of October 4, 2012.This information is current as

ReleaseαInhibition of Neutrophil-Dependent TNF-

Protects during Acute Kidney Injury via Adora2b Adenosine Receptor Signaling

Tak, Holger K. Eltzschig and Eric T. ClambeyAlmut Grenz, Jae-Hwan Kim, Jessica D. Bauerle, Eunyoung

ol.1201651http://www.jimmunol.org/content/early/2012/09/26/jimmun

published online 1 October 2012J Immunol 

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The Journal of Immunology

Adora2b Adenosine Receptor Signaling Protects during AcuteKidney Injury via Inhibition of Neutrophil-Dependent TNF-aRelease

Almut Grenz,* Jae-Hwan Kim,† Jessica D. Bauerle,‡ Eunyoung Tak,*

Holger K. Eltzschig,* and Eric T. Clambey*

Renal ischemia is among the leading causes of acute kidney injury (AKI). Previous studies have shown that extracellular adenosine is

a prominent tissue-protective cue elicited during ischemia, including signaling events through the adenosine receptor 2b (Adora2b).

To investigate the functional role of Adora2b signaling in cytokine-mediated inflammatory pathways, we screened wild-type and

Adora2b-deficient mice undergoing renal ischemia for expression of a range of inflammatory cytokines. These studies demon-

strated a selective and robust increase of TNF-a levels in Adora2b-deficient mice following renal ischemia and reperfusion. Based

on these findings, we next sought to understand the contribution of TNF-a on ischemic AKI through a combination of loss- and

gain-of-function studies. Loss of TNF-a, through either Ab blockade or study of Tnf-a–deficient animals, resulted in significantly

attenuated tissue injury and improved kidney function following renal ischemia. Conversely, transgenic mice with overexpression

of TNF-a had significantly pronounced susceptibility to AKI. Furthermore, neutrophil depletion or reconstitution of Adora2b2/2

mice with Tnf-a–deficient neutrophils rescued their phenotype. In total, these data demonstrate a critical role of adenosine

signaling in constraining neutrophil-dependent production of TNF-a and implicate therapies targeting TNF-a in the treatment

of ischemic AKI. The Journal of Immunology, 2012, 189: 000–000.

Acute kidney injury (AKI) is clinically defined as an abruptreduction in kidney function (e.g., a decrease in glo-merular filtration rate [GFR]) over minutes to days (1).

AKI can result from multiple etiologies, including sepsis andsurgical procedures (both of the kidney and other tissues includingthe heart) (2–5). Although the rate of mortality associated withAKI varies depending on the specific combination of etiology withAKI, even moderate levels of kidney injury are associated with a70% greater risk of death than in patients without evidence of AKI(3). Notably, individuals who survive AKI are at an increased riskfor long-term sequelae, including progression to chronic kidneydisease and end-stage renal disease (6, 7). Despite the signifi-cant medical challenge of AKI, therapeutic approaches to preventor treat AKI are limited; to date, the majority of clinical trialsfor AKI have failed (8, 9). Because of this unmet medical need,there is significant interest in further understanding the path-ogenesis of AKI and developing novel therapeutic approachesfor AKI.

Although there are multiple etiologies for AKI, a commonoccurrence contributing to AKI is obstruction of renal blood flow,which results in renal ischemia (10). The pathogenesis of AKIfollowing renal ischemia is multifactorial, involving the impairedfunction and apoptosis of tissue-resident epithelial and endo-thelial cells, followed by the resulting inflammatory cascade thatsequentially recruits neutrophils, monocytes, and T cells to thepostischemic kidney (10). The outcome of AKI is regulated bythe balance of pathogenic mechanisms (e.g., prolonged ischemia,cell death, and the elicitation of proinflammatory cytokines includingTNF-a) relative to protective mechanisms (e.g., extracellular aden-osine, heme oxygenase, and the production of proresolving leu-kotrienes) (10).Among tissue-protective factors elicited during ischemia, there

