Carbon monoxide protects the kidney through thecentral circadian clock and CD39Matheus 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; bHillhurstBiopharmaeuticals 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 StateUniversity, Atlanta, GA 30303; and fLaboratory of Transplantation Immunobiology, Department of Immunology, Institute of Biomedical Sciences, Universityof 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 September22, 2017)

Ischemia reperfusion injury (IRI) is the predominant tissue insultassociated with organ transplantation. Treatment with carbon mon-oxide (CO) modulates the innate immune response associatedwith IRIand accelerates tissue recovery. The mechanism has been primarilydescriptive and ascribed to the ability of CO to influence inflamma-tion, cell death, and repair. In a model of bilateral kidney IRI in mice,we elucidate an intricate relationship between CO and purinergicsignaling involving increased CD39 ectonucleotidase expression, de-creased expression of Adora1, with concomitant increased expressionof Adora2a/2b. This response is linked to a >20-fold increase in ex-pression of the circadian rhythm protein Period 2 (Per2) and a fivefoldincrease in serum erythropoietin (EPO), both of which contributeto abrogation of kidney IRI. CO is ineffective against IRI in Cd39−/−

and Per2−/− mice or in the presence of a neutralizing antibody toEPO. Collectively, these data elucidate a cellular signaling mecha-nism whereby CO modulates purinergic responses and circadianrhythm to protect against injury. Moreover, these effects involveCD39- and adenosinergic-dependent stabilization of Per2. As COalso increases serum EPO levels in human volunteers, these findingscontinue to support therapeutic use of CO to treat IRI in associationwith organ transplantation, stroke, and myocardial infarction.

heme oxygenase | circadian rhythm | DAMPS | innate immunity |adenosine

Ischemia reperfusion injury (IRI) is obligatory and unavoidablein patients who undergo an organ transplant. The sudden un-

availability of oxygen and glucose initiate a cascade of eventsincluding activation of tissue leukocytes and endothelium. Sim-ilarly, the reestablishment of blood flow to an ischemic organelicits a second set of events that include rapid reactive oxygenspecies (ROS) generation, leukocyte infiltration, and additionalmechanical injury. The severity of the IRI and the relative healthof the organ is speculated to contribute to long-term graft sur-vival (1–4). While a number of therapeutic approaches havebeen tested, including a variety of preservation solutions, so-phisticated organ transport apparatus, anti-inflammatory agents,live donors, and even ischemic preconditioning, there has beenlittle change in IRI (5–7). What is perhaps even more importantis that when a solution to IRI is identified, organs that areotherwise considered too risky to use could be rescued and assuch impact the number of transplants that could be performedand decrease an otherwise continuously growing waiting list.Given the impossibility of transplanting an organ without someamount of ischemic time, a focus on interventions that mayprotect the organ before harvest as well as promoting faster re-covery and repair after reperfusion is warranted (8–12).Heme oxygenase-1 (HO-1) is a member of a stress response

gene family and is considered a protective gene. HO-1 catalyzesthe breakdown of heme to bilirubin. In so doing, three productsare generated and include carbon monoxide (CO), biliverdin,and iron. When HO-1 activity is increased, there is a potent pro-

tective phenotype that results. Biliverdin and CO are accepted asthe primary underlying bioactive molecules that provide potentprotective benefits to the cell by modulating apoptosis, inflam-mation, and proliferation (13–15). Administration of CO or bili-verdin can, in most cases, recapitulate that observed with HO-1itself. Treatment with CO at low concentrations imparts potentprotection in numerous models of disease, including transplantation(16–18), colitis (19), sepsis (20), and lung injury (21). CO has beenwell-studied and characterized to prevent IRI in small and largeanimal models (22–24), which in turn resulted in the first clinicaltrial where CO was administered to kidney transplant recipientsintraoperatively (https://clinicaltrials.gov/).Circadian rhythms are critical determinants of organ function

and susceptibility to injury dictated by a family of proteins col-lectively known as the “clock genes.”Disruption of one or more ofthese genes increases susceptibility to tissue injury (25). IRI leadsto impairment of the circadian clock (26) and is independent ofhypoxia. Adenosine-elicited A2b-mediated Period 2 (Per2) stabi-lization modulates adaptation to ischemic injury of the heart inmice (27). In fact, tolerance to ischemic injury has been shown tobe dependent upon the time of day that the injury occurs (28).Adenosine from the breakdown of extracellular ATP by

CD39 and CD73 (ecto-5′-nucleotidase) is generated during tissue

Significance

Tissue injury caused by lack of blood flow results in a series ofadaptive responses of the body to ensure survival. Cellularproduction of carbon monoxide (CO) preserves organ functionand promotes healing. How this occurs has remained elusive.Here we demonstrate using a model of ischemia reperfusioninjury (IRI) of the kidney, mimicking kidney transplant, thatsafe administration of CO protects against IRI. Remarkably, thisoccurs through specific modulation of a gene that regulatesenergy metabolism (CD39) and one that controls circadianrhythm (Period 2). Collectively, we define here an innovativesignaling pathway linking the brain and the kidney vis a vis agas molecule. These data may have important therapeuticconsequences for transplant recipients and victims of stroke.

Author contributions: M.C.-C., D.G., C.J.H., B.W., S.C.R., and L.E.O. designed research;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-uted new reagents/analytic tools; M.C.-C., J.-L.L., N.O.S.C., S.C.R., and L.E.O. analyzed data;and M.C.-C., C.J.H., N.O.S.C., S.C.R., and L.E.O. wrote the paper.

Conflict of interest statement: L.E.O. is a scientific consultant for Hillhurst Biopharmaceuticalsand has stock options. E.G. is a founder of Hillhurst Biopharmaceuticals and owns stock.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: lotterbe@bidmc.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1716747115/-/DCSupplemental.

Published online February 20, 2018.

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hypoxia as an adaptive response. In previous work, we have shownthat CO enhances functional expression of adenosine receptorsand stabilization of HIF1α in macrophages (29, 30). Employing amurine model of kidney IRI, we have now tested the hypothesisthat CO protects against kidney IRI through CD39 and adenosineA2b-mediated receptor stabilization of Per2. Additionally, eryth-ropoietin (EPO), a critical effector protein regulated in a circadianmanner, is required for CO-induced renal protection. Collectively,our findings may be related in part to the response of the cellularO2 sensors that drives expression and stabilization of genes thatregulate cellular bioenergetics.

Materials and MethodsAnimals. Isogenic male C57BL/6 mice (WT and Per2−/−) were purchased fromCharles River or Jackson Labs (at 25–30 g). CD39-knockout mice (Cd39−/−) werebred at the BIDMC as described previously (31). All animals had access to waterand food ad libitum. All animal care, housing and procedures were approved bythe Beth Israel Deaconess Medical Center (BIDMC) Institutional Animal Care andUse Committee (IACUC) and were in accordance with the Association for theAssessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.

