeutic complement inhibition is a major focus for novel drug development. Of upstream targets, factor D (FD) is appealing because it circulates in plasma at low concentrations and has a single function: to cleave factor B to generate C3 convertase of the alternative pathway (AP). Mice with a targeted deletion of factor H (FH; Cfh–/– mice) develop C3 glomerulopathy (C3G) due to uncontrolled AP activity. To assess the impact of FD inhibition, we studied Cfh–/– Cfd–/– mice. We show that C3G in Cfh–/– mice is not rescued by removing FD. We used serum from Cfh–/– Cfd–/– mice to demonstrate that residual AP function occurs even when both FD and FH are missing and that hemolytic activity is present due to the action of C3(H2O). We propose that uncontrolled tick-over leads to slow activation of the AP in Cfh–/– Cfd–/– mice and that a minimal threshold of FH is necessary if tissue deposition of C3 is to be prevented. The FD/FH ratio dictates serum C3 level and renal C3b deposition. In C3G patients with chronic renal disease, the FD/FH ratio correlates inversely with C3 and C5 serum levels, suggesting that continuous AP control may be difficult to achieve by targeting FD.

Authors

Yuzhou Zhang, Adam Keenan, Dao-Fu Dai, Kristofer S. May, Emily E. Anderson, Margaret A. Lindorfer, John B. Henrich, Gabriella R. Pitcher, Ronald P. Taylor, Richard J.H. Smith

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Figure 6

Mechanism of action underlying complement activation in the absence of FH and FD.

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Mechanism of action underlying complement activation in the absence of F...
(A) Sepharose C3b deposition (SC3D) assay. No SC3D is observed using serum from Cfh–/– (n = 4) or Cfd–/– (n = 7) mice; however, C3b deposition is restored when human FD (huFD) is added to the serum of Cfd–/– but not Cfh–/– mice. A small amount of C3b deposition is detectable using serum from Cfh–/– Cfd–/– mice (n = 11); addition of huFD does not improve the deposition, while addition of huFH quenches it. Serum collected for WT mice (n = 7) is used as a control. (B) Hemolytic assay using rabbit erythrocytes (RaE). Consistent with the SC3D results, there are moderate levels of hemolysis using serum from Cfh–/– Cfd–/– mice. The addition of huFD does not alter the hemolysis, while the addition of huFH suppresses it. Data in each group are derived from serum collected from 5 individual mice. (C) Addition of C1-Inh at high concentrations to Cfh–/– Cfd–/– serum (n = 6, mean ± SD) prevents residual hemolysis, while addition of the plasma kallikrein inhibitor PKSI-527 (n = 4) has no effect, consistent with C3(H2O)-associated residual AP activity in the serum of Cfh–/– Cfd–/– mice. (D) Addition of human C3(MA) to Cfh–/– Cfd–/– serum increases hemolysis of RaE, but the addition of C3 does not (n = 5, mean values are used in box plots). (E) Injection of C3(MA) in Cfh–/– Cfd–/– mice increases subsequent serum-mediated ex vivo hemolysis of RaE, while injection of C3 does not increase hemolysis (n = 4 for both groups). (F) Glomerular C3 deposition is also increased with the injection of C3(MA) (n = 4) compared with deposition with C3 injection (n = 4). (G) Local iC3(H2O) deposition over time. Supernatants from homogenized kidney tissues of Cfh–/– Cfd–/– mice (n = 2 for each age group) show increased iC3(H2O) with age as resolved on a Western blot probed with an anti-C3a antibody (β-actin, loading control). *P < 0.05 by 2-tailed paired t test for D and E or unpaired 2-tailed t test for F.