is clear evidence that the generation and signaling of extracellularadenosine can function as a primary mechanism that limits ischemictissue injury and inflammation (11, 12). These suppressive functionsof extracellular adenosine signaling are exemplified by seminalstudies by Sitkovsky et al. (13), in which the adenosine receptor2a (Adora2a) was identified as a potent anti-inflammatory receptorin vivo. During tissue ischemia, there is a transient accumulationof extracellular adenosine, generated by the stepwise degradationof precursor molecules (nucleotides including ATP, ADP, or AMP)(14, 15). Extracellular adenosine signals through four distinctadenosine receptors (Adora1, Adora2a, Adora2b, Adora3) (16, 17),and studies show that hypoxia also enhances adenosine signalingby transcriptional increases of adenosine receptor levels (15). Al-though the mechanisms by which extracellular adenosine mediateprotective effects remain only partially understood, adenosine re-ceptor signaling can potently limit inflammation by influencing bothparenchymal and bone marrow-derived compartments (11, 18). Inthe context of Adora2b, the focus of this research, multiple studieshave demonstrated that adenosine generation and signaling viaAdora2b protects against ischemic injury in the heart (19, 20),liver (21), and intestine (22, 23).

*Mucosal Inflammation Program, Department of Anesthesiology, University of Col-orado Denver, Aurora, CO 80045; †Department of Anesthesiology, Korea UniversityCollege of Medicine, Seoul 136-701, Korea; and ‡Department of Anesthesiology,Perioperative, and Pain Medicine, Brigham and Women’s Hospital, Harvard MedicalSchool, Boston, MA 02115

Received for publication June 14, 2012. Accepted for publication August 23, 2012.

This work was supported by the Juvenile Diabetes Research Foundation and anAmerican Heart Association grant (to A.G.), National Institutes of Health GrantsR01-HL092188, R01-DK083385, and R01-HL098294, and a grant from the Crohn’sand Colitis Foundation of America (to H.K.E.).

Address correspondence and reprint requests to Dr. Eric T. Clambey, Mucosal In-flammation Program, Department of Anesthesiology, University of Colorado Denver,12700 East 19th Avenue, Mailstop B112, Research Complex 2, Room 7118, Aurora,CO 80045. E-mail address: eric.clambey@ucdenver.edu

Abbreviations used in this article: Adora2b, adenosine receptor 2b; AKI, acute kid-ney injury; ARE, AU-rich element; GFR, glomerular filtration rate.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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Previous studies identified that extracellular adenosine genera-tion and signaling through adenosine receptors can potently limitAKI (24–29). Although protective effects of adenosine involvesignaling through multiple adenosine receptors (including Adora1,Adora2a, and Adora2b), each of which influences discrete celltypes and phases of AKI (as reviewed in Refs. 26, 30), thehypoxia-inducible nature of Adora2b (also known as the A2Badenosine receptor) places it as a prominent pathway affordingrenovascular protection during ischemia (31). Recent studies haveshown that one protective mechanism elicited by Adora2b sig-naling is to restore optimal blood flow to the postischemic kidney(32). In this study, we sought to investigate the contribution ofAdora2b in limiting inflammation and tissue damage during AKI,with a particular focus on leukocyte regulation. These studiesdemonstrated that Adora2b has a critical role in specifically reg-ulating TNF-a production and that Adora2b restricts tissue dam-age mediated by neutrophils via this pathway.

Materials and MethodsMice generation and breeding

All mice were housed in a 12-h light/dark cycle and were gender-, age-, andweight-matched between 12 and 16 wk. In transcript and pharmacologicalstudies, C57BL/6J mice obtained from The Jackson Laboratory were used.Mice deficient in TNF-a (33) were obtained from The Jackson Laboratory(B6;129S-Tnftm1Gkl/J, stock number 003008). TNFD AU-rich elements(ARE) mice (34) were kindly provided by Dr. Jesus Rivera-Nieves(University of California, San Diego, La Jolla, CA) and generated, vali-dated, and characterized as previously described (35). Adora2b-deficient(Adora2b2/2) mice were characterized as previously described (31).

Murine model for renal ischemia

Mice underwent right nephrectomy followed by left renal artery ischemia(0, 10, 30, and 40 min of ischemia) using a hanging weight system, aspreviously described (24, 25, 31, 36). Briefly, a right nephrectomy isperformed, and then the left kidney is carefully isolated and the left adrenalgland dissected away. The kidney is placed in a lucite cup, and a 7–0 nylonsuture is threaded under the renal artery. Weights are attached at both endsof the suture, and ischemia is performed for indicated time points. Ischemiais confirmed by color change of the kidney from red to pale white. At theend of the designated ischemic time period, the weights are resupported,and reperfusion ensues. Inulin clearance was performed 1 h followingreperfusion.