CO Exposure. CO exposure as an inhaled gas was achieved by placingmice intoa sealed Plexiglas chamber at 250 parts per million (ppm) with compressed air(32). Mice were exposed to CO for 1 h before surgery and then returned toroom air. Controls received air only for the same time period. BW-101 asa CO prodrug (33) was prepared by solubilizing in solutol and used at a doseof 100 mg/kg, i.p. HBI-002 as a liquid CO formulation was orally dosed at0.2 mg/kg. HBI-002 was provided by Hillhurst Biopharmaceuticals Inc.

Pharmacologic Agents. ZM241385 (A2a receptor antagonist) and MRS1754(A2b receptor antagonist) were obtained from Tocris Bioscience and given ina dose of 5 mg/kg i.p. 30 min before CO treatment and 6 h after reperfusion.Recombinant erythropoietin (rEPO) was obtained from eBioscience, andanimals were treated in a dose of 5,000 IU/kg i.p. EPO blocking antibody(R&D) was reconstituted with 1 mL of sterile PBS, and each mouse received80 μL (40 μg per animal, i.p.) immediately before CO and 6 h after reper-fusion. IgG nonspecific antibody (R&D) was used as the control.

Experimental Model of Renal IRI. Surgery was performed as previously de-scribed (34). Briefly, mice were anesthetized with Ketamine-Xylazine, amidline incision was made, and both renal pedicles were cross-clamped for45 min. During the procedure, animals were kept well hydrated with salineand at a constant temperature (∼37 °C) through a heating pad device.Subsequently, microsurgery clamps were removed, the abdomen closed, andanimals placed in single cages, warmed by indirect light until completelyrecovered from anesthesia. Animals were kept under adjustable conditionsuntil sacrifice—namely, 2, 6 and 24 h after renal reperfusion. For thenephrectomy experiment, mice were anesthetized, and bilateral nephrec-tomy was performed after renal pedicle occlusion. In this set of experiments,animals were killed 6 h after CO treatment.

Analysis of Renal Function. Serum creatinine and blood urea nitrogen (BUN)were used for evaluation of renal function. Blood samples were collected atthe indicated time points from the heart, immediately before induced death.Serum samples were analyzed on an IDEXX Catalyst DX analyzer (IDEXXLaboratories).

In Vitro Hypoxia-Reoxygenation Model. LLCPK-1 cells were grown to sub-confluency, serum-starved overnight, and exposed to hypoxia (0.1% O2/5%CO2, balance N) for 16 h followed by reoxygenation (21% O2/5% CO2/bal-ance N2 and/or 21% O2/5% CO/250 ppm CO/balance N2) for 8 h. A group ofcells received 250 ppm CO for 4 h before hypoxia. Control cells were exposedto either normoxia or 250 ppm CO for the same duration.

Cell Death. LLCPK-1 were subjected to a regimen of 16-h anoxia and 8-hreoxygenation (±250 ppm CO) to determine cell death. At each time point,both adherent and suspended cells were harvested and labeled with pro-pidium iodide as a marker of cell death and to determine cell cycle and DNAcontent by FACS.

Cytotoxicity Assay. Crystal violet staining was used to assess the number ofsurviving cells under either normoxic or hypoxic conditions, with or withoutCO pretreatment. At the end of treatment, the number of surviving cells was

determined by the crystal violet stainingmethod. Briefly, surviving cells in six-well plates were washed twice with PBS, and then crystal violet solution(Sigma-Aldrich) was added to the cells. After washing with water, plates wereleft to dry overnight. On the following day, a solution of 10% acetic acid wasadded to each well, and the number of surviving cells was determined bymeasuring absorbance at 562 nm.

ROS Measurement. Intracellular ROS generation was assessed using 2′,7′-dichlorofluorescin diacetate (DCF-DA; 10 mM), and microscopy was per-formed. Cells on coverslips were perfused under controlled O2 and COconditions in a flow-through chamber at 37 °C on an inverted fluorescentmicroscope. Images were acquired with an Olympus camera (excitation,488 nm; emission, 535 nm).

Gene Expression. Total RNA was extracted from homogenized tissues usingRNeasy Mini Kit (Qiagen). For cDNA synthesis, 2 μg of total RNA was used andtranscribed with SuperScript II Reverse Transcriptase (Invitrogen) and ran-dom primers (Invitrogen). The real-time PCR was performed in duplicatewith the Cyber Green PCR Master Mix (BioRad) with the following primers:

Beta-Actin: forward (F)-ACTGGCATTGTCATGGACTC, revers (R)-GCACAGCTTCTCCTTGATGT;

TNF: F-CCTCCCTCTCATCAGTTCTATGG, R-TGTCCCTTGAAGAGACCTGG;

IL-10: F-CCAAGCCTTATCGGAAATGA, R-TTTTCACAGGGGAGAAAT;

PER2: F-GGTGGACAGCCTTTCGATTA, R-AGGGCCTACCCTGACTTTGT;

Adora1 (A1): F-AGAACCACCTCCACCCTTCT, R-TACTCTGGGTGGTGGTCACA;

Adora2a (A2a): F-GAAGCAGATGGAGAGCCAAC, R-GAGAGGATGATGGCCAGGTA;

Adora2b (A2b): F-TGCATGCCATCAACTGTATC, R-TGGAAACTGTAGCGGAAGTC.

The cycling conditions were as following: 95 °C for 15 s, 56 °C for 40 s.

TNF, cGMP, and EPO Measurement. Protein levels of cAMP, TNF, and EPO weredetermined by ELISA (Enzo and R&D) according to the manufacturer’s in-structions. cAMP content was normalized to protein concentration andexpressed as picomole of cAMP/milligram of total protein.

ATP Measurements. ATP levels were determined using a fluorimetric reactionassay (Biovision) according to the manufacturer’s instructions.

Western Blotting. Kidney samples were sonicated in tissue lysis buffer (5 MNaCl, 5 mM EDTA, 1% Triton, 10 mM Tris•Cl, pH 7.4) and clarified by cen-trifugation for 10 min at 17,709 × g. The supernatants were aliquoted andstored at −80 °C. Protein concentration was measured using the bicincho-ninic acid (BCA) method following the manufacturer’s protocol, as describedin Pierce BCA protein assay kit (Pierce). Thirty micrograms of protein extractwere added on SDS/PAGE, transferred to nitrocellulose, and detected usingSuperSignal (Thermo). HIF-1α antibody was purchased from Novus (CO).

Histologic Analysis. During animal sacrifice, one of the kidneys was removed,and after sectioning and removing the renal capsule, it was cut sagittally intwo approximately symmetrical fragments. The material was fixed in 10%buffered formalin until paraffin blocks were assembled. After processing, atleast one cut of each fragment was studied in hematoxylin and eosin (H&E)staining. Immunohistochemical staining for infiltrating cells was performedon 5-μm sections using the HRP method with primary antibodies specific formacrophages (F4/80) or neutrophils/granulocytes (GR1; BD Bioscience). An-tigenic recovery was initially performed with 10 mM citrate buffer, pH 6.0.Then blocking solution (horse serum, 7%) was added to for 30 min. Primaryvalidated antibodies were then added (dilution 1:100 of F4/80 and 1:400 ofGR1), and the slides were incubated overnight. The slides were then washed,and a solution of H2O2 was added for 10 min, followed by further washing.Secondary antibody (with 1 h incubation) was then added, followed byapplication of the avidin/biotin complex. After these steps, hematoxylincounterstaining was performed. For analysis, the slides were observed in ablinded fashion under light microscopy, and 25 randomly selected fields perslide were evaluated and enumerated for positively stained cells (100–200 cells counted per field) and compared to naïve control kidney stainingand secondary antibody controls. This area of interest covered nearly theentire outer medullary regions of each section and thus is highly represen-tative of the extent of positive-stained cells. We used this method ofquantitative analyses previously (32).

