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Detrimental effects of adenosine signaling in sickle cell disease

Abstract

Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine A2B receptor (A2BR)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the A2BR has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease.

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Figure 1: Increased adenosine levels contribute to sickling and hemolysis in SCD transgenic mice.
Figure 2: In vivo effects of PEG-ADA treatment on multiple organ damage and renal dysfunction in SCD transgenic mice.
Figure 3: PEG-ADA treatment attenuates hypoxia-reoxygenation–induced acute sickle crisis in SCD transgenic mice.
Figure 4: Excess adenosine acts through A2BRs to induce 2,3-DPG and subsequent sickling in SCD transgenic mice.
Figure 5: Adenosine levels are elevated in individuals with SCD and A2BR-mediated elevation of 2,3-DPG concentrations is required for hypoxia-induced human erythrocyte sickling.

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Acknowledgements

Supported by US National Institute of Health grants DK077748 (to Y.X.), DK083559 (to Y.X.), HL070952 (to M.R.B.) and HL092188 (to H.K.E.) and by China National Science Foundation Scholarship Council 2008637068 (to J.W.). We thank T. Krahn (Bayer HealthCare AG) for the adenosine receptor A2BR agonist BAY 60-6583. Adenosine receptor–deficient mice were obtained from the following sources: A1R-deficient mice (J. Schnermann, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health); A2AR-deficient mice (J.-F. Chen, Boston University School of Medicine); A2BR-deficient mice (M.R. Blackburn, University of Texas–Houston Medical School); and A3R-deficient mice (M. Jacobson, Merck Research Laboratories).

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Y.Z. carried out the measurement of adenosine and 2,3-DPG in humans and mice, analysis of sickling, hemolysis and lifespan of mouse RBCs, histological analysis of multiple tissues and image quantification, ELISA analysis of inflammatory cytokines in the lung homogenates and immunostaining of lung tissues with neutrophil markers, human erythrocyte culture and analysis of sickling under hypoxic conditions, and immunostaining of A2BRs on human and mouse RBCs, and contributed to generation of figures. Y.D. conducted PEG-ADA purification, treatment of mice with PEG-ADA or PSB1115 and proteinuria measurement, and isolation of multiple organs and blood from mice. J.W. was involved in the purification of PEG-ADA and treatment of mice with PEG-ADA or PSB1115, and performed heme content measurement and histological analysis of kidneys. W.Z. was involved in the purification of PEG-ADA; treated mice with PEG-ADA or PSB1115 and contributed to immunostaining of lung tissues with neutrophil markers. A.G. treated normal human erythrocyte cultures with A2BR or A2AR agonists. D.C.A. and M.V.M. conducted metabolomic screens in blood of wild-type and SCD transgenic mice. L.C.-D. provided expertise in confocal analysis of A2BR expression on RBCs. D.E.L. provided expertise in flow cytometry to measure the lifespan of RBCs. W.Z. assisted with urine osmolality analysis. H.S., L.T. and G.L. provided expertise in hemolytic disorders and kidney dysfunction. H.K.E. assisted A.G. with experiments on the effects of A2AR and A2BR agonists on 2,3-DPG induction in normal RBCs. R.E.K. provided expertise in adenosine signaling and helped edit the manuscript; M.R.B. provided mice deficient in each of the four types of adenosine receptors; H.S.J. provided expertise in hemolytic disorders, procured human subjects' approval and maintained the database of de-identified human subject information. Y.X. was the principal investigator, oversaw the design of experiments and interpretation of results, wrote and organized the manuscript, including the text and figures, and edited the manuscript.

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Correspondence to Yang Xia.

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Zhang, Y., Dai, Y., Wen, J. et al. Detrimental effects of adenosine signaling in sickle cell disease. Nat Med 17, 79–86 (2011). https://doi.org/10.1038/nm.2280

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