Inulin clearance

Inulin clearance was measured 1 h after renal ischemia (40 min) as de-scribed previously (31). Briefly, mice were anesthetized using 50 mg/kgi.p. pentobarbital. Animals were then placed on a temperature-controlledoperating table to keep rectal temperature at 37˚C. The right jugular veinwas cannulated for continuous infusion. Blood samples were taken viaretro-orbital vein plexus puncture. A catheter was placed in the urinarybladder for timed urine collection after removal of the right kidney. Aftersurgery, mice received a bolus of 0.45% sodium chloride solution in anamount equal to 20% body weight. Continuous infusion was maintained ata rate of 800 ml/h/25 g body weight, and FITC-labeled inulin (0.75 g/100ml; Sigma-Aldrich) was added to the infusion for evaluation of whole-kidney GFR as described before (37, 38). After stabilization of the animalsfor 20 min, 20-min timed urine collections were performed. Blood wasobtained in the middle of every urine collection period for measurement ofFITC-inulin. Concentration of inulin in plasma and urine was performedby measurement of wavelength using a spectophotometer (Biotek Synergy2), and GFR was calculated by standard formulas.

Renal histology

Kidneys were excised and harvested 24 h following 40 min of renal ischemia.Renal tissues were fixed in 4.5% buffered formalin, dehydrated, and em-bedded in paraffin. Sections (3 mm) were stained with H&E (31). Exami-nation and scoring of three representative sections of each kidney (n = 4–6for each condition) were carried out blinded.

Cytokine ELISA

To quantify renal cytokine content, we performed cytokine ELISA andfollowed the manufacturer’s instructions (Meso Scale Discovery).

Transcriptional analysis

Total RNA was isolated from mouse kidneys using the TRIzol Reagentaccording to the manufacturer’s instructions (Invitrogen). Therefore, frozentissue was homogenized in TRIzol Reagent (Invitrogen) and chloroform.After spinning at 12,000 3 g for 15 min, the aqueous phase was removed,and the RNAwas precipitated with isopropanol. RNAwas pelleted, washedwith ethanol, treated with DNAse, and the concentration was quantified.The PCR reactions contained 1 mM sense and 1 mM antisense oligonu-cleotides with SYBR Green (Bio-Rad). Each target sequence was ampli-fied using increasing numbers of cycles of 94˚C for 1 min, 58˚C for 0.5min, and 72˚C for 1 min. Quantification of transcript levels was measuredby real-time RT-PCR (iCycler; Bio-Rad). Primers for TNF were: forward59-CCC ACT CTG ACC CCT TTA CT-39 and reverse 59-TTT GAG TCCTTG ATG GTG GT-39.

Ab-based studies

To neutralize TNF-a in vivo, mice were treated with infliximab (Remicade;Jannsen Biotech), a TNF-a–specific mAb therapy, at a dose of 10 mg/kgbody weight by i.p. injection 60 min prior renal ischemia. To depleteneutrophils, mice were injected with 150 mg anti-Gr1 Ab (clone RB6-8C5;BioXCell). This Ab results in .90% depletion of neutrophils for at least24 h (data not shown and Ref. 39).

Neutrophil adoptive transfer

Neutrophils were enriched from Adora2b2/2 or Tnf-a2/2 mice using mag-netic bead-based enrichment (Ly6G beads; Miltenyi Biotec) for positiveenrichment for neutrophils. A total of 33 106 enriched neutrophils were thenadoptively transferred by i.v. transfer into the indicated recipient mice.

Statistical analysis

A grading scale of 0–4, as outlined by Jablonski et al. (40), was used for thehistopathological assessment of proximal tubular damage. These renalinjury score data are given as median and range; all other data are pre-sented as mean 6 SD. Renal injury was analyzed with the Kruskal-Wallistest, with follow-up pairwise comparisons by Wilcoxon Mann–WhitneyU test. Jablonski index results are shown as box-and-whisker plots. Thelines within the boxes show the median, the bounds of the boxes show theupper and lower median, and the whiskers extend out to the data’s smallestand largest number. For all other outcomes, data were compared by two-factor ANOVA with Bonferroni’s posttest or by two-tailed Student t testwhen appropriate. Data are expressed as mean6 SD. A p value,0.05 wasconsidered statistically significant. For all statistical analyses, GraphPadPrism 5.0 software (GraphPad) for Windows XP was used.

Study approval

All animal protocols were in accordance with the United States Guidelinesof the International Animal Care and Use Committee for use of livinganimals and approved by the Institutional Animal Care and Use Committeeof the University of Colorado guidelines for animal care.

