Hemopexin as an Inhibitor of Hemolysis-Induced Complement Activation

Abstract

Hemopexin is the main plasmatic scavenger of cell-free heme, released in the context of intravascular hemolysis or major cell injury. Heme is indispensable for the oxygen transport by hemoglobin but when released outside of the erythrocytes it becomes a danger-associated molecular pattern, contributing to tissue injury. One of the mechanisms of pro-inflammatory action of heme is to activate the innate immune complement cascade. Therefore, we hypothesized that injection of hemopexin will prevent hemolysis-induced complement activation. Human plasma-derived hemopexin is compatible with the heme clearance machinery of the mice. 100 or 500 mg/kg of hemopexin was injected in C57Bl/6 mice before treatment with phenylhydrazine (inducer of erythrocytes lysis) or with PBS as a control. Blood was taken at different timepoints to determine the pharmacokinetic of injected hemopexin in presence and absence of hemolysis. Complement activation was determined in plasma, by the C3 cleavage (western blot) and in the kidneys (immunofluorescence). Kidney injury was evaluated by urea and creatinine in plasma and renal NGAL and HO-1 gene expression were measured. The pharmacokinetic properties of hemopexin (mass spectrometry) in the hemolytic mice were affected by the target-mediated drug disposition phenomenon due to the high affinity of binding of hemopexin to heme. Hemolysis induced complement overactivation and signs of mild renal dysfunction at 6 h, which were prevented by hemopexin, except for the NGAL upregulation. The heme-degrading capacity of the kidney, measured by the HO-1 expression, was not affected by the treatment. These results encourage further studies of hemopexin as a therapeutic agent in models of diseases with heme overload.

Circulating FH Protects Kidneys From Tubular Injury During Systemic Hemolysis

“…Immunofluorescence staining of C3 revealed usual physiological traces of C3 fragment deposits surrounding the tubular cells in the WT mice, which disappeared in the hepatoFH −/− mice due to the complement consumption (8,21). No enhancement of the C3 fragments deposits were detected in the tubulointerstitium upon PHZ injection neither in the WT mice [in agreement with our previous studies (8,25)] nor in the hepatoFH −/− mice (data on day 2, Figure 4C). In contrast, the C3 fragments deposits were seen in glomeruli, which increased in both strains after PHZ treatment in a similar manner (Figures 4C,E).…”

Complement activation is a crucial driver of acute kidney injury in rhabdomyolysis

“…The injected hemopexin dose was the maximal well-tolerated in heme-overload. 21 The induction of rhabdomyolysis was efficient since CK and ASAT were not different in GLY mice +/-hemopexin. However, renal function (BUN) improved in hemopexin injected mice (p=0,01) (Fig.…”

Hemopexin Modulates Expression of Complement Regulatory Proteins in Rat Glomeruli

“…This was shown in mice with drug-induced extensive hemolysis and concurrent complement activation. Intravenous HPX treatment prevented extent of complement activation and renal dysfunction [1].…”

“…Subsequent oxidation of unbound Hb releases heme, which is rapidly bound by hemopexin (HPX). Saturation/depletion of HPX increases circulating levels of bioavailable (free) heme, which can cause oxidative organ stress/injury and inflammation [1].…”