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Hypoxia Staining. Hypoxia levels were evaluated in kidney tissue and cells.Animals and cells were treated with hypoxyprobe (Hypoxyprobe Inc.) at60 mg/kg and 400mM, respectively, 1 h before harvesting. For tissue sections,slides were stained following the protocol described above. The detection ofhypoxia in the cells was done by immunofluorescence. Briefly, the cells weregrown on slides and washed 2× with PBS, and a 2% paraformaldehyde so-lution was added. Thereafter, a further wash was performed, and a 0.5%triton solution was added. After further washing, blocking solution (7%horse serum) was added for 30 min, followed by addition of the primaryantibody (diluted 1:100 in PBS) with overnight incubation. The sections werethen incubated for 1 h in secondary antibody and labeled with the TexasRedfluorophore (Abcam). The nuclear dye (Hoechst-Life Technologies) was thenadded, and the slides were analyzed on a fluorescence microscope (Zeiss).

Statistical Analyses. Results are expressed at means ± SD. Statistical analyseswere performed using the Student’s t test and ANOVA. P values less than0.05 were considered significant.

ResultsCO Attenuates Renal Dysfunction Caused by IRI.Others and we havedemonstrated in rat and pig models that inhaled CO given beforeischemia and reperfusion is protective. Therefore, our first ex-periment was to validate the model. Pretreatment with CO (in-haled, orally, or via a CO prodrug) before IRI surgery preventedkidney injury as determined by decreased levels in serum creat-

inine and BUN, which correlated with less inflammatory cellinfiltrate and preservation of kidney architecture (Fig. 1 A–C).IRI-induced monocyte and neutrophil infiltration measured bystaining with F4/80 and GR1, respectively, was markedly reducedin mice treated with CO (Fig. 1 D–F). Inflammatory cytokinesare elevated as a hallmark of IRI. We observed a significant in-crease in the proinflammatory mediator TNF in air-treatedcontrols, which was significantly abrogated in the presence ofCO (Fig. 2A, P < 0.05). As has been shown in other models(35–37), CO exposure simultaneously increased expression ofthe anti-inflammatory cytokine IL-10 (Fig. 2B). These resultssuggest that protection afforded by CO is in part attributable tomodulation of the inflammatory response. Whether these datasuggest an overall mechanism by which CO protects against IRIor if they simply reflect the health of the kidney is unclear.Testing treatment with CO beginning either 1 h or 4 h afterreperfusion did not offer significant benefit. This is not con-cerning since pretreating the donor or recipient are feasibleoptions. Vera et al. (38) showed similar protective effectsagainst IRI-induced kidney injury with a CO releasing molecule(CORM-3). Taken together, these data show that exposure toCO gas modulated cytokine expression to favor a protectivephenotype with decreased infiltration of granulocytes and

Fig. 1. CO prevents kidney IRI in mice. (A and B) Mice were treated with inhaled CO (iCO), oral CO (HBI-002), or a CO-prodrug (BW-101) 1 h before a 45-minbilateral kidney ischemia. Serum creatinine (A) and BUN (B) were measured 24 h after reperfusion. Results represent mean ± SD of 5–10 mice per group. *P <0.001 vs. iCO, P < 0.05 vs. HBI-002, and P < 0.01 vs. BW-101. (C) Representative H&E-stained sections from control kidney and from mice subjected to IRI treatedwith Air or iCO as above. Images are representative of 10 sections from five mice. Arrows indicate leukocyte infiltration. (D–F) Quantitation of F4/80- and GR1-positive staining for macrophage and neutrophils, respectively. Results represent mean ± SD of 6–8 sections from five mice in each group. **P < 0.05 vs. CO.

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macrophages and maintenance of renal function otherwise lostin air-treated controls.

CO Prevents Hypoxia/Reoxygenation (H/R)-Induced Cellular Stress.Exposure of cells to a period of hypoxia followed by transfer toa normoxia atmosphere results in a very similar cellular responseto that observed with IRI cytokine expression, ROS generationand cell death. Parallel experiments in vitro in kidney epithelial

cells showed that exposure to CO reduced (H/R)-induced ele-vations in ROS generation and completely prevented cell death(Fig. 3 A–C). As observed in the kidney, CO also reduced ele-vations in TNF, MCP-1, and IL-1β expression (Fig. S1).Previous studies in the liver and lung showed that exposure to

CO can paradoxically reduce tissue hypoxia, likely involving theability of CO to displace cellular O2 stores (39–41). We thereforetested the effects of CO on kidney epithelial cells exposed toH/R and in vivo in a kidney subjected to ischemia/reperfusion(I/R). CO effectively reduced cell and tissue hypoxia measuredwith the O2-sensitive hypoxyprobe (Fig. 3 D and E). The lack ofhypoxia observed in H/R and I/R corresponded to the reductionin ROS shown in Fig. 3A. Further, we measured ATP in H/Rsamples and observed a significant decrease in ATP (0.9 ±0.14 nmol) by H/R versus normoxic controls (1.25 ± 0.10 nmol,P < 0.01; Fig. 3F). CO restored ATP levels to 1.12 ± 0.04 nmol,which could be explained by enhancing oxidative phosphoryla-tion given the results of the hypoxyprobe experiments. CO mayact to reduce oxygen consumption, thereby making more available;generate more ATP via regulation of cytochrome oxidase activity;or influence O2 availability by displacement from intracellularstores. Work is ongoing toward delineating the biochemicaleffects low concentrations of CO impart on overall cellular

Fig. 2. CO modulates cytokine expression in the kidney after IRI. (A and B)Tissue expression of TNF and IL-10 mRNA in the kidney in naïve mice or kidneysfrom mice 24 h after IRI treated with either Air or CO. CO blocked TNF andsimultaneously enhanced IL-10 expression. Results represent mean ± SD of5 per group. *P < 0.05 vs. CO; **P < 0.01 vs. control and IRI + Air.

Fig. 3. Effects of CO on promoting kidney epithelial cell bioenergetics and viability in response to H/R conditions. (A) Exposure to CO (250 ppm) prevented H/R-induced elevations in DCF fluorescence, as a marker for reactive oxygen species production. Images are representative of at least three independent experimentsin triplicate. (B and C) LLCPK-1 kidney epithelial cell viability measured by crystal violet (B) or propidium iodine incorporation (C) was determined in response toH/R in the presence and absence of CO (250 ppm). Results represent mean ± SD of 4–6 samples in triplicate. *P < 0.02 vs. normoxia or H/R + CO. (D and E) COreduced tissue hypoxia measured with the O2-sensitive hypoxyprobe. Cells and animals were treated with hypoxyprobe at 400 mM and 60 mg/kg 1 h beforeharvesting, and cells and tissue sections from H/R and IRI, respectively, were stained as described inMaterials andMethods. (F) ATP levels in LLCPK-1 cells shows COreversing an H/R-induced decrease in ATP at 24 h of H/R. Results represent mean ± SD of three independent experiments in triplicate. *P < 0.05, **P < 0.02.

