Carbon Monoxide Protects the Kidney Through the Central Circadian Clock and CD39
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Carbon monoxide protects the kidney through the central circadian clock and CD39 Matheus Correa-Costaa, David Galloa, Eva Csizmadiaa, Edward Gompertsb, Judith-Lisa Lieberuma, Carl J. Hausera,c, Xingyue Jid,e, Binghe Wangd,e, Niels Olsen Saraiva Câmaraf, Simon C. Robsonc, and Leo E. Otterbeina,1 aTransplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; bHillhurst Biopharmaeuticals Inc., Montrose, CA 91020; cTransplant Institute, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; dDepartment of Chemistry, Georgia State University, Atlanta, GA 30303; eCenter for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303; and fLaboratory of Transplantation Immunobiology, Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, 05508-900, Sao Paulo, Brazil Edited by Gregg L. Semenza, Johns Hopkins University School of Medicine, Baltimore, MD, and approved January 24, 2018 (received for review September 22, 2017) Ischemia reperfusion injury (IRI) is the predominant tissue insult tective phenotype that results. Biliverdin and CO are accepted as associated with organ transplantation. Treatment with carbon mon- the primary underlying bioactive molecules that provide potent oxide (CO) modulates the innate immune response associated with IRI protective benefits to the cell by modulating apoptosis, inflam- and accelerates tissue recovery. The mechanism has been primarily mation, and proliferation (13–15). Administration of CO or bili- descriptive and ascribed to the ability of CO to influence inflamma- verdin can, in most cases, recapitulate that observed with HO-1 tion, cell death, and repair. In a model of bilateral kidney IRI in mice, itself. Treatment with CO at low concentrations imparts potent we elucidate an intricate relationship between CO and purinergic protection in numerous models of disease, including transplantation signaling involving increased CD39 ectonucleotidase expression, de- (16–18), colitis (19), sepsis (20), and lung injury (21). CO has been creased expression of Adora1, with concomitant increased expression well-studied and characterized to prevent IRI in small and large > of Adora2a/2b. This response is linked to a 20-fold increase in ex- animal models (22–24), which in turn resulted in the first clinical pression of the circadian rhythm protein Period 2 (Per2) and a fivefold trial where CO was administered to kidney transplant recipients increase in serum erythropoietin (EPO), both of which contribute −/− intraoperatively (https://clinicaltrials.gov/). to abrogation of kidney IRI. CO is ineffective against IRI in Cd39 Per2−/− Circadian rhythms are critical determinants of organ function and mice or in the presence of a neutralizing antibody to and susceptibility to injury dictated by a family of proteins col- EPO. Collectively, these data elucidate a cellular signaling mecha- lectively known as the “clock genes.” Disruption of one or more of nism whereby CO modulates purinergic responses and circadian these genes increases susceptibility to tissue injury (25). IRI leads rhythm to protect against injury. Moreover, these effects involve CD39- and adenosinergic-dependent stabilization of Per2. As CO to impairment of the circadian clock (26) and is independent of also increases serum EPO levels in human volunteers, these findings hypoxia. Adenosine-elicited A2b-mediated Period 2 (Per2) stabi- continue to support therapeutic use of CO to treat IRI in association lization modulates adaptation to ischemic injury of the heart in with organ transplantation, stroke, and myocardial infarction. mice (27). In fact, tolerance to ischemic injury has been shown to be dependent upon the time of day that the injury occurs (28). heme oxygenase | circadian rhythm | DAMPS | innate immunity | Adenosine from the breakdown of extracellular ATP by ′ adenosine CD39 and CD73 (ecto-5 -nucleotidase) is generated during tissue schemia reperfusion injury (IRI) is obligatory and unavoidable Significance Iin patients who undergo an organ transplant. The sudden un- availability of oxygen and glucose initiate a cascade of events Tissue injury caused by lack of blood flow results in a series of including activation of tissue leukocytes and endothelium. Sim- adaptive responses of the body to ensure survival. Cellular ilarly, the reestablishment of blood flow to an ischemic organ production of carbon monoxide (CO) preserves organ function elicits a second set of events that include rapid reactive oxygen and promotes healing. How this occurs has remained elusive. species (ROS) generation, leukocyte infiltration, and additional Here we demonstrate using a model of ischemia