ResultsAdora2b2/2 mice have a selective and profound induction ofTNF-a during AKI

Previous studies had implicated Adora2b signaling in tissue pro-tection from ischemia and reperfusion injury (32, 41, 42). Based onthese studies, we exposed wild-type C57BL/6J (Adora2b+/+) andAdora2b2/2 mice to ischemic kidney injury using a well-established hanging-weight model (36); following reperfusion,kidney function was assessed by measuring the GFR. As expected,Adora2b2/2 mice had more pronounced kidney injury, as measuredby an impaired GFR following renal ischemia (Fig. 1A). Next, wesought to define how Adora2b regulates inflammatory cytokineprofiles following renal ischemia, comparing Adora2b+/+ andAdora2b2/2 mice subjected to AKI. Ischemic kidney tissue wasthen screened for the relative abundance of a panel of cytokines. Inthis screen, we found that Adora2b2/2 mice had a pronounced in-crease in TNF-a protein, whereas other cytokines (IL-1, IL-2, IL-4,and IL-12) had less pronounced differences in expression betweenAdora2b+/+ and Adora2b2/2 mice (Fig. 1B). These data identifyelevated levels of TNF-a in Adora2b2/2 mice as one potentialpathogenic player contributing to the worse AKI in these mice.

2 Adora2b REGULATES NEUTROPHIL TNF IN RENAL ISCHEMIA

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Adora2b-dependent regulation of TNF-a occurs followingprolonged periods of ischemia

To better understand the contribution of Adora2b-dependent in-hibition of TNF-a production, we defined the kinetics of TNF-aregulation by Adora2b. To do this, we exposed Adora2b+/+ andAdora2b2/2 mice to various periods of ischemia followed bymeasurement of TNF-a transcript levels. Animals exposed to aslittle as 10 or 30 min of ischemia had no difference in TNF-alevels between Adora2b+/+ and Adora2b2/2 mice (Fig. 2). Incontrast, with 40 min of renal ischemia, Adora2b2/2 mice hadincreased levels of TNF-a relative to their wild-type counterparts(Fig. 2). Given the temporal regulation of TNF-a following pro-longed ischemia, we focused our further studies on 40 min ofischemia, the time at which there is a maximal difference betweenAdora2b+/+ and Adora2b2/2 mice.

Ab blockade of TNF-a limits AKI following renal ischemia inAdora2b2/2 mice

Based on the worse AKI in Adora2b-deficient mice (31, 32)and identification of elevated TNF-a in Adora2b2/2 mice, wetested the outcome of blocking TNF-a during renal ischemia inAdora2b2/2 mice through treatment of animals with infliximab(Remicade; Janssen), a TNF-a–specific mAb therapy used for thetreatment of inflammatory diseases including rheumatoid arthri-tis and inflammatory bowel disease (43). In untreated animals,infliximab treatment had no effect on renal histology (Fig. 3A–C).Infliximab-treated Adora2b2/2 mice subjected to renal ischemiahad reduced renal injury relative to control-treated animals (Fig.3A–C). Mice treated with infliximab before renal ischemia hadreduced kidney injury as demonstrated by reduced acute tubularnecrosis, less destruction of the brush border, and attenuated hy-aline cast formation (Fig. 3B). Semiquantitative histological anal-ysis demonstrated a reduction in the Jablonski index with infliximabtreatment (Fig. 3C). These data identify that TNF-a elicited duringrenal ischemia in Adora2b2/2 mice contributes to the enhancedseverity of AKI in these animals.

Ab blockade of TNF-a limits AKI in wild-type mice followingrenal ischemia

The previous studies focused primarily on the role of TNF-a inthe enhanced tissue injury in Adora2b2/2 mice. To extend thesefindings to the context of wild-type C57BL/6J mice, we admin-istered the TNF-a–blocking Ab (infliximab) to either control miceor mice experiencing renal ischemia. Infliximab treatment did notalter baseline renal histology in control animals not subjected toischemia (Fig. 3D–F). However, when animals were subjected torenal ischemia, infliximab-treated animals had pronounced histo-logical protection from renal ischemia with less tubular acutenecrosis, reduced brush border injury, and attenuated cast forma-tion (Fig. 3D–F).

