Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs
Felicitas S. Boretti, … , Abdu I. Alayash, Dominik J. Schaer
Felicitas S. Boretti, … , Abdu I. Alayash, Dominik J. Schaer
Published July 20, 2009
Citation Information: J Clin Invest. 2009;119(8):2271-2280. https://doi.org/10.1172/JCI39115.
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Research Article

Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs

Abstract

Release of hemoglobin (Hb) into the circulation is a central pathophysiologic event that contributes to morbidity and mortality in chronic hemolytic anemias and severe malaria. These toxicities arise from Hb-mediated vasoactivity, possibly due to NO scavenging and localized tissue oxidative processes. Currently, there is no established treatment that targets circulating extracellular Hb. Here, we assessed the role of haptoglobin (Hp), the primary scavenger of Hb in the circulation, in limiting the toxicity of cell-free Hb infusion. Using a canine model, we found that glucocorticoid stimulation of endogenous Hp synthesis prevented Hb-induced hemodynamic responses. Furthermore, guinea pigs administered exogenous Hp displayed decreased Hb-induced hypertension and oxidative toxicity to extravascular environments, such as the proximal tubules of the kidney. The ability of Hp to both attenuate hypertensive responses during Hb exposure and prevent peroxidative toxicity in extravascular compartments was dependent on Hb-Hp complex formation, which likely acts through sequestration of Hb rather than modulation of its NO- and O2-binding characteristics. Our data therefore suggest that therapies involving supplementation of endogenous Hb scavengers may be able to treat complications of acute and chronic hemolysis, as well as counter the adverse effects associated with Hb-based oxygen therapeutics.

Authors

Felicitas S. Boretti, Paul W. Buehler, Felice D’Agnillo, Katharina Kluge, Tony Glaus, Omer I. Butt, Yiping Jia, Jeroen Goede, Claudia P. Pereira, Marco Maggiorini, Gabriele Schoedon, Abdu I. Alayash, Dominik J. Schaer

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Figure 4

Hp prevents extravascular Hb oxidative processes and oxidative tissue damage.

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Hp prevents extravascular Hb oxidative processes and oxidative tissue da...
(A) Representative bright-field images show Perls-stained (iron) kidney sections with DAB intensification and corresponding renal 4-HNE immunohistochemistry (tissue protein-bound 4-HNE) from Hb-exposed guinea pigs in the absence or presence of Hp (original magnification, ×100 [top panels]; ×400 [bottom panels]). Greater staining intensity for both iron and 4-HNE is evident at 24 hours after Hb exposure in non-Hp–treated animals. Scale bars: 200 μm (top panels); 20 μm (bottom panels). (B) Quantitative digital image analysis of total staining intensity of 30 random kidney section images from 3 Hb and 3 Hb-Hp–treated animals (mean ± SEM). (C) UV-visible spectra of Hb in urine collected 2 hours after free Hb transfusion. Each line represents the data of 1 individual animal. The derived concentrations of heme species are shown in the right panel as mean ± SEM (n = 5). (D) MALDI-MS analysis of oxidation-induced Hb globin chain cross-linking in guinea pig urine. Representative spectra of urine from Hb-transfused (left panel) and Hb-Hp–transfused animals (right panel) are shown. While no major signal of non-modified Hb can be detected at the expected m/z of human Hb α– and β–globin chains, peaks representing extensively adducted and cross-linked globin chains can be observed at m/z 23.9, 34.3, and 44.3 kDa. None of these masses appears in the spectra of Hb-Hp–treated animals.