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function and metabolism. Control animals treated with COor air in the absence of H/R or I/R showed no increase inhypoxyprobe positivity, ROS generation, or cell death. Thesedata suggest that CO acts in part to reduce the severity ofcellular and tissue hypoxia that ultimately contributes in partto attenuation of kidney damage that occurs at the timeof reoxygenation.

CO Increases CD39 and Modulates Adenosine Receptor Expression inthe Kidney. We previously described the effects of CO on aden-osine receptor expression in endotoxin-stimulated macrophages yethad no in vivo correlate (29). Adenosine levels are increased inresponse to tissue hypoxia and are important in renal cytoprotection(42, 43). The abrogation of cellular and tissue hypoxia by CO de-scribed above directed us to next look at the effects of CO on tissuelevels of the 5′ectonucleotidase CD39 and the adenosine receptorsA1 and A2 in the kidney subjected to IRI. CD39 is known to beup-regulated in response to changes in tissue O2 levels dictatedby oxygen sensors (44). Following reperfusion, we observed a 20-fold up-regulation of CD39 by 2 h that returned to baseline by6 h. Animals treated with CO showed enhanced expression ofCD39 50 ± 3% over IRI alone at 2 h, which remained nearlyeightfold higher at 6 h before returning to baseline at 24 h (Fig.4A). The increase in CD39 expression correlated with a threefolddown-regulation in the expression of A1 in the kidney followingreperfusion at 2, 6, and 24 h and conversely a twofold up-regulation of A2a and A2b receptor expression at these sametime points (Fig. 4 B–D). Kidneys harvested from control naïvemice exposed to CO showed up-regulation of A2b, no change inA2a, and a down-regulation of A1 expression over kidneys fromnaïve air controls (Fig. S2). Collectively, these data suggest thatexposure to CO by modulating adenosine receptor expressionlevels influences susceptibility to injury by differentially alteringsensitivity to adenosine.

CO Requires CD39 to Prevent Kidney IRI. Up-regulation ofCD39 and the A2b receptor by both IRI and CO suggested animportant role in the injury response. We next tested the ability ofCO to prevent IRI in Cd39−/− mice. The IRI injury in the absenceof CD39 was modest yet significantly elevated over wild-type

controls (P < 0.05). However, unlike the protection observed byCO inWTmice, CO was completely ineffective in Cd39−/−mice inreducing IRI-induced elevations in serum creatinine, BUN, andTNF levels (Fig. 5 A–C). Pharmacologic inhibition of A2 receptorsusing Zm241385 plus MRS1754 mirrored the results observed inthe Cd39−/− mice. The protection afforded by CO to prevent IRI,as measured by serum creatinine, BUN, and TNF, was lost inthese mice with decreased adenosine generation with Cd39 de-letion or A2b/A2a inhibition (Fig. 5 D–F).

CO Regulates Per2 via CD39 and A2 Receptor Signaling. Adenosinebinding to the A2b receptor has been shown to increase ex-pression of the circadian rhythm molecule Per2, which directs aprotective response to cardiac ischemia (27). It is also knownthat CO can regulate circadian rhythm gene expression, specif-ically NPAS2 (45), which regulates the period genes (46). Giventhe above, we next tested if CO modulated Per2 expression viaA2 receptor signaling. Data presented in Fig. 6A show that

Fig. 4. Effects of IRI and CO on CD39 and A2 receptor expression. (A–D)Expression levels of CD39, A1, A2a, and A2b were measured by PCR overtime after reperfusion. Animals treated with CO increased expression ofCD39 and the A2 receptors but inhibited IRI-induced increases in A1 receptorexpression. Results represent mean ± SD of 4–6 mice per group at each timepoint. *P < 0.02 vs. Air.

Fig. 5. CD39 activity and A2 receptor signaling are required for CO to preventkidney IRI in mice. (A–C) CO blocks IRI-induced elevations in creatinine andBUN and tissue levels of TNF, but these effects are lost in Cd39−/− mice. Resultsrepresent mean ± SD of 4–6 mice per group. Moreover, animals lackingCD39 and subjected to IRI showed enhanced severity of injury compared withWT mice with IRI regardless of CO. (D–F) Similar to Cd39−/− mice, WT micetreated with selective inhibitors of A2a and A2b receptors, Zm241385 andMRS1754, respectively, were also nonresponsive to the protection afforded byCO against IRI. Serum creatinine, BUN, and tissue TNF levels were similar tothat observed in Cd39−/− mice. Results represent mean ± SD of 4–6 mice pergroup. *P < 0.05 vs. control and CO-WT; **P < 0.001 vs. other groups.

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animals subjected to kidney IRI and exposed to CO have a sig-nificant increase in Per2 expression at 6 and 24 h versus nochange in air-exposed IRI alone-treated mice. This expressionrequired A2 receptor activity, as inhibition with Zm241385 plusMRS1754 completely abrogated Per2 expression in CO-treatedmice subjected to kidney IRI (Fig. 6B). The increase in A2receptor expression by CO has been demonstrated in macro-phages where A2 receptors were increased on the cell mem-brane. This resulted in potent activation of intracellular signaltransduction that resulted in a more tolerogenic profile andlower production of TNF (22, 29).The increased expression of Per2 led us to test whether absence

of Per2 would abrogate the reno-protective effects of CO. Per2−/−

mice subjected to the standard 45-min IRI did not survive theinsult, and CO was unable to provide protection. We thereforereduced the IRI to 30 min, which was not sufficient to induceinjury in WT mice but resulted in significant injury in the Per2−/−

mice with elevations in serum BUN without mortality. The in-creased BUN in the Per2−/− was unaffected by CO treatment (Fig.6C). Characterization of the Per2 circadian expression profileshowed that Per2 expression in naïve mice peaked in the kidney inthe late afternoon (Zeitgeber 9, ZT9; Fig. S3). All studies de-scribed above were performed in the morning (ZT3) whenPer2 expression was lowest. To further validate a role for Per2 inIRI, we subjected a cohort of WTmice to IRI (45 min) at ZT9 andobserved significantly less IRI versus IRI performed at ZT3 (Fig.6D). Collectively, we identify Per2 as a cytoprotective gene im-portant in protecting against kidney IRI. Such modulation, knownas “immunochronotherapy,” aids in tissue recovery and elimina-tion of potentially harmful intracellular molecules (47).Adenosine A2 receptors are coupled to stimulatory G protein,

and their activation leads to increased levels of cAMP and reg-ulation of Per2. With the increase in A2 receptor expression andelevated levels of CD39 and ATP by CO, we next looked at A2-linked cAMP activation. Exposure to CO resulted in significantly

increased levels of cAMP and HIF1α levels in the kidney afterIRI (Fig. 7). The elevation in cAMP, HIF1α, and Per2 is im-portant in protection against IRI (27). Eckle and coworkersdemonstrated that A2b-dependent Per2 stabilization promotesHIF-dependent gene regulation; Per2 is also known to stabilizeHIF-1α expression, as does CO (27, 30, 39). The protective roleof HIF-1α in renal ischemia has been described previously andthought to be important for a faster recovery of the injured tissue(39, 46). Indeed, Hill and coworkers (48, 49) showed that re-nal IRI in HIF-1α+/− animals showed significant worsening ofrenal dysfunction, while pharmacologically increasing the half-life of HIF-1α led to improved renal function and reducedinflammation.

