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Macrophage extracellular trap formation promoted by platelet activation is a key mediator of rhabdomyolysis-induced acute kidney injury

Abstract

Rhabdomyolysis is a serious syndrome caused by skeletal muscle injury and the subsequent release of breakdown products from damaged muscle cells into systemic circulation1. The muscle damage most often results from strenuous exercise, muscle hypoxia, medications, or drug abuse and can lead to life-threatening complications, such as acute kidney injury (AKI)1. Rhabdomyolysis and the AKI complication can also occur during crush syndrome, an emergency condition that commonly occurs in victims of natural disasters, such as earthquakes, and man-made disasters, such as wars and terrorism2. Myoglobin released from damaged muscle is believed to trigger renal dysfunction in this form of AKI. Recently, macrophages were implicated in the disease pathogenesis of rhabdomyolysis-induced AKI3,4, but the precise molecular mechanism remains unclear. In the present study, we show that macrophages released extracellular traps (ETs) comprising DNA fibers and granule proteins in a mouse model of rhabdomyolysis. Heme-activated platelets released from necrotic muscle cells during rhabdomyolysis enhanced the production of macrophage extracellular traps (METs) through increasing intracellular reactive oxygen species generation and histone citrullination. Here we report, for the first time to our knowledge, this unanticipated role for METs and platelets as a sensor of myoglobin-derived heme in rhabdomyolysis-induced AKI. This previously unknown mechanism might be targeted for treatment of the disease. Finally, we found a new therapeutic tool for prevention of AKI after rhabdomyolysis, which might rescue some sufferers of this pathology.

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Figure 1: METs observed in the renal tubules of mice subjected to rhabdomyolysis-induced AKI contribute to disease pathogenesis.
Figure 2: Rhabdomyolysis-induced AKI depends on platelets, macrophages, and neutrophils.
Figure 3: Ablation of Mac-1 eliminates METs and ameliorates rhabdomyolysis-induced AKI.
Figure 4: CitH3 and METs are induced by heme-activated platelets via Mac-1, ROS, and PADs.

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Acknowledgements

We thank the Collaborative Research Resources, School of Medicine, Keio University for technical assistance. We gratefully thank T. Shimosawa (University of Tokyo) for animal care assistance. This work was supported in part by a grant-in-aid from Ministry of Education, Culture, Sports, Science and Technology of Japan (15K09459 (J.H.) and 16K19497 (K.O.)). T.N.M. was supported by National Heart, Lung and Blood Institute (NLHBI), National Institutes of Health (HL065095). J.H. was also supported by grants from Research on Rare and Intractable Diseases, Ministry of Health, Labour and Welfare, Japan (nannti-ippann-004) and the study group for strategic exploration of drug seeds for antineutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis and construction of clinical evidence from Japan Agency for Medical Research and development, AMED under grant number JP17ek0109104.

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Contributions

K.O. performed the majority of the experiments, analyzed the data, and wrote the manuscript. T.N.M. provided the Mac-1−/− mice and critical discussion of this work and revised the manuscript editorially. M. Kamiya and Y.U. contributed to the imaging of METs and the preparation of new reagents. M. Kurosawa contributed to mouse care and FACS analysis. A.S., K.Y., and K. Hase provided the Padi4−/− mice. K. Homma and J.S. contributed to clinical study of the subjects with rhabdomyolysis. H.M. conducted a part of the mouse experiments using some inhibitors. T.H. contributed the experiments using Lf. M.H. contributed to clinical concepts behind this study and laboratory management. J.H. designed the research, wrote the manuscript, and supervised the study. All authors reviewed the manuscript.

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Correspondence to Junichi Hirahashi.

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The authors applied for a new patent entitled “An inhibitor of leukocyte extracellular traps.” The application number is 14/783,295, and the patent was submitted to Meunier Carlin Curfman LLC.

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Okubo, K., Kurosawa, M., Kamiya, M. et al. Macrophage extracellular trap formation promoted by platelet activation is a key mediator of rhabdomyolysis-induced acute kidney injury. Nat Med 24, 232–238 (2018). https://doi.org/10.1038/nm.4462

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