A Adenosine Receptor–Mediated Induction of IL-6...

A2B Adenosine Receptor–Mediated Induction of IL-6Promotes CKD

Yingbo Dai,*† Weiru Zhang,*‡ Jiaming Wen,*† Yujin Zhang,* Rodney E. Kellems,*and Yang Xia*

*Department of Biochemistry and Molecular Biology, The University of Texas–Houston Medical School, Houston,Texas; †Department of Urology, The Third Xiangya Hospital of Central South University, Changsha, Hunan, China;and ‡Deparment of Nephrology, The First Xiangya Hospital of Central South University, Changsha, Hunan, China

ABSTRACTChronic elevation of adenosine, which occurs in the setting of repeated or prolonged tissue injury, canexacerbate cellular dysfunction, suggesting that it may contribute to the pathogenesis of CKD. Here,mice with chronically elevated levels of adenosine, resulting from a deficiency in adenosine deaminase(ADA), developed renal dysfunction and fibrosis. Both the administration of polyethylene glycol–modi-fied ADA to reduce adenosine levels and the inhibition of the A2B adenosine receptor (A2BR) attenuatedrenal fibrosis and dysfunction. Furthermore, activation of A2BR promoted renal fibrosis in both miceinfused with angiotensin II (Ang II) and mice subjected to unilateral ureteral obstruction (UUO). Thesethree mouse models shared a similar profile of profibrotic gene expression in kidney tissue, suggestingthat they share similar signaling pathways that lead to renal fibrosis. Finally, both genetic and pharma-cologic approaches showed that the inflammatory cytokine IL-6 mediates adenosine-induced renalfibrosis downstream of A2BR. Taken together, these data suggest that A2BR-mediated induction of IL-6contributes to renal fibrogenesis and shows potential therapeutic targets for CKD.

J Am Soc Nephrol 22: 890–901, 2011. doi: 10.1681/ASN.2010080890

Chronic kidney disease (CKD) is a worldwide pub-lic health problem. Twenty-six million Americanadults have CKD, and it is the ninth leading cause ofdeath in the United States, with poor outcomes andhigh cost.1 One of the major progressive featuresseen in CKD is renal fibrosis, a condition represent-ing one of the largest challenges in nephrology be-cause it ends with chronic renal failure (CRF).2– 4

The only treatment today for CRF is dialysis or kid-ney transplantation, thus making CRF one of themost expensive diseases to treat on a per-patientbasis.5,6 Reactive treatments rarely restore normalkidney function, and preventive approaches tolimit renal fibrosis are lacking because of the poorunderstanding of the pathogenesis of CKD and itsprogression to renal fibrosis.

Ischemia and hypoxia have long been consid-ered to be associated with CKD.7,8 One of the best-known signaling molecules to be induced under hy-poxic conditions is adenosine.9,10 Adenosine signals

through the engagement of G-protein– coupled cellsurface receptors A1R, A2AR, A2BR, and A3R11. Onone hand, adenosine protects tissues like thebrain,12,13 kidney,14 and heart15 from acute isch-emic damage, thereby exhibiting chemoprotectiveproperties.16 –19 On the other hand, in the setting ofrepeated or prolonged tissue injury, chronic eleva-tion of adenosine becomes detrimental by promot-ing or exacerbating tissue injury and dysfunc-tion.9,11 Of note, adenosine is known to be elevatedin CKD patients.20 However, the role of chronic el-

Received August 29, 2010. Accepted January 11, 2011.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Yang Xia, Department of Biochemistry andMolecular Biology, University of Texas–Houston Medical School.Phone: 713-500-5039; Fax: 713-500-0652; Email: [email protected]

Copyright © 2011 by the American Society of Nephrology

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890 ISSN : 1046-6673/2205-890 J Am Soc Nephrol 22: 890–901, 2011

evation of adenosine in the pathogenesis of CKD remains un-identified, and underlying mechanisms for renal fibrosis arepoorly studied.

In this study, we report an important role for increasedadenosine in CKD in three distinct mouse models: adenosinedeaminase (ADA)-deficient mice, a well-accepted animalmodel to study the consequences of enhanced adenosine sig-naling, angiotensin II (Ang II)-infused mice and surgically in-duced unilateral ureteral obstruction (UUO) in mice, twovaluable models to study renal fibrosis. In each of these threemodels, we show that A2BR-mediated IL-6 induction underliesrenal fibrosis. These findings immediately offer novel thera-peutic strategies for CKD.