EPO Mediates CO-Induced Protection Against IRI. Stabilization ofHIF-1α results in translocation to the nucleus to promote tran-scription of a number of target genes including endothelin andEPO. The remarkable effects of CO on HIF1α described aboveand in previous reports and the fact that increased carbox-yhemoglobin leads to elevations in EPO led us to next test thehypothesis that EPO was the penultimate target gene involved inthe protective effects observed with CO against IRI. Exposure ofnaïve mice to CO led to increased serum EPO levels within 2 h(Fig. 8A). The increased serum EPO partially originated fromthe kidney as CO-exposed binephrectomized animals alsoexhibited significant elevations in serum EPO, which we posit isliver-derived (Fig. 8B). Assessment of EPO pharmacodynamicsshowed that administration of recombinant mouse EPO wassimilar with or without CO exposure (Fig. S4). The CO-inducedincrease in serum EPO was also observed in animals subjected toIRI compared with air-treated mice subjected to IRI (Fig. 8A).Interestingly, CO-treated Per2−/− mice showed an up-regulationof serum EPO, compared with nontreated littermates (Fig. 8C),suggesting that CO could increase EPO through an indirectmanner, or perhaps Per2 lies downstream of EPO signaling.Neutralization of EPO with an anti-mouse EPO antibody

completely abrogated the effects of CO to protect against kidneyIRI versus IgG control (Fig. 9 A–C). CO-induced increases incAMP levels remained unchanged in the antibody-treated ani-mals, suggesting that the upstream A2–HIF1α/Per2 signaling was

Fig. 6. Expression of the circadian rhythm gene Per2 is required for CO toprotect the kidney against IRI and is dependent on A2 receptor activation.(A) Expression levels of Per2 over time in the kidney in response to IRI ± CO.CO markedly increases Per2 in conjunction with IRI versus no change ob-served in IRI+Air-treated mice. *P < 0.05 vs. Air. (B) Blockade of A2 receptorsprevents the CO-induced increase in Per2 expression. *P < 0.05 vs. Air. (C) COis unable to prevent IRI-induced elevations in serum BUN in Per2−/− mice.*P < 0.05 vs. other groups. (D) Since Per2 cycles throughout the day andpeaks at ZT9, we tested susceptibility to kidney IRI compared with ZT3 whenPer2 expression is low (Fig. S3). Mice that underwent IRI at ZT9 showed lesssevere IRI measured by elevations in BUN versus those subjected to IRI at ZT3.Results represent mean ± SD of 4–6 mice per group. *P < 0.05, **P < 0.001.

Fig. 7. A2 receptor signaling is activated in CO-treated mice and associatedwith increased expression of HIF-1α. (A) Mice subjected to kidney IRI ± COwere killed at different times after reperfusion, and cAMP levels weremeasured by ELISA. *P < 0.01 vs. Air. (B) Kidney lysates were evaluated atdifferent time points after reoxygenation for HIF-1α expression as indicated.Results represent mean ± SD of 4–6 mice per group.

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still operational (Fig. 9D). Finally, we tested the effects of CD39on the signaling cascade that resulted in EPO up-regulation.CD39-null mice exposed to CO showed no increase in Per2and EPO expression (Fig. 9 E and F). Collectively we delineate asignaling cascade for CO in the kidney that involves increases inATP and CD39 that ultimately leads to Per2-dependent up-regulation of EPO and protection against IRI.

DiscussionResults obtained in this study demonstrate that CO protects thekidney against IRI, at least in part by increasing circulating levelsof EPO. Further mechanistic investigation of the increase in

EPO found expression to be regulated and completely de-pendent on purinergic signaling initiated by stabilization of ATPproduction and increased expression of CD39, an ecto-nucleoside triphosphate diphosphohydrolase/ectonucleotidase.The resulting generation of adenosine correlated with a markedincrease in CD39 and CD73. Downstream of the A2 receptor, wefind increased cAMP and Per2 expression and stabilization ofHIF1α and likely other O2 sensors that in large part wouldregulate EPO expression. Curiously, both extracellular ATP(eATP) and hypoxia, in the setting of inflammation might triggeraryl hydrocarbon receptor (AHR) inactivation by HIF1α. In areciprocal manner, the presence of CD39, which might be in-duced by AHR, would deplete eATP and suppress HIF1α-mediated activity by generating adenosine in tandem with CD73.Several studies have tried to unravel the cytoprotective role of

EPO in models of renal injury. The kidneys are particularly sen-sitive to high glucose levels, and EPO administration preventedrecurrent dysfunction during transient hyperglycemia. While un-able to prevent tubular necrosis, treatment with EPO was able toattenuate apoptosis and glomerular dysfunction (50). Perhaps thebest described mechanism supporting the beneficial properties ofEPO is its potent anti-inflammatory effects. In a model of acutekidney injury (AKI) in response to sepsis, treatment with EPOresulted in lower renal expression of TLR4, NF-κB, CD68, and abattery of proinflammatory molecules compared with the vehiclecontrols. In contrast, levels of EPOR were elevated after treat-ment (51–53). Hu and colleagues have shown that treatment withEPO before ischemic injury promoted an improvement in renalfunction with a reduction in tubular necrosis, which correlated todecreased neutrophil infiltration, lower expression of proin-flammatory chemokines, and decreased translocation of NF-κBinto the nucleus (54). As to Per and A2A versus A2B functionality,it has been shown that A2A and A2B interact to promote ex-pression of a chimeric receptor. A2B can only be expressed withA2A and would thus explain the Per2 data (55).There are several studies that have demonstrated that EPO

regulates the expression of HO-1. In a model of experimental

Fig. 8. CO exposure increases serum EPO levels. (A) Serum EPO levels wereevaluated in naive, Air-, and CO-treated mice ± IRI. Blood samples werecollected at different times after reoxygenation. *P < 0.05 vs. Air; **P <0.001 vs. Air. (B) EPO levels were measured in naive and bilateral nephrec-tomized mice. *P < 0.05. (C) EPO levels were measured in per2−/− mice. *P <0.05 vs. baseline. Results represent mean ± SD of 4–6 mice per group.

Fig. 9. EPO is associated with a better outcome after CO treatment, and CD39 is an important mediator of the CO-induced protection in the IRI model. Micewere subjected to IRI ± CO in the presence or absence of anti-EPO blocking antibody or an IgG control antibody and were evaluated by Serum Creatinine (A),BUN (B), kidney TNF (C), and cAMP levels (D). *P < 0.05 vs. the other groups. (E and F) Per2 mRNA and serum EPO levels in WT and CD39 KO mice, subjected toIRI ± CO. *P < 0.02 vs. all of the other groups. Results represent mean ± SD of 5 mice per group.