Genetic ablation of TNF-a limits AKI following renal ischemia

Based on the efficacy of Ab blocking against TNF-a in amelioratingAKI, we next tested the outcome of AKI in mice genetically defi-cient in TNF-a (Tnf2/2). When Tnf2/2 mice were subjected to40 min of ischemia, Tnf2/2 mice had reduced AKI, as measured by

FIGURE 1. Adora2b2/2 mice have a selective and profound induction

of TNF-a during AKI. (A) GFR following 40 min of renal ischemia and

1 h of reperfusion in wild-type (WT; black bars) and Adora2b2/2 (white

bars) mice (n = 4–6). (B) Measurement of TNF-a, IL-1, IL-2, IL-4, and IL-

12 protein levels after 40 min of renal ischemia and 2 h of reperfusion from

WT and Adora2b2/2 mice by ELISA (n = 3–4).2I, Animals not subjected

to renal ischemia; +I, animals subjected to renal ischemia. KW, Kidney

weight.

FIGURE 2. Adora2b-dependent regulation of TNF-a

occurs following prolonged periods of ischemic injury.

Measurement of TNF-a transcript levels in wild-type

(WT, black bars) and Adora2b2/2 (white bars) mice

after different ischemia times (0, 10, 30, and 40 min)

and 2 h of reperfusion (n = 2).

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improved kidney function (characterized by a higher GFR) andimproved tissue integrity as analyzed by histology (Fig. 4). Thesegenetic data provide compelling evidence for a critical role for el-evated TNF-a as a primary driver of renal injury during ischemia/reperfusion injury in mice.

Mice with genetic overexpression of TNF-a have exacerbatedAKI following renal ischemia

Given the correlation among Adora2b deficiency, increased TNF-alevels, and worse AKI, we tested what consequence elevatedlevels of TNF-a in normal mice might have on AKI. To do this,we analyzed AKI in TNFDARE mice, a mouse model in which theARE that confers TNF-a mRNA instability has been geneticallyablated through homologous recombination (34). Notably, thesemice have elevated levels of TNF-a both in the resting state and

following an inflammatory insult (34). When TNFDARE micewere exposed to 40 min of ischemia, TNFDARE mice had sig-nificantly worse AKI than wild-type controls, measured by re-duced GFR (Fig. 5A), and worse tissue integrity by histology (Fig.5B, 5C). Based on these studies, mice with a genetic lesion thatresults in elevated TNF-a levels have worse AKI. These geneticdata demonstrate that appropriate control of TNF-a protein ex-pression levels are required to mitigate excessive tissue injuryduring ischemia/reperfusion and AKI in mice.

Neutrophils contribute to the increased AKI ofAdora2b-deficient mice

Given the important role of both TNF-a and neutrophils in ischemicinjury, we next sought to test the contribution of neutrophils to the

FIGURE 3. TNF-a contributes to AKI following renal ischemia in wild-

type (WT) and Adora2b2/2 mice. (A) GFR following 40 min of renal

ischemia and 1 h of reperfusion in Adora2b2/2 untreated (black bar) or

treated with the anti-TNF Ab infliximab (white bars, 10 mg/kg body

weight i.p. 60 min prior renal ischemia; n = 4–6). (B) Renal histology

(H&E staining) following 40 min of renal ischemia and 24 h of reperfusion

(original magnification 3400; one of three to five representative slides is

shown) in Adora2b2/2 animals untreated (black bar) or treated with the

anti-TNF Ab infliximab (white bars). (C) Quantification of renal tissue

injury using the Jablonski index (n = 3–5). (D) GFR following 40 min of

renal ischemia and 1 h of reperfusion in WT mice untreated (black bar)

or treated with the anti-TNF Ab infliximab (white bars, 10 mg/kg body

weight i.p. 60 min prior renal ischemia; n = 4–6). (E) Renal histology

(H&E staining) following 40 min of renal ischemia and 24 h of reperfusion

in WT mice without (black bars) or with (white bars) infliximab treatment

(original magnification 3400; one of three to five representative slides is

shown). (F) Quantification of renal tissue injury in WT mice without (black

bars) or with (white bars) infliximab treatment by using the Jablonski index

(n = 3–5). 2I, Animals not subjected to renal ischemia; +I, animals sub-

jected to renal ischemia. KW, Kidney weight.

FIGURE 4. Genetic ablation of TNF-a limits AKI following renal is-

chemia. (A) GFR following 40 min of renal ischemia and 1 h of reper-

fusion in wild-type (WT; black bars) and Tnf-a2/2 gene-targeted mice

(white bars) (n = 4–6). (B) Renal histology (H&E staining) following 40

min of renal ischemia and 24 h of reperfusion (original magnification

3400; one of three to five representative slides is shown). (C) Quantifi-

cation of renal tissue injury using the Jablonski index (n = 3–5). 2I,

Animals not subjected to renal ischemia; +I, animals subjected to renal

ischemia. KW, Kidney weight.