RESULTS

Excess Renal Adenosine Contributes to KidneyDysfunction in ADA�/� miceTo determine in vivo significance of increased adenosine inCKD, we took advantage of ADA-deficient mice (ADA�/�).ADA catalyzes the irreversible deamination of adenosine to

produce inosine. As a result of ADA deficiency, we found thatthe mice accumulate high levels of adenosine in the kidney(Figure 1A). For this reason, ADA�/� mice serve as a valuableanimal model to determine the direct role of excess adenosinein pathogenesis of CKD. To determine the contribution ofadenosine in ADA�/� mice, enzyme therapy was carried out bythe intraperitoneal injection of polyethylene glycol–modifiedADA (PEG-ADA), a safe drug successfully used to treat bothADA-deficient humans and mice to lower elevated adenosinelevels.21–23 We found that the adenosine levels in kidneys ofADA�/� mice receiving the high dose (HD) regimen of PEG-ADA were similar to those in kidney tissues of the ADA� con-trols. However, adenosine levels in kidney tissues of mice onthe low dose (LD) regimen of PEG-ADA were significantlyhigher than those of HD-treated mice and the controls (Figure1A). We also observed that adenosine levels in the controlADA� mice with HD PEG-ADA treatment were not signifi-cantly reduced below that of control ADA� mice without PEG-ADA treatment. Thus, PEG-ADA treatment is a useful exper-imental strategy for controlling the endogenous level ofadenosine present in kidney tissue of ADA-deficient mice.

Next, we analyzed renal function in each group by measuring

Figure 1. Increased adenosine in kidney tissues contributes to renal dysfunction and fibrosis in ADA-deficient mice. (A) Modulation ofrenal adenosine levels in ADA�/� after different dosages of PEG-ADA treatment. Adenosine levels in the kidney tissues were measuredby HPLC. (B) Twenty-four-hour urine was collected by metabolic cages, and urinary albumin and creatinine were measured.(C) Histologic analysis of kidneys from control and ADA�/� mice on LD or HD regimens of PEG-ADA. H&E staining and Masson’strichrome staining showed significant vascular damage and fibrosis in glomeruli and interstitial tissues from ADA-deficient mice on theLD regimen. Vascular damage and renal fibrosis were prevented by treatment with a HD regimen of PEG-ADA from birth (HD group).Scale bars, 50 �m. (D) Quantitative image analysis showed LD PEG-ADA–treated mice exhibited increased collagen staining, whereasADA-deficient mice maintained on HD PEG-ADA enzyme therapy from birth have significantly less collagen staining. (E) Collagencontent in both control and ADA�/� mice with or without PEG-ADA treatment. Data are expressed as mean � SEM (n � 6). *P � 0.05versus control mice. **P � 0.05 versus ADA-deficient mice with LD PEG-ADA treatment.

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J Am Soc Nephrol 22: 890–901, 2011 Adenosine Signaling in Chronic Renal Fibrosis 891

albumin content in 24-hour collected urine, an accurate assess-ment of renal function. In ADA-deficient mice on LD PEG-ADAtreatment, the ratio of urinary albumin to creatinine in 24-hourcollected urine was significantly increased compared with con-trols (Figure 1B). In contrast, HD PEG-ADA treatment signifi-cantly attenuated the increased proteinuria seen in LD PEG-ADA–treated ADA�/� mice (Figure 1B). HD PEG-ADAtreatment had no significant effect on adenosine levels and kidneyfunction in the control mice. Thus, these findings provide the firstin vivo evidence that chronic elevation of kidney adenosine is as-sociated with renal dysfunction in an intact animal.

Increased Renal Adenosine Underlies Renal Damage andFibrosis in ADA�/� MiceOne of the major features associated with CKD is renal fibrosis.To determine the critical role of persistentaccumulated adenosine in the progressionof renal fibrosis, histologic studies wereconducted to characterize the renal fibrosisin each group of mice described above.Analysis of hematoxylin-eosin (H&E)-stained sections from mice with LD PEG-ADA treatment showed extensive renaldamage (Figure 1C). The majority of theglomeruli seen in these mice showed de-creased Bowman’s space, decreased capil-lary lumen, and mesangial hypercellularity(Figure 1C, top). Masson’s Trichromestaining showed significant fibrosis inboth glomeruli and interstitial areas be-tween tubules (Figure 1C, middle andbottom). Quantitative image analysisshowed significantly increased collagenstaining in kidneys of LD PEG-ADA–treated ADA-deficient mice (Figure 1D).Consistent with histologic studies, totalcollagen measurements of the kidneys ofADA�/� mice with LD PEG-ADA treat-ment was significantly elevated (Figure 1E).In contrast, the HD-treated ADA�/� miceshowed a significant reduction in renal fi-brosis, collagen staining, and total collagencontent relative to LD PEG-ADA–treatedADA�/� mice (Figure 1, C–E). Taken to-gether, these results suggest that increasedadenosine contributes to renal fibrosis andthat chronic reduction of adenosine levelsby PEG-ADA enzyme therapy attenuatesthe development of renal fibrosis inADA�/� mice.

