Therapeutic C3 inhibitor Cp40 abrogates complement activation induced by modern hemodialysis filters

Abstract

Approximately 350,000 individuals in the United States rely on maintenance hemodialysis treatment because of end-stage renal disease. Despite improvements in dialysis technology, the mortality rate for patients treated with maintenance dialysis is still exceptionally high, with a 5-year survival rate of only 35%. Many patients succumb to conditions resulting at least in part from the chronic induction of inflammation. Among the triggers of inflammation, the complement system is of particular importance, being a well-appreciated mediator of inflammatory processes that is involved in many pathologic states. Here we used a refined pre-clinical model of hemodialysis in cynomolgus monkeys to confirm that even modern, polymer-based hemodialysis filters activate complement and to evaluate the potential of Cp40, a peptidic C3 inhibitor, to attenuate hemodialysis-induced complement activation. Our data show marked induction of complement activation even after only a single session of hemodialysis. Importantly, complete inhibition of complement activation was achieved in response to two distinct Cp40 treatment regimens. Further, we show that application of Cp40 during hemodialysis resulted in increased levels of the anti-inflammatory cytokine IL-10, indicating that Cp40 may be a potent and cost-effective treatment option for attenuating chronic inflammatory conditions in dialysis-dependent patients.

Keywords: C3; Complement; Compstatin; Cp40; Hemodialysis.

Copyright © 2014 Elsevier GmbH. All rights reserved.

Figures

Figure 1

Modern hemodialysis filters induce complement activation. (A) Schematic diagram of hemodialysis circuit. Access lines were set up on the femoral veins of a cynomolgus monkey (Macaca fascicularis) for blood outflow and inflow. Blood was sampled from two access ports (outflow & inflow sampler) located on each blood line to obtain samples before and after filtering. Cp40 and heparin were introduced into the system immediately prior to blood circulation via a third port. Sterile bicarbonate buffer was used as dialysate. (B) Hemodialysis was performed in cynomolgus monkeys for a period of four hours and blood samples were collected prior to, during, and after blood circulation as indicated. (C) Complement C3 activation was measured in plasma samples by ELISA and represented as optical density (O.D.) values. Three sessions of hemodialysis were performed with a 2-day interval between each session. The figures depict data from blood samples collected from the inflow sampler (Fig. 1A). Similar results were obtained with samples collected from the outflow sampler.

Figure 2

Cp40 prevents hemodialysis-induced complement activation. (A) Hemodialysis was performed in cynomolgus monkeys for a period of four hours and blood samples were collected prior to, during, and after blood circulation as indicated. Cp40 was injected as an i.v. bolus of 2 mg/kg followed by an i.v. infusion of 4 μg/kg/min. (B) Blood samples were collected from a monkey that was untreated (black line) or treated with Cp40 (gray line) and the activation of complement C3 was measured in plasma samples by ELISA, represented as optical density (O.D.) values. The figure depicts data from blood samples collected from the inflow sampler (Fig. 1A). Similar results were obtained with samples collected from the outflow sampler. (C) Levels of Cp40 were measured by mass spectrometry in the plasma of a monkey treated with Cp40. Samples were collected from both samplers as indicated in Fig. 1A.

Figure 3

A single dose of Cp40 at the beginning of hemodialysis is sufficient to prevent complement activation. (A) Hemodialysis was performed in cynomolgus monkeys for a period of four hours and blood samples were collected prior to, during, and after blood circulation as indicated. Cp40 was injected as an i.v. bolus of 2 mg/kg. (B) Blood samples were collected from untreated (black line) and Cp40-treated (gray line) monkeys and the activation of complement C3 was measured in plasma samples by ELISA, represented as a percentage of total C3 activation (%). The figures depict data from blood samples collected from the inflow sampler (Figure 1A). Similar results were obtained with samples collected from the outflow sampler. (C) Levels of Cp40 were measured by mass spectrometry in the plasma of monkeys treated with Cp40. Samples were collected from both samplers as indicated in Fig. 1A.

Figure 4

The use of Cp40 during hemodialysis induces increased plasma levels of the anti-inflammatory cytokine IL-10. Hemodialysis was performed in cynomolgus monkeys for a period of four hours and blood samples were collected prior to, during, and after blood circulation. Cp40 was injected as an i.v. bolus of 2 mg/kg (Animals 4 and 5) or as an i.v. bolus of 2 mg/kg followed by i.v. infusion of 4 μg/kg/min (Animal 3). Blood samples were collected from untreated (black line) or Cp40-treated (gray line) monkeys and plasma levels of IL-10 were measured by multiplex ELISA.