FIGURE 5. Mice with genetic overexpression of TNF-a have exacer-

bated AKI following renal ischemia. (A) GFR following 40 min of renal

ischemia and 1 h of reperfusion in wild-type (WT; black bars) and

TNFDARE (white bars) gene-targeted mice (n = 4–6). (B) Renal histology

(H&E staining) following 40 min of renal ischemia and 24 h of reperfusion

(original magnification 3400; one of three to five representative slides is

shown). (C) Quantification of renal tissue injury using the Jablonski index

(n = 3–5). 2I, Animals not subjected to renal ischemia; +I, animals sub-

jected to renal ischemia. KW, Kidney weight.

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worse AKI in Adora2b2/2 mice. To do this, Adora2b2/2 mice weresubjected to Ab-mediated depletion of neutrophils, followingtreatment with an anti-Gr1 Ab (39). Following deletion of neu-trophils, Adora2b2/2 mice were subjected to renal ischemia. Inthese studies, we found that neutropenic mice had improved renalfunction (measured by increased GFR relative to controls, Fig. 6A)and reduced histological damage (Fig. 6B, 6C). Further, when weanalyzed TNF-a levels in animals depleted of neutrophils, we foundthat neutrophil depletion resulted in a profound decrease in theamount of TNF-a detected during AKI (Fig. 6D). These data in-dicate that neutrophils contribute to the exacerbated AKI followingrenal ischemia in Adora2b2/2 mice. Moreover, these data identifyneutrophils as a major source of TNF-a during AKI followingprolonged renal ischemia.To investigate the role of neutrophil-derived TNF-a as a factor

contributing to Adora2b2/2 AKI, we next performed adoptivetransfer studies of magnetically enriched neutrophils from eitherAdora2b2/2 or Tnf2/2 mice into Adora2b2/2 recipients previ-ously depleted of neutrophils. Notably, whereas adoptive transferof neutrophils from Adora2b2/2 mice into Adora2b2/2 mice hadimpaired renal function (as measured by GFR, Fig. 6E), adoptive

transfer of neutrophils from Tnf2/2 mice was associated withattenuated AKI and a relatively improved GFR (Fig. 6E). Basedon these data, neutrophil-derived TNF-a is an important contrib-utor to AKI in Adora2b2/2 mice. In total, these studies demon-strate a central role for TNF-a as a molecular mediator of tissueinjury during renal ischemia in both wild-type and Adora2b2/2

mice.

DiscussionThe pathogenesis of AKI following renal ischemia is a complexprocess, involving the impaired function and apoptosis of tissue-resident epithelial and endothelial cells, coupled with a resultinginflammatory cascade that recruits multiple leukocyte subsets to thepostischemic kidney (10). The outcome of renal ischemia is tightlyregulated by the relative balance of pathogenic versus protectivemechanisms (10). One potent protective mechanism elicited duringischemia is extracellular adenosine generation (generated by thestepwise degradation of extracellular ATP through the extracellularenzymes CD39 and CD73) and signaling through adenosine re-ceptors (24–26). Notably, evidence for the potent anti-inflammatoryproperties mediated by adenosine receptor signaling came frompioneering studies by Sitkovsky’s group (13, 44), in which Adora2asignaling was identified as a primary anti-inflammatory pathwaythat constrains tissue injury during inflammation and ischemiain multiple organ systems. Although the adenosine receptor canbe a potent, tissue-protective pathway, it is important to note thatexcess extracellular adenosine signaling, whether in the contextof adenosine deaminase deficiency or in states of repeated ische-mic events (e.g., sickle cell), can induce profound pathologies(45–47).In the context of AKI following renal ischemia, the adenosine