A2BR Activation Contributes to RenalDysfunction and Fibrosis in ADA�/�

MiceTo evaluate the role of adenosine recep-

tor (AR) signaling in adenosine-mediated renal fibrosis seenin ADA�/� mice, we first measured AR expression profilesunder LD and HD PEG-ADA therapy protocols. Kidneysfrom control (ADA�) mice � HD PEG-ADA treatmentwere also examined. Quantitative RT-PCR showed that allfour ARs were expressed in kidneys of control and ADA�/�

mice in the presence or absence of PEG-ADA therapy (Fig-ure 2, A–D). However, only A2BR expression was signifi-cantly increased in ADA�/� mice maintained on LD PEG-ADA treatment compared with that of control and ADA�/�

mice on the HD regimen of PEG-ADA (Figure 2, A–D).Thus, elevated A2BR expression is associated with elevatedadenosine. Because fibroblast cells play an important role intissue fibrosis, we isolated renal fibroblasts and determinedthe distribution of adenosine receptor transcripts. The RT-

Figure 2. A2BR activation is responsible for elevated adenosine-induced kidney dysfunctionand fibrosis in ADA-deficient mice. (A–D) Four AR mRNA levels in ADA�/� mice. QuantitativeRT-PCR measurement of four AR mRNAs in the control and ADA�/� mice with or withoutPEG-ADA treatment. (E and F) PSB1115 (an A2BR antagonist) treatment attenuated renaldysfunction and fibrosis in ADA�/� mice. Proteinuria (E) and total collagen content of thekidneys (F) of the controls and ADA�/� mice with LD PEG-ADA treatment with or withoutPSB1115 injection. Data are expressed as mean � SEM (n � 6). *P � 0.05 versus controlmice. **P � 0.05 versus ADA-deficient mice on LD PEG-ADA therapy.


892 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 890–901, 2011

PCR results showed that the A2BR is the predominant ARexpressed in the primary wild-type renal fibroblast cells(Supplementary Figure 1). To test the importance of A2BRsignaling in renal fibrosis, we injected LD-treated ADA�/�

mice with the A2BR antagonist, PSB1115 (1.5mg/kg per day)for 8 weeks. The PSB1115 injections significantly decreasedproteinuria and renal fibrosis (collagen content) in thesemice (Figure 2, E and F). Collectively, these findings providein vivo evidence for the important role of A2BR signaling inadenosine-mediated renal fibrosis and dysfunction inADA�/� mice.

Increased Adenosine Contributes to RenalDysfunction, Kidney Damage, and Fibrosis inAng II–Infused Mice via A2BR SignalingTo assess the general significance of high adenosine in thepathophysiology of CKD, we chose to investigate the poten-tial contribution of excess adenosine to the renal fibrosisand dysfunction in Ang II–infused mice, a well-acceptedanimal model of renal fibrosis.24,25 Adenosine levels in kid-neys of Ang II–infused mice were significantly higher thanthose of controls (Figure 3A). To determine whether theincreased adenosine contributes to renal fibrosis and dys-function in these mice, we tested the effect of PEG-ADAtherapy (2.5 units/wk) on these features. Similar to ADA�/�

mice, treatment of Ang II–infused mice with PEG-ADA sig-nificantly improved kidney function characterized by de-creased proteinuria (Figure 3C). Histologic studies showedthat chronic Ang II infusion induced remarkable kidneydamage in the mice similar to that seen in ADA�/� mice,including decreased Bowman’s space, decreased capillarylumen, and mesangial hypercellularity in the glomerularcompartment (Figure 4A). Trichrome staining showed re-markable increased collagen staining in both glomeruli andinterstitial tissues in Ang II–infused mice compared withcontrols (Figure 4A). Of significance, PEG-ADA treatmentattenuated both kidney damage and renal fibrosis seen inthese mice (Figure 4A). Quantitative image analysis showedsignificantly increased collagen staining in kidneys of AngII–infused mice (Figure 4B), and PEG-ADA treatment sig-nificantly reduced collagen staining in these mice. Consis-tent with histologic studies, total collagen measurements ofkidneys of Ang II–infused mice were significantly elevated,and PEG-ADA treatment significantly reduced Ang II–in-duced collagen production (Figure 4C). These findingsshowed a previously unrecognized role for increased aden-osine in Ang II–induced kidney fibrosis.

Similar to ADA-deficient mice, we found that A2BR ex-pression was significantly elevated in the kidneys of AngII–infused mice and that A2BR expression was reduced byPEG-ADA treatment (Figure 3B). Next, to test whether ele-vated adenosine-mediated renal fibrosis and dysfunction inAng II–infused mice is via A2BR signaling, we used bothpharmacologic and genetic approaches. We found thatPSB1115 treatment or genetic deletion of A2BR significantly

decreased Ang II–induced proteinuria (Figure 3C) and re-nal fibrosis measured by collagen staining (Figure 4, A andB) and total collagen content (Figure 4C), indicating thatA2BR signaling contributes to renal dysfunction and fibrosisin Ang II–infused mice. Taken together, these findings pro-

Figure 3. Elevated adenosine in kidney tissues underlies renaldysfunction in Ang II–infused mice via A2BR signaling.(A) Adenosine levels in the kidneys of control and Ang II–infused mice in the presence or absence of PEG-ADA.(B) Quantitative RT-PCR measurement of renal A2BR mRNAlevels of control and Ang II–infused mice in the presence orabsence of PEG-ADA. (C) Albumin and creatinine concentra-tions were measured in 24-hour collected mouse urine of thecontrol and Ang II–infused mice with or without PEG-ADA orPSB1115 treatment and A2BR-deficient mice with or withoutAng II infusion. Data are expressed as mean � SEM (n � 6).#P � 0.05 versus control mice. ##P � 0.05 versus Ang II–infused mice without PEG-ADA or PSB1115.



vide strong in vivo evidence that increased adenosine, viaA2BR signaling, contributes to CKD induced by chronic AngII infusion.