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autoimmune encephalomyelitis (EAE), EPO administration ledto increased expression of HO-1 and modulation of adaptiveimmunity, leading to a suppression of the inflammatory response(56). Another study showed that the increased expression of HO-1 by EPO occurs through the activation of PI3K pathways,MAPK, and Nrf2 (57). Finally, Burger et al. (58) showed thatEPO-induced up-regulation of HO-1 was responsible for thereduction of apoptosis of cardiomyocytes after IRI becauseblockade of HO-1 or administration of EPO to Hmox1−/− miceresulted in loss of EPO protection. Based on these data, wepropose a cycle or feed-forward loop amplification in the kidneywhere HO-1, likely via CO generation, leads to increased ex-pression of EPO as defined by the signaling described above. TheEPO, in turn, imparts protection but requires the presence ofHO-1, perhaps as continuous protection.We have published previously that exposure of macrophages to

CO resulted in increased expression of the A2a receptor and asignificant increase in sensitivity to adenosine (29). Given the roleof CD39 in the generation of adenosine, we tested Cd39−/− animalsto examine whether CO-mediated ectonucleotidase signaling wasimportant in the protection afforded by CO and therein the effectson EPO expression. Indeed, lack of CD39 resulted in worse renalfunction after ischemia and reperfusion, with increased serumlevels of creatinine and BUN and expression of TNF. CO wasunable to rescue Cd39−/− animals, likely explained by a poor EPOresponse. Finally, with decreased pericellular adenosine levels, weobserved no increase in cAMP or Per2 compared with wild-typelittermates exposed to Air. HIF1α has been shown to affect cir-cadian expression of Per2 in kidney cells, and inhibition in HIF1αdecreased the amplitude of the circadian rhythm of the Per2 pro-moter (59). Adamovich et al. (60) recently reported that oxygen,via HIF1α, is a resetting cue for circadian clocks and includesregulation of transcript levels of Rev-erbα and Per2. The requisitecompetition that CO has for similar binding sites also bound byoxygen in numerous heme-containing proteins such as Rev-erbα, itis plausible that CO targets similar signaling pathways directly orindirectly via HIF1α activity. Perhaps the most compelling andelegant data linking HIF1α and Per2 were reported by Eckle et al.(27), who showed that stabilization of Per2 is controlled by HIF1αand that this effect limited ischemic damage of the heart. Further,they show that Per2 could be stabilized with exposure of the mouseto intense light. With regard to organ transplantation, perhaps therecipient or the organ itself, during the preservation time and whilein transit, could be exposed to light to improve function in con-junction with optimal protective rhythms. Whether AKI has a cir-cadian rhythmicity is unclear, but studies in the heart and lung haveclearly demonstrated that the dyssynchrony or reorganization of thecentral clock in the brain can impact susceptibility to cardiovasculardisease and lung injury (61, 62). The association between AKI andcircadian rhythm has not been well studied and validated. How-ever, it has been substantiated by literature evidence showing theclear impact of the central and local circadian clocks on sodiumregulation and susceptibility to organ injury in individuals with lownocturnal blood pressures versus those with high nocturnal pres-sures. Understanding how and when an organ might be mostsensitive to injury may offer innovative therapeutic opportunities.Methodologies for continuously measuring real-time kidney func-tion in animals has been described and will be useful in models ofIRI and AKI.Due to its physicochemical characteristics, CO easily crosses the

plasma membrane and acts as a potent intracellular messenger.The genetic regulation of CD39 appears to be exerted by thetranscription factor Sp1, a member of a family referred to as Sp/XKLF, which also regulates VEGF and cystathionine-β-synthase(44). Furthermore, ischemic injury leads to a reduction in ex-pression of Sp1 in the renal tissue (63). There is one reportshowing that treatment with CO leads to increased expression ofSp1 and overexpression of a mutant form of this factor abrogated

its transcriptional activity induced by CO (64). Liao et al. (65)showed that CD39 expression is also regulated by increased cy-toplasmic levels of cAMP, which involves activation of the PKA/CREB, PKA/PI3K/ATF2, and PKA/ERK/ATF2 pathways. Be-cause we also observed an increase in cAMP levels in the kidneysof our treated animals, we conclude that the regulation of CD39may occur in part via activation of this second messenger (65).Both ATP and adenosine are important in protection against renalinjury (66). It is also worth noting that renal tubular, vascularendothelial, as well as mesangial cells express ectonucleotidaseson their surfaces with enzymatic properties that have high simi-larity to CD39. Finally, during an ischemic injury, the enzymaticcapacity to convert ATP to ADP and then to AMP is reduced byup to 71% (67–69) Thus, these results support our findings thatCO-induced up-regulation of CD39 on renal cells is a criticalevent necessary in protecting the kidney from injury.Cell regeneration after an ischemic insult is favored by the

presence of energy sources capable of accelerating such re-parative process. Thus, the availability of ATP is crucial for anefficient recovery. In this light, CO exposure leads to an increasein protein levels of PGC-1α and mitochondrial biogenesis (70–72), which has been observed by others and both of which areregulated by adenosine signaling (73, 74). The greater number ofmitochondria in the animals exposed to CO could explain thecontinued presence of ATP that is otherwise lost in response toIR. Mitochondrial dysfunction is a mediator of a variety of cel-lular insults and a common element in the initiation of variousdiseases. The importance of mitochondrial biogenesis is reflec-ted in its ability to increase the activity of metabolic pathwayssuch as fatty acid oxidation, in addition to increasing the anti-oxidant defense mechanisms, mitigating damage from aging,tissue hypoxia, excess glucose, or fatty acids, which contribute tothe pathogenesis of acute and chronic renal injuries (70, 71). Arecently published study showed that the use of compounds thatmay promote an increased capacity for mitochondrial biogenesisin the cell could be a promising therapeutic target in the future(72). Without the loss in ATP, cells can proliferate, as evidencedby increased ERK expression, better viability, and normal cellcycle pattern, suggesting that the presence of CO favors tissuehomeostasis, promoting faster recovery when necessary.Based on our findings, we conclude that CO promotes a

beneficial effect on ischemic renal injury by a mechanism de-pendent on purinergic signaling and Per2 expression. Treat-ment with CO generates an increased expression of CD39 andtype 2 adenosine receptors, which once activated lead to acascade of events that stabilizes HIF-1α via Per2, allowing itstranscriptional activity to be more sustainable. One of thetarget genes for HIF1α is EPO, which is important in protectionof the kidney. Collectively, our work provides a critical mech-anism by which CO is able to protect the kidney in a model ofIRI. Translation to organ transplant utilizing a pretreatmentregimen is certainly viable when considering treating the donorand/or recipients before harvest or implantation, as has beendemonstrated in large and small animal models (23, 24). Inpigs, having COHb peaking at the time the renal vessels areunclamped is very effective at reducing delayed graft function(23). Additional postoperative dosing with CO as well astreatment of the donor and graft itself during preservation mayoffer additional benefits. Given that clinical trials for CO areongoing, these findings lend insight into the cellular and mo-lecular mechanisms of action. We further conclude that theprotective effects of CO treatment also extend to cellularmetabolic changes, which improve energy charge, functionalcapacity, and as such, favor tissue recovery.