receptors Adora1 (A1), Adora2a (A2a), and Adora2b (A2b) haveeach been demonstrated to play protective roles to limit AKI (27,28, 31). In contrast, Adora3 (A3) has a detrimental role in thiscontext, such that Adora3-deficient mice are protected againstrenal ischemia-induced AKI (48). Although multiple adenosinereceptors participate in protection of the kidney against ischemia,it is notable that different adenosine receptors are thought to elicitdistinct mechanisms of protection, ranging from primary modu-lation of parenchymal cells within the kidney (via Adora1 sig-naling) (26) to inhibition of the recruitment and activation ofmultiple bone marrow-derived cell types including neutrophils,NK T cells, and T cells (via Adora2a signaling) (11, 49, 50). Inthis context, it is worth noting that Adora2a signaling has previ-ously been demonstrated to regulate TNF-a production fromisolated human neutrophils in vitro and in murine neutrophils in anair pouch model of inflammation (49). Adora2a can also influencethe extent of adenosine receptor signaling beyond its own signalingcapacity. In particular, careful studies by Sitkovsky’s laboratory (51)have demonstrated that optimal cell-surface expression of Adora2brelies on coexpression with Adora2a, revealing an unanticipatedinterconnectedness between these two receptors. Whether theAdora2b-dependent regulation observed in this current study isinfluenced by integration with Adora2a-dependent signaling dur-ing AKI remains to be determined. In the context of AKI fol-lowing ischemia/reperfusion, Adora2a constrains tissue damageboth by limiting neutrophil transmigration and inhibiting T cells(29, 52).Although Adora2a is thought to primarily target leukocytes in its

anti-inflammatory actions, Adora2b signaling appears to be moremultifactorial, with examples of Adora2b inhibition in both pa-renchymal and leukocyte subsets. Adora2b is expressed bymultiplecell types and tissues, including expression in the vasculature and inleukocyte subsets such as macrophages (31, 53). To date, studies

FIGURE 6. Neutrophils contribute to the increased AKI of Adora2b2/2

mice. (A) GFR following 40 min of renal ischemia and 1 h of reperfusion

in Adora2b2/2 mice without (black bars) or with neutrophil depletion via

anti-Gr1 Ab treatment (white bars) 24 h prior to renal ischemia (n = 3–5).

(B) Renal histology (H&E staining) following 40 min of renal ischemia

and 24 h of reperfusion (original magnification 3400; one of three to five

representative slides is shown). (C) Quantification of renal tissue injury

using the Jablonski index (n = 3–5). (D) TNF-a protein levels following 40

min of renal ischemia and 2 h of reperfusion by ELISA. (E) Neutrophils

were isolated from either Adora2b2/2 mice (black bars) or Tnf-a2/2 mice

(white bars) and injected into Adora2b2/2 mice that had been previously

depleted of neutrophils by anti-Gr1 Ab treatment. GFR was performed

following 40 min of renal ischemia and 1 h of reperfusion in the recipient

Adora2b2/2 mice. KW, Kidney weight; n.s., no statistically significant

difference.

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analyzing the protective effects of Adora2b in limiting AKI havefocused primarily on a renovascular function for Adora2b inlimiting AKI (31, 32); this focus was based largely on bonemarrow chimera studies that demonstrated Adora2b can mediateprotective effects when it is expressed on nonhematopoietic cells(31). Notably, however, there is clear evidence that Adora2b sig-naling can regulate diverse components of the inflammatory re-sponse, including neutrophils, macrophages, dendritic cells, andeven lymphocytes (53–57). In this manuscript, we have focused oneffector mechanisms regulated by Adora2b within the hemato-poietic system, with a focus on the role of Adora2b following alonger ischemic insult (40 min relative to previous studies using30 min of ischemia). In these studies, we found that Adora2b hasa pronounced effect, limiting the production of TNF-a followingrenal ischemia, and that neutrophil-derived TNF-a contributes toAKI in Adora2b2/2 mice. These data are consistent with reportsidentifying that Adora2b limits TNF-a production from macro-phages (53–55), as well as a recent report demonstrating Adora2bcan restrict TNF-a production from neutrophils in the context ofmyocardial ischemia (58).Though our studies have focused on the protective role of

adenosine receptor signaling in AKI following renal ischemia, it isimportant to note that the etiology of AKI can profoundly influencethe contribution of adenosine receptor signaling in either protectingagainst or exacerbating AKI. For example, studies by Lee et al. (27)have clearly shown that the Adora1 adenosine receptor has di-vergent effects in AKI with different modes of renal injury. Inthe context of renal ischemia, Adora1 has a protective role, andAdora1-deficient mice have worse AKI following renal ischemia(27). Conversely, in the context of nephrotoxic and radiocontrastagent-induced AKI, Adora1 has a detrimental role such thatAdora1-deficient mice are protected against this form of AKI (59).These data exemplify the complex interplay between AKI etiologyand adenosine receptor signaling to ultimately influence kidneyfunction.Our data in this study, and elsewhere, demonstrate a protective