reperfusion mechanical injury. The severity of the IRI and the relative health injury (IRI) of the kidney, mimicking kidney transplant, that of the organ is speculated to contribute to long-term graft sur- safe administration of CO protects against IRI. Remarkably, this vival (1–4). While a number of therapeutic approaches have occurs through specific modulation of a gene that regulates been tested, including a variety of preservation solutions, so- energy metabolism (CD39) and one that controls circadian phisticated organ transport apparatus, anti-inflammatory agents, rhythm (Period 2). Collectively, we define here an innovative live donors, and even ischemic preconditioning, there has been signaling pathway linking the brain and the kidney vis a vis a little change in IRI (5–7). What is perhaps even more important gas molecule. These data may have important therapeutic is that when a solution to IRI is identified, organs that are consequences for transplant recipients and victims of stroke. otherwise considered too risky to use could be rescued and as Author contributions: M.C.-C., D.G., C.J.H., B.W., S.C.R., and L.E.O. designed research; such impact the number of transplants that could be performed M.C.-C., D.G., E.C., J.-L.L., and X.J. performed research; E.C., E.G., X.J., and B.W. contrib- and decrease an otherwise continuously growing waiting list. uted new reagents/analytic tools; M.C.-C., J.-L.L., N.O.S.C., S.C.R., and L.E.O. analyzed data; Given the impossibility of transplanting an organ without some and M.C.-C., C.J.H., N.O.S.C., S.C.R., and L.E.O. wrote the paper. amount of ischemic time, a focus on interventions that may Conflict of interest statement: L.E.O. is a scientific consultant for Hillhurst Biopharmaceuticals protect the organ before harvest as well as promoting faster re- and has stock options. E.G. is a founder of Hillhurst Biopharmaceuticals and owns stock. covery and repair after reperfusion is warranted (8–12). This article is a PNAS Direct Submission. Heme oxygenase-1 (HO-1) is a member of a stress response Published under the PNAS license. gene family and is considered a protective gene. HO-1 catalyzes 1To whom correspondence should be addressed. Email: [email protected]. the breakdown of heme to bilirubin. In so doing, three products This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are generated and include carbon monoxide (CO), biliverdin, 1073/pnas.1716747115/-/DCSupplemental. and iron. When HO-1 activity is increased, there is a potent pro- Published online February 20, 2018. E2302–E2310 | PNAS | vol. 115 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1716747115 Downloaded by guest on September 29, 2021 hypoxia as an adaptive response. In previous work, we have shown determined by the crystal violet staining method. Briefly, surviving cells in six- PNAS PLUS that CO enhances functional expression of adenosine receptors well plates were washed twice with PBS, and then crystal violet solution and stabilization of HIF1α in macrophages (29, 30). Employing a (Sigma-Aldrich) was added to the cells. After washing with water, plates were murine model of kidney IRI, we have now tested the hypothesis left to dry overnight. On the following day, a solution of 10% acetic acid was that CO protects against kidney IRI through CD39 and adenosine added to each well, and the number of surviving cells was determined by measuring absorbance at 562 nm. A2b-mediated receptor stabilization of Per2. Additionally, eryth- ropoietin (EPO), a critical effector protein regulated in a circadian ROS Measurement. Intracellular ROS generation was assessed using 2′,7′- manner, is required for CO-induced renal protection. Collectively, dichlorofluorescin diacetate (DCF-DA; 10 mM), and microscopy was per- our findings may be related in part to the response of the cellular formed. Cells on coverslips were perfused under controlled O2 and CO O2 sensors that drives expression and stabilization of genes that conditions in a flow-through chamber at 37 °C on an inverted fluorescent regulate cellular bioenergetics. microscope. Images were acquired with an Olympus camera (excitation, 488 nm; emission, 535 nm). Materials and Methods − − Animals. Isogenic male C57BL/6 mice (WT and Per2 / ) were purchased from Gene Expression. Total RNA was extracted from homogenized tissues using − − Charles River or Jackson Labs (at 25–30 g). CD39-knockout mice (Cd39 / )were RNeasy Mini Kit (Qiagen). For cDNA synthesis, 2 μg of total RNA was used and bred at the BIDMC as described previously (31). All animals had access to water transcribed with SuperScript II Reverse Transcriptase (Invitrogen) and ran- and food ad libitum. All animal care, housing and procedures were approved by dom primers (Invitrogen).