ACKNOWLEDGMENTS. The studies were supported by NIH Award R44DK111260-01 and Department of Defense Award W81XWH-16-0464 (to L.E.O.)and the Sao Paulo Research Foundation (FAPESP) Grant 2011/19581-8 (to M.C.-C.).

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1. Eltzschig HK, Eckle T (2011) Ischemia and reperfusion–Frommechanism to translation.Nat Med 17:1391–1401.

2. Chertow GM, Burdick E, Honour M, Bonventre JV, Bates DW (2005) Acute kidneyinjury, mortality, length of stay, and costs in hospitalized patients. J Am Soc Nephrol16:3365–3370.

3. Jang HR, Ko GJ, Wasowska BA, Rabb H (2009) The interaction between ischemia-reperfusion and immune responses in the kidney. J Mol Med (Berl) 87:859–864.

4. Munshi R, Hsu C, Himmelfarb J (2011) Advances in understanding ischemic acutekidney injury. BMC Med 9:11.

5. Bonventre JV, Weinberg JM (2003) Recent advances in the pathophysiology of is-chemic acute renal failure. J Am Soc Nephrol 14:2199–2210.

6. Thurman JM (2007) Triggers of inflammation after renal ischemia/reperfusion. ClinImmunol 123:7–13.

7. Ysebaert DK, et al. (2000) Identification and kinetics of leukocytes after severe is-chaemia/reperfusion renal injury. Nephrol Dial Transplant 15:1562–1574.

8. Hwang HS, et al. (2013) Pretreatment with paricalcitol attenuates inflammation inischemia-reperfusion injury via the up-regulation of cyclooxygenase-2 and prosta-glandin E2. Nephrol Dial Transplant 28:1156–1166.

9. Kinsey GR, et al. (2012) Autocrine adenosine signaling promotes regulatory T cell-mediated renal protection. J Am Soc Nephrol 23:1528–1537.

10. Ko GJ, et al. (2011) Blocking Fas ligand on leukocytes attenuates kidney ischemia-reperfusion injury. J Am Soc Nephrol 22:732–742.

11. Wang PH, et al. (2008) Bradykinin [corrected] B1 receptor antagonism is beneficial inrenal ischemia-reperfusion injury. PLoS One 3:e3050.

12. Yang SH, et al. (2011) Sulfatide-reactive natural killer T cells abrogate ischemia-reperfusion injury. J Am Soc Nephrol 22:1305–1314.

13. Wegiel B, Nemeth Z, Correa-Costa M, Bulmer AC, Otterbein LE (2014) Hemeoxygenase-1: A metabolic nike. Antioxid Redox Signal 20:1709–1722.

14. Jarmi T, Agarwal A (2009) Heme oxygenase and renal disease. Curr Hypertens Rep 11:56–62.15. Wagener FA, et al. (1999) Differential effects of heme oxygenase isoforms on heme

mediation of endothelial intracellular adhesion molecule 1 expression. J PharmacolExp Ther 291:416–423.

16. Amano MT, Camara NO (2013) The immunomodulatory role of carbon monoxideduring transplantation. Med Gas Res 3:1–15.

17. Ozaki KS, et al. (2012) Carbon monoxide inhibits apoptosis during cold storage andprotects kidney grafts donated after cardiac death. Transplant Int 25:107–117.

18. Wang H, et al. (2012) Activation of peroxisome proliferator-activated receptor γprolongs islet allograft survival. Cell Transplant 21:2111–2118.

19. Onyiah JC, et al. (2013) Carbon monoxide and heme oxygenase-1 prevent intestinalinflammation in mice by promoting bacterial clearance. Gastroenterology 144:789–798.

20. MacGarvey NC, et al. (2012) Activation of mitochondrial biogenesis by hemeoxygenase-1-mediated NF-E2-related factor-2 induction rescues mice from lethalStaphylococcus aureus sepsis. Am J Respir Crit Care Med 185:851–861.

21. Zuckerbraun BS, et al. (2006) Carbon monoxide reverses established pulmonary hy-pertension. J Exp Med 203:2109–2119.

22. Hou J, et al. (2013) 5-Aminolevulinic acid combined with ferrous iron induces carbonmonoxide generation in mouse kidneys and protects from renal ischemia-reperfusioninjury. Am J Physiol Renal Physiol 305:F1149–F1157.

23. Hanto DW, et al. (2010) Intraoperative administration of inhaled carbon monoxidereduces delayed graft function in kidney allografts in Swine. Am J Transplant 10:2421–2430.

24. Caumartin Y, et al. (2011) Carbon monoxide-releasing molecules protect againstischemia-reperfusion injury during kidney transplantation. Kidney Int 79:1080–1089.

25. Curtis AM, Bellet MM, Sassone-Corsi P, O’Neill LA (2014) Circadian clock proteins andimmunity. Immunity 40:178–186.

26. Kung TA, et al. (2007) Rapid attenuation of circadian clock gene oscillations in the ratheart following ischemia-reperfusion. J Mol Cell Cardiol 43:744–753.

27. Eckle T, et al. (2012) Adora2b-elicited Per2 stabilization promotes a HIF-dependentmetabolic switch crucial for myocardial adaptation to ischemia. Nat Med 18:774–782.

28. Durgan DJ, et al. (2010) Short communication: Ischemia/reperfusion tolerance is time-of-day-dependent: Mediation by the cardiomyocyte circadian clock. Circ Res 106:546–550.

29. Haschemi A, et al. (2007) Cross-regulation of carbon monoxide and the adenosineA2a receptor in macrophages. J Immunol 178:5921–5929.

30. Chin BY, et al. (2007) Hypoxia-inducible factor 1alpha stabilization by carbon mon-oxide results in cytoprotective preconditioning. Proc Natl Acad Sci USA 104:5109–5114.

31. Beldi G, et al. (2008) Regulated catalysis of extracellular nucleotides by vascular CD39/ENTPD1 is required for liver regeneration. Gastroenterology 135:1751–1760.

32. Otterbein LE, et al. (2003) Carbon monoxide suppresses arteriosclerotic lesions asso-ciated with chronic graft rejection and with balloon injury. Nat Med 9:183–190.

33. Ji X, et al. (2016) Toward carbon monoxide-based therapeutics: Critical drug deliveryand developability issues. J Pharm Sci 105:406–416.

34. Correa-Costa M, et al. (2012) Transcriptome analysis of renal ischemia/reperfusion injuryand its modulation by ischemic pre-conditioning or hemin treatment. PLoS One 7:e49569.

35. Kawanishi S, et al. (2013) Inhalation of carbon monoxide following resuscitationameliorates hemorrhagic shock-induced lung injury. Mol Med Rep 7:3–10.

36. Piantadosi CA, et al. (2011) Heme oxygenase-1 couples activation of mitochondrialbiogenesis to anti-inflammatory cytokine expression. J Biol Chem 286:16374–16385.