role for Adora2b in limiting acute, ischemic tissue injury (23, 31,58). In these contexts, the hypoxia-inducible nature of Adora2bplaces it as a central mediator affording tissue-protective effectsduring ischemia (31). However, it is important to note that in othercontexts, Adora2b signaling has been associated with proin-flammatory roles, such as the induction of IL-6 in dendritic cells(60). Moreover, depending on the duration and context of Adora2bsignaling, this receptor can be a primary driver of pathobiology.Notably, rigorous studies from the laboratories of Xia and Blackburn(47, 61, 62) have clearly demonstrated that Adora2b is respon-sible for pathogenic outcomes in priaprism, sickle cell disease,and bleomycin-induced lung fibrosis.The outcome of AKI is regulated at multiple stages, from

mechanisms that function rapidly (within minutes to hours of in-jury) to the later involvement of components of the adaptive im-mune response including T cells that can contribute to tissue injuryand repair days after the initial insult (10). In this larger context,it is important to note that Adora2b induces multiple protectiveeffects during ischemic injury. For example, by analyzing the roleof Adora2b within the renal vasculature, we recently showed thatAdora2b promotes optimal postischemic blood flow within thekidney, thereby ensuring maximal return of blood flow, tissueoxygenation, and the removal of waste products from the ischemickidney (32). This mechanism functions through cross talk ofAdora2b with ENT1, the equilibrative nucleoside transporter, atransporter protein that controls the extent and timing of extra-cellular adenosine. Based on the data presented in this study,demonstrating that Adora2b-regulated TNF-a directly impacts

AKI, it is worth noting that TNF-a can have profound effects onthe vasculature, raising the possibility that the mechanism iden-tified in this study may integrate with the Adora2b–ENT1 pathwayto regulate postischemic blood flow to the kidney (32). AlthoughAdora2b signaling can mediate protective effects during renalischemia [using either nephrectomy and unilateral ischemia orsimultaneous, bilateral renal ischemia (32)], it remains to be seenwhat contribution Adora2b has in the context of AKI followingnephrotoxin exposure [e.g., cisplatin (63)] or following sepsis[a context in which Adora2b deficiency is beneficial in limitingbacterial colonization (55)]. This is an important question forfuture investigation because different forms of AKI are char-acterized by distinct kinetics and effector mechanisms (e.g.,ischemia/reperfusion, nephrotoxin, or sepsis) (2, 10, 63). A notablecontrast between our model and that of cisplatin-induced AKIis that parenchymal cells are the prominent source of TNF-a(64, 65), in contrast to our renal ischemia model in which neu-trophils are a prominent source of TNF-a. Because Adora2b canmediate protective effects on both parenchymal and bone marrow-derived cell types, it is possible that the contribution of Adora2bsignaling in AKI will vary depending on the etiology of AKI understudy.Beyond the kidney, Adora2b has been shown to mediate ad-

ditional tissue-protective effects in models of acute inflammation.For example, Adora2b signaling can elicit cardioprotective effectsthrough the circadian rhythm protein Period2, which in turn pro-motes metabolic adaptation in the ischemic heart (41). In acutecolitis and following hypoxia, Adora2b also functions as an es-sential receptor for the anti-inflammatory properties of netrin-1,a neuronal guidance molecule (66). Adora2b signaling can alsoregulate the superoxide burst in neutrophils (67), which wouldlimit neutrophil-derived production of potentially toxic reactiveoxygen species. Finally, Adora2b has been shown to limit theinflammatory properties of macrophages and T cells (53, 55, 57).Given that these cell types can influence regulate AKI (52, 68, 69),Adora2b-mediated protection in AKI may arise by altering thefunction of multiple leukocyte subsets.The multiple etiologies of AKI, combined with the paucity

of therapeutic interventions, continue to be a significant unmetmedical challenge. Given the weight of evidence that extracellularadenosine generation and signaling are tissue-protective duringAKI, this pathway is of significant therapeutic interest. To opti-mally harness the potential of Adora2b in limiting AKI, it will beimportant for future studies to define how the multiple protectivemechanisms of Adora2b integrate to induce maximal protectionduring AKI. In addition, it will be equally important to determinehow to therapeutically activate the beneficial effects of Adora2bsignaling without triggering deleterious consequences of prolongedAdora2b signaling (47, 70).

AcknowledgmentsWe thank Dr. Jesus Rivera-Nieves for kindly providing the TNFDARE

mice and members of the Eltzschig laboratory for critical discussion.

DisclosuresThe authors have no financial conflicts of interest.

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