Elevated Adenosine Contributes to Increased RenalFibrotic Gene Expression in ADA�/� Mice and AngII–Infused Mice via A2BR SignalingTo determine the common intermediates for renal fibrosis, weexamined the expression of fibrotic mediators in the renal tis-sue of both ADA�/� mice and Ang II–infused mice. We foundthat the expression of pro-collagen I, IL-6, and plasminogenactivator inhibitor-1 (Pai-1) mRNAs was significantly in-creased in the kidneys of ADA�/� mice maintained on the LDregimen of PEG-ADA and Ang II–infused mice (Figure 5).Thus, ADA�/� mice and Ang II–infused mice share a similarfibrotic gene expression profile in kidney tissue, suggestingthat they share similar signaling pathways for progression torenal fibrosis. Unexpectedly, we found that PEG-ADA treat-ment significantly decreased elevated profibrotic gene expres-sion in the kidneys of both ADA�/� mice and Ang II–infusedmice (Figure 5), These results show that elevated adenosinecontributes to increased expression of fibrotic marker genesand plays an important role in renal fibrosis.

Among the four adenosine receptors, we found that A2BRwas the one elevated (Figures 2, A–D, and 3B) and contributed

to renal fibrosis in both ADA�/� mice and Ang II–infusedmice (Figures 1 and 4). Similar to PEG-ADA treatment, wefound that PSB1115 successfully lowered the expression ofpro-collagen I, IL-6, and Pai-1 mRNA in the kidney tissues ofADA�/� mice (Figure 5, A–F). Likewise, we found thatPSB1115 treatment or genetic deletion of A2BR significantlyreduced expression of the fibrotic genes in the kidneys of AngII–infused mice (Figure 5, A–F). Altogether, these findings in-dicate that A2BR signaling is responsible for excess adenosine-induced fibrotic gene expression in the kidney.

Genetic Deletion of A2BR Attenuates UUO-InducedRenal FibrosisTo further determine the general role of A2BR activation inprogression of renal fibrosis, we evaluated renal response ofwild-type (WT) and A2BR-deficient mice to UUO,26,27 awell-accepted experimental procedure to induce renal fi-brosis. We found that the genetic deletion of A2BR in micereduced UUO-induced vascular damage measured by H&Estaining (Figure 6A, top), renal fibrosis measured by colla-gen staining (Figure 6A, bottom), and quantified by imag-ine analysis (Figure 6B) and collage production (Figure 6C).Similar to ADA�/� and Ang II–infused mice, pro-collagen I,IL-6, and Pai-1 mRNA levels were significantly increased inthe surgically manipulated kidney of WT mice with UUO

Figure 4. Increased adenosine is responsible for renal fibrosis in Ang II–infused mice via A2BR signaling. (A) H&E and Trichrome stainingshowed that Ang II–infused mice without PEG-ADA or PSB1115 treatment showed kidney damage and fibrosis. PEG-ADA enzymetherapy, PSB1115 treatment, or genetic deletion of A2BR significantly reduced vascular damage and fibrosis. Scale bars, 50 �m.(B) Quantitative image analyses showed Ang II–infused mice exhibited increased collagen staining in the kidney. PEG-ADA enzymetherapy, PSB1115 treatment, or genetic deletion of A2BR significantly decreased collagen staining in the kidney of Ang II–infused mice.(C) Collagen content in the kidney was significantly increased in Ang II–infused mice. PEG-ADA enzyme therapy, PSB1115 therapy, orgenetic deletion of A2BR significantly decreased collagen content in the kidneys of Ang II–infused mice. Data are expressed as mean �SEM (n � 6). #P � 0.05 versus control mice. ##P � 0.05 versus Ang II–infused mice without PEG-ADA therapy or PSB1115 treatment.



(Figure 6, D–F). However, genetic deletion of A2BR in miceled to a significant reduction of UUO-induced the fibroticgene expression (Figure 6, D–F). Thus, these findings pro-vide strong in vivo evidence that A2BR plays an importantrole in UUO-induced chronic renal fibrosis featured withenhanced fibrotic gene expression.

IL-6 Is a Common Profibrotic Mediator Responsible forExcess Adenosine-Induced Collagen Production in theKidney via A2BR SignalingIt is difficult to determine specific factors and signaling path-ways involved in adenosine-mediated renal fibrosis in intactanimals in vivo. In an effort to decipher specific molecules in-volved in adenosine-mediated renal fibrosis, we performedexperiments using kidney organ cultures. Specifically, we iso-lated kidneys from WT mice and four adenosine receptor–deficient mice and incubated renal explants in the presence orabsence of 5�-N-ethylcarboxamindo-adenosine (NECA), a po-tent nonmetabolized adenosine analog (20 �M), for 24 hours.We found that NECA directly induced collagen secretion andthat genetic deletion of A2BR abolished NECA-induced colla-gen secretion, suggesting that A2BR is likely the major AR con-tributing to renal fibrosis by increasing collagen production in

the kidney (Figure 7A). Next, we found that NECA increasedthe IL-6 mRNA expression and pro-collagen I mRNA levels byfourfold and twofold, respectively (Figure 7, B and C). Thisinduction was completely abolished by the A2BR-specific an-tagonist, MRS1754 (Figure 7, B and C). Consistent with thepharmacologic findings, we found that genetic deletion ofA2BR abolished the NECA-induced pro-collagen I and IL-6mRNA production in the kidney cultures (Figure 7, B and C).Taken together, the pharmacologic and genetic studies showedthat A2BR contributes to adenosine-induced fibrotic gene ex-pression in cultured kidneys.

IL-6 is a profibrotic mediator28 –31 and several studies impli-cate that IL-6 functions downstream of A2BR activation as aprofibrotic mediator of adenosine-induced fibrosis.14,32,33 Be-cause IL-6 gene expression was elevated in the kidney of ADA-deficient mice, Ang II–infused mice, and UUO mice, we spec-ulated that IL-6 is a common intermediate responsible foradenosine-induced pro-collagen expression in kidney tissuesseen in these three mouse model of renal fibrosis. To test thishypothesis, we treated the isolated kidney explants with NECA(20 �M) for 24 hours in the presence and absence of neutral-izing IL-6 antibody or isotype control antibody. We found thatthe NECA-induced pro-collagen I mRNA expression was abol-

Figure 5. Elevated adenosine in kidney tissues stimulates increased expression of fibrotic marker genes via A2BR activation. (A–C) Theexpression of fibrotic marker genes in kidney tissue of control and ADA�/� mice maintained on an LD regimen of PEG-ADAenzyme therapy in the presence or absence of PSB1115 or on an HD regimen of PEG-ADA. HD PEG-ADA enzyme therapy forADA�/� mice or PSB1115 treatment for ADA�/� mice on LD PEG-ADA treatment prevented the increased expression of fibroticmediators. Data are expressed as mean � SEM (n � 4 to 5). *P � 0.05 versus control mice. **P � 0.05 versus ADA�/� mice withLD PEG-ADA therapy. Collagen I mRNA (A), IL-6 mRNA (B), and Pai-1 mRNA (C) in control and PEG-ADA or PSB1115-treatedADA�/� mice. (D–F) Ang II–infused mice exhibited increased expression of fibrotic mediators in kidney tissue. PEG-ADA, PSB1115treatment, or genetic deletion of A2BR inhibited the increased expression of fibrotic marker genes in Ang II–infused mice. Dataare expressed as means � SEM (n � 5 to 6). #P � 0.05 versus control mice. ##P � 0.05 versus Ang II–infused mice withoutPEG-ADA or PSB1115 therapy.



ished by IL-6 neutralizing antibody but not by isotype controlantibody (Figure 7D). These findings provide direct evidencethat adenosine-induced IL-6 production contributes to renalfibrosis associated with matrix protein production.

DISCUSSION

A role for adenosine signaling in renal fi-brosis in CKD was initially shown by thestriking renal fibrosis and dysfunction ob-served in ADA�/� mice, features similar tothose seen in patients with CKD. Our initialfinding was extended and confirmed byanalysis of Ang II–infused mice and UUOmice, two well-accepted models for renalfibrosis. These findings led us to furtherdiscover that IL-6 is a common profibroticsignaling molecule responsible for adenos-ine-mediated renal fibrosis by induction ofprocollagen gene expression via A2BR acti-vation. Overall, our analysis of renal fibro-sis and kidney dysfunction in these threemouse models provides strong support forthe novel view that A2BR-mediated IL-6signaling contributes to CKD and immedi-ately suggests novel adenosine-based ther-apies in the disease.

ADA-deficient mice were originally con-structed to serve as an animal model of severecombined immunodeficiency, reflecting themost highly studied feature of ADA defi-ciency in humans. In agreement with expec-tations, we showed that ADA-deficient micehave a combined immunodeficiency charac-terized by a reduction in T, B, and NK cells.34

Although immunodeficiency is the moststudied feature of ADA deficiency, additionalfeatures including hepatocellular impair-ment, pulmonary insufficiency, neurologicdisturbances, kidney pathology, adrenal ab-normalities, and skeletal deformities werealso observed in ADA�/� mice.35 Most ofthese features had been described earlier inADA-deficient children in the years beforethe advent of enzyme therapy.21–23 Before theadvent of PEG-ADA therapy, most ADA-de-ficient children died within the first 2 years oflife.21–23 Analysis of tissues taken at autopsyfor eight patients with ADA-deficient severecombined immune deficiency showed nu-merous nonlymphoid alterations, includingrenal, adrenal, bone, and cartilage.36 It isnoteworthy that the renal findings we de-scribe here for ADA�/� mice are reminiscentof those described earlier for ADA-deficientchildren, including mesangial sclerosis and

hypercellularity.36 Thus, our findings about the important role ofelevated adenosine in renal fibrosis in ADA-deficient mice arestrongly supported by the renal impairment observed in ADA-deficient children.

Figure 6. Genetic deletion of A2BR attenuates UUO-induced renal fibrosis in mice.(A) H&E staining and Masson’s trichrome staining and (B) quantitative imageanalyses showed significant reduction of UUO-induced vascular damage and fibro-sis in glomeruli and interstitial tissues in A2BR-deficient mice compared with WTmice. (C) Quantitative RT-PCR measurement of renal A2BR mRNA levels of sham-operated and UUO-manipulated WT mice. (D) Collagen content in kidneys of WTand A2BR-deficient mice with sham-operation or UUO manipulation. (E–G) CollagenI mRNA (E), IL-6 mRNA, (F), and Pai-1 mRNA (G) in in the kidney of WT andA2BR-deficient with sham-operation or UUO manipulation. Data are expressed asmean � SEM (n � 5 to 6). $P � 0.05 versus sham-operated WT mice. $$P � 0.05versus UUO-manipulated WT mice.



The renin angiotensin system is a signaling cascade control-ling arterial BP and salt balance.37,38 In both humans and ani-mals with renal dysfunction, circulating Ang II is elevated.39

Intriguingly, adenosine is reported to be elevated in Ang II–infused animals, a well-accepted animal model of CKD.24,25

However, the pathologic role of elevated adenosine in Ang II–induced CKD is unknown. Our initial discovery of the contrib-utory role of elevated adenosine to CKD in ADA-deficientmice led us to further show that increased adenosine also playsan important role in Ang II–induced renal fibrosis and dys-function in vivo. Of note, we found that renal damage andfibrosis in Ang II–infused mice were significantly but not com-pletely ameliorated by either PEG-ADA or PSB1115 treatment.These findings indicate that excess adenosine contributes di-rectly to renal fibrosis and damage via A2BR activation. How-ever, because renal pathology was not completely inhibited by

PEG-ADA or PSB1115, it is also possiblethat adenosine signaling may not accountfor all of Ang II–induced renal damage andfibrosis and that some of the detrimentaleffects of Ang II are independent of ele-vated adenosine. More importantly, we ex-tended and confirmed the important roleof A2BR in renal fibrosis in the UUO mousemodel of renal fibrosis. However, unlikeADA deficiency, the Ang II infusion andUUO-induced renal fibrosis mouse modelsare not characterized by widespread eleva-tion of adenosine (data not shown). Thus,the association of chronic elevated adeno-sine and A2BR signaling that we report herefor Ang II–infused mice and UUO mice isnot likely to be related to systemic actionsof adenosine but rather local effects ofadenosine on kidney fibrosis. Overall, ourfindings showed adenosine signaling as anovel mechanism for renal fibrosis andkidney dysfunction in general and therebyidentified this signaling pathway as a prom-ising therapeutic target for the treatmentand prevention of progressive renal fibrosisin CKD.

Here we showed that renal A2BR geneexpression is significantly elevated in ADA-deficient mice, Ang II–infused mice, andthe UUO model. These findings suggestthat there are common causative factors re-sponsible for elevated A2BR expression inthese mouse models of renal fibrosis. A2BRexpression is known to be elevated underhypoxic conditions.10,40 One of the tran-scription factors known to be responsiblefor enhanced A2BR gene expression underhypoxic condition is hypoxia inducible fac-tor (HIF).41 Another transcription factor

underlying increased A2BR expression is the cAMP responseelement binding protein CREB.10,40 Intriguingly, the A2BR is aGs-coupled receptor that signals through adenylyl cyclase ac-tivation, leading to increased cAMP production and the acti-vation of CREB. Thus, activation of A2BR itself functions as apositive feedback to further enhance A2BR gene expression. Inview of this regulatory network and our findings that selectiveelevation of A2BR gene expression was observed in the kidneysof all three models of renal fibrosis, we speculate that hypoxia-mediated HIF stabilization and A2BR-mediated CREB activa-tion function together as a malicious cycle underlying upregu-lation of A2BR expression in renal fibrosis.

The progressive nature of renal fibrosis seen in ADA-defi-cient mice, Ang II–infused mice, and the UUO mouse modelsuggests that adenosine may activate common pathways thatmaintain or promote the progression from renal damage to

Figure 7. IL-6 is a common signaling molecule downstream of A2B adenosine receptoractivation responsible for excess adenosine-mediated renal fibrosis by increased pro-collagen production. (A) Analysis of collagen secretion from cultured kidney explantsisolated from WT and four adenosine receptor-deficient mice in response to NECAtreatment. Data are expressed as mean � SEM. @P � 0.05 versus without treatment(n � 4). @@P � 0.05 versus WT treated with NECA. (B) NECA increased the expressionof pro-collagen I mRNA levels in kidney explants, which was completely abolished bythe A2BR-specific antagonist, MRS1754, or genetic deletion of A2BR. @P � 0.05 versuswithout treatment. @@P � 0.05 versus treatment with NECA (n � 4 to 6). (C) NECA-induced IL-6 mRNA expression in kidney explants was inhibited by the A2BR-specificantagonist, MRS1754, or genetic deletion of A2BR. Data are expressed as mean �SEM. @P � 0.05 versus without treatment. @@P � 0.05 versus treatment with NECA(n � 4 to 6). (D) NECA-induced expression of pro-collagen I mRNA levels in kidneyexplants was abolished by the presence of IL-6 neutralizing antibody but not isotypecontrol antibody. @P � 0.05 versus without treatment. @@P � 0.05 versus treatmentwith NECA (n � 4 to 6).



kidney fibrosis. All of these animal models are useful in exam-ining common mechanisms and potential therapeutic possi-bilities by targeting adenosine mediated fibrosis, as well as in-vestigating the cellular signaling pathways involved in theprogression, maintenance, and resolution of renal fibrosis. Weused both in vivo and in vitro approaches to characterize aden-osine levels, adenosine receptors, and signaling pathways in-volved in renal fibrosis. Using PEG-ADA enzyme therapy, weshowed that increased adenosine is an important contributingfactor for renal fibrosis in ADA-deficient and Ang II–infusedmice. Using an A2BR-specific antagonist, PSB1115, we furthershowed that activation of the A2BR is a major factor contribut-ing to renal fibrosis and dysfunction in both mouse models.Moreover, using A2BR-deficient mice, we further showed thatA2BR is required for UUO-induced renal fibrosis. Of note, wefound that all three mouse models share similar expressionprofiles of mediators of renal fibrosis, such as IL-6, PAI-1, andprocollagen, suggesting that renal fibrosis in these three mousemodels shares common mechanistic pathways that may be di-rectly or indirectly influenced via A2BR signaling. Using phar-macologic and genetic approaches, we determined that aden-osine functions through the A2BR to stimulate procollagenexpression. Finally, we showed that IL-6, a potent profibroticfactor, induced by adenosine in the kidney via A2BR activation,contributes to increased procollagen production, a major fi-brotic protein. Taken together, our studies showed that ade-nosine-induced A2BR activation resulted in increased produc-tion of IL-6. The resulting increase in IL-6 productioncontributes to renal fibrosis by stimulating increased procolla-gen synthesis.

Although prolonged chronic exposure to high levels ofadenosine is harmful to tissue, as we showed here for kidneys,the work of Grenz et al.14 showed that A2BR signaling is impor-tant for protecting the kidney from acute injury after ischemicpreconditioning. Subsequently, Rosenberger et al.42 showedthat HIF is responsible for hypoxia-mediated acute inflamma-tory response (including the induction of IL-6) by induction ofnetrin-1, which has an anti-inflammatory role via A2BR activa-tion. In contrast, under conditions of chronically elevatedadenosine concentrations, we showed here that persistentA2BR activation is detrimental because of the induction of pro-collagen and other profibrotic mediators via IL-6 signaling.Overall, these findings support an emerging view that, al-though A2BR signaling is protective during acute injury, pro-longed exposure to a high adenosine concentration is detri-mental.

There is an enormous knowledge gap in understanding theprogression of renal fibrosis in CKD. ADA-deficient mice haveserved as valuable experimental system to conduct in vivo bio-chemical screens to identify aspects of mammalian physiologyand development that are impaired by abnormally high aden-osine signaling. For example, ADA-deficient mice were instru-mental in discovering the detrimental role of elevated adeno-sine in priapism (prolonged penile erection),11 pulmonarydisease,35 and now renal fibrosis. After our initial discovery of

the role of adenosine signaling in renal fibrosis and function inADA-deficient mice, we subsequently extended these findingsto more widely accepted models of renal fibrosis involvingchronic Ang II infusion and UUO mouse models. In all threemouse models studied here, we showed that elevated A2BR-mediated IL-6 signaling underlies the progression of renal fi-brosis and renal dysfunction. These findings will not only pro-vide new insight into the pathogenesis of CKD but also openup the possibility of preventing and treating this challengingdisease with PEG-ADA enzyme therapy to reduce adenosinelevels and A2BR antagonists to block detrimental signaling.Thus, interference with adenosine signaling is likely a novel,effective, and safe mechanism-based strategy to treat and pre-vent renal fibrosis. If adenosine-induced renal fibrosis can beprevented by PEG-ADA or AR antagonists, the progression ofthis disease could be halted.

CONCISE METHODS

MiceADA-deficient mice were generated and genotyped as described pre-

viously.34,35,43 Control mice, designated ADA�, were littermates that

were heterozygotes for the null Ada allele. Heterozygous mice do not

display a phenotype. All mice were initially on a mixed 129sV/

C57BL/6J background and subsequently backcrossed at least 10 gen-

erations on the C57BL/6 background. All phenotypic comparisons

were performed among littermates. Four adenosine receptor-defi-

cient mutants were also backcrossed at least 10 generations onto the

C57BL/6 background and were genotyped according to established

protocols.44 Ang II was delivered at a rate of 1.5 mg/kg body weight

per day into 12-week-old C57BL/6J mice with osmotic minipumps

(Alzet model 2001; Alza, Palo Alto, CA) implanted subcutaneously in

the nape of the neck for 2 weeks. All mice were maintained and housed

in accordance with National Institute of Health guidelines and with

the approval of the Animal Care and Use Committee at the University

of Texas Health Science Center at Houston.

ADA Enzyme TherapyPEG-ADA was generated by the covalent modification of purified

bovine ADA with activated PEG as described previously.45– 47 Differ-

ent dosages of PEG-ADA were delivered weekly by intraperitoneal

injection to reduce adenosine levels. Specifically, the ADA-deficient

mice were maintained on high dose (HD) enzyme therapy at 5 U/wk

for at least 8 weeks to allow for normal kidney development. At 8

weeks of age, the dose of PEG-ADA for some mice was tapered down

over a 4-week period to a low dosage of 0.625 U/wk (2.5 U for 2 weeks,

1.25 U for 2 weeks, and then 0.625 U for the remainder of the exper-

iment). This dosing protocol was designated an low dose (LD) PEG-

ADA treatment regimen. Some ADA-deficient mice were treated with

HD PEG-ADA (5 U/wk) from birth for 16 weeks. This dosing proto-

col was designated an HD PEG-ADA treatment regimen. For all ex-

periments, ADA� mice treated with or without an HD of PEG-ADA

at 5 U/wk were used as the controls.



For Ang II–infused mice, a group of mice was injected with 2.5 U

PEG-ADA weekly for 2 weeks. This dosing protocol was designated

Ang II–infused mice with PEG-ADA treatment regimen (Ang

II�PEG-ADA). Other Ang II–treated mice were injected with normal

saline for 2 weeks; this group was designated as Ang II without PEG-

ADA treatment regimen (Ang II). Age-matched C57Bl/6 mice treated

with or without PEG-ADA at 2.5 U/wk were used as the controls.

Treatment with the A2BR Antagonist PSB1115ADA-deficient mice on LD PEG-ADA treatment with kidney dys-

function and renal fibrosis or WT mice were divided into two groups:

one group was injected with 200 �g of PSB1115 (A2B receptor antag-

onist; Tocris Bioscience, St. Louis, MO) in PBS, daily for 2 weeks.

PSB1115 treatment was initiated at 8 weeks of age for ADA-deficient

mice that were on the LD PEG-ADA regimen. Note that the LD regi-

men was achieved by providing HD PEG-ADA for the first 8 weeks of

life to allow for normal development, followed by a gradual reduction

of PEG-ADA therapy to the LD level over an additional 4-week pe-

riod. At 8 weeks of age, some of the LD PEG-ADA–treated ADA-

deficient mice began to receive daily injections of PSB1115 for an

additional 8 weeks. A control group of ADA� was injected with saline

or HD PEG-ADA.

UUOTwelve- to 16-week-old male mice were anesthetized with ketamine

and xylazine. Mice were placed on a heating pad at 37°C. The left

ureter was identified through a midline incision and ligated with 3-0

sutures at two sites near the renal hilum. Sham-operated mice (the

same operation without ureter ligation) were also produced and used

as controls. Kidney tissue samples collected at day 14 after UUO were

used for histology, collagen content measurements, and a real-time

PCR analysis.

Quantification of Kidney Adenosine LevelsMice were anesthetized, and the kidneys were rapidly removed and

frozen in liquid nitrogen. Adenine nucleosides were extracted from

frozen kidneys using 0.4 N perchloric acid, and adenosine was sepa-

rated and quantified using reverse-phase HPLC as described previ-

ously.34,43

UrinalysisUrinary samples were collected for 24 hours using a metabolic cage

(Nalgene). We quantified urinary albumin by ELISA (Exocell) and

measured urinary creatinine by a picric acid colorimetric assay kit

(Exocell). We used the ratio of urinary albumin to urinary creatinine

as an index of urinary protein as described.48,49

Histologic AnalysisMice were anesthetized, and the kidneys were isolated and pressure-

infused with 4% paraformaldehyde in PBS and fixed overnight at 4°C.

Fixed tissues were rinsed in PBS, dehydrated through graded ethanol

washes, and embedded in paraffin. Five-micrometer sections were

collected on slides and stained with H&E or Masson’s trichrome, ac-

cording to the manufacturer’s instructions (Shardon-Lipshaw).

Morphometric Analysis of the Renal Fibrosis inMasson