37. Pulido JN, et al. (2011) Inhaled carbon monoxide attenuates myocardial inflammatory cyto-kine expression in a ratmodel of cardiopulmonary bypass. J Extra Corpor Technol 43:137–143.

38. Vera T, Henegar JR, Drummond HA, Rimoldi JM, Stec DE (2005) Protective effect ofcarbon monoxide-releasing compounds in ischemia-induced acute renal failure. J AmSoc Nephrol 16:950–958.

39. Faleo G, et al. (2008) Carbon monoxide ameliorates renal cold ischemia-reperfusioninjury with an upregulation of vascular endothelial growth factor by activation ofhypoxia-inducible factor. Transplantation 85:1833–1840.

40. Fujita T, et al. (2001) Paradoxical rescue from ischemic lung injury by inhaled carbonmonoxide driven by derepression of fibrinolysis. Nat Med 7:598–604.

41. Zuckerbraun BS, et al. (2005) Carbon monoxide prevents multiple organ injury in amodel of hemorrhagic shock and resuscitation. Shock 23:527–532.

42. Bauerle JD, Grenz A, Kim JH, Lee HT, Eltzschig HK (2011) Adenosine generation andsignaling during acute kidney injury. J Am Soc Nephrol 22:14–20.

43. Vallon V, Mühlbauer B, Osswald H (2006) Adenosine and kidney function. Physiol Rev86:901–940.

44. Eltzschig HK, et al. (2009) Central role of Sp1-regulated CD39 in hypoxia/ischemiaprotection. Blood 113:224–232.

45. Gilun P, et al. (2013) Carbon monoxide-mediated humoral pathway for the trans-mission of light signal to the hypothalamus. J Physiol Pharmacol 64:761–772.

46. Stow LR, Gumz ML (2011) The circadian clock in the kidney. J Am Soc Nephrol 22:598–604.

47. Scheiermann C, Kunisaki Y, Frenette PS (2013) Circadian control of the immune sys-tem. Nat Rev Immunol 13:190–198.

48. Bonventre JV, Yang L (2011) Cellular pathophysiology of ischemic acute kidney injury.J Clin Invest 121:4210–4221.

49. Hill P, et al. (2008) Inhibition of hypoxia inducible factor hydroxylases protects againstrenal ischemia-reperfusion injury. J Am Soc Nephrol 19:39–46.

50. Hochhauser E, et al. (2008) Recombinant human erythropoietin attenuates hepatic injuryinduced by ischemia/reperfusion in an isolated mouse liver model. Apoptosis 13:77–86.

51. Caetano AM, et al. (2011) Erythropoietin attenuates apoptosis after ischemia-reperfusion-induced renal injury in transiently hyperglycemic Wister rats.Transplant Proc 43:3618–3621.

52. Souza AC, et al. (2012) Erythropoietin prevents sepsis-related acute kidney injury inrats by inhibiting NF-κB and upregulating endothelial nitric oxide synthase. Am JPhysiol Renal Physiol 302:F1045–F1054.

53. Rodrigues CE, et al. (2012) Effects of continuous erythropoietin receptor activator insepsis-induced acute kidney injury and multi-organ dysfunction. PLoS One 7:e29893.

54. Hu L, et al. (2012) Erythropoietin ameliorates renal ischemia and reperfusion injuryvia inhibiting tubulointerstitial inflammation. J Surg Res 176:260–266.

55. Moriyama K, Sitkovsky MV (2010) Adenosine A2A receptor is involved in cell surfaceexpression of A2B receptor. J Biol Chem 285:39271–39288.

56. Chen SJ, et al. (2010) Erythropoietin enhances endogenous haem oxygenase-1 andrepresses immune responses to ameliorate experimental autoimmune encephalo-myelitis. Clin Exp Immunol 162:210–223.

57. Genc K, Egrilmez MY, Genc S (2010) Erythropoietin induces nuclear translocation ofNrf2 and heme oxygenase-1 expression in SH-SY5Y cells. Cell Biochem Funct 28:197–201.

58. Burger D, Xiang F, Hammoud L, Lu X, Feng Q (2009) Role of heme oxygenase-1 in thecardioprotective effects of erythropoietin during myocardial ischemia and re-perfusion. Am J Physiol Heart Circ Physiol 296:H84–H93.

59. Okabe T, et al. (2014) The impact of HIF1α on the Per2 circadian rhythm in renalcancer cell lines. PLoS One 9:e109693.

60. Adamovich Y, Ladeuix B, Golik M, Koeners MP, Asher G (2017) Rhythmic oxygen levelsreset circadian clocks through HIF1α. Cell Metab 25:93–101.

61. Haspel JA, et al. (2014) Circadian rhythm reprogramming during lung inflammation.Nat Commun 5:4753.

62. Khaper N, et al. (2017) Implications of disturbances in circadian rhythms for cardio-vascular health: A new frontier in free radical biology. Free Radic Biol Med, S0891-5849(17)31199-1.

63. Wu N, Siow YL, O K (2010) Ischemia/reperfusion reduces transcription factor Sp1-mediated cystathionine beta-synthase expression in the kidney. J Biol Chem 285:18225–18233.

64. Lin HH, Lai SC, Chau LY (2011) Heme oxygenase-1/carbon monoxide induces vascularendothelial growth factor expression via p38 kinase-dependent activation of Sp1.J Biol Chem 286:3829–3838.

65. Liao H, Hyman MC, Baek AE, Fukase K, Pinsky DJ (2010) cAMP/CREB-mediated tran-scriptional regulation of ectonucleoside triphosphate diphosphohydrolase 1 (CD39)expression. J Biol Chem 285:14791–14805.

66. Szoleczky P, et al. (2012) Identification of agents that reduce renal hypoxia-reoxygenation injury using cell-based screening: Purine nucleosides are alternativeenergy sources in LLC-PK1 cells during hypoxia. Arch Biochem Biophys 517:53–70.

67. Ribeiro MC, et al. (2012) Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions. Biochim Biophys Acta 1820:2030–2036.

68. Candinas D, et al. (1996) Loss of rat glomerular ATP diphosphohydrolase activityduring reperfusion injury is associated with oxidative stress reactions. ThrombHaemost 76:807–812.

69. Kishore BK, et al. (2005) Expression of NTPDase1 and NTPDase2 in murine kidney:Relevance to regulation of P2 receptor signaling. Am J Physiol Renal Physiol 288:F1032–F1043.

70. Lancel S, et al. (2009) Carbon monoxide rescues mice from lethal sepsis by supportingmitochondrial energetic metabolism and activating mitochondrial biogenesis.J Pharmacol Exp Ther 329:641–648.

71. Tran M, et al. (2011) PGC-1α promotes recovery after acute kidney injury duringsystemic inflammation in mice. J Clin Invest 121:4003–4014.

72. Piantadosi CA, Suliman HB (2012) Redox regulation of mitochondrial biogenesis. FreeRadic Biol Med 53:2043–2053.

73. Wills LP, et al. (2012) The β2-adrenoceptor agonist formoterol stimulates mitochon-drial biogenesis. J Pharmacol Exp Ther 342:106–118.

74. Motterlini R, Otterbein LE (2010) The therapeutic potential of carbon monoxide. NatRev Drug Discov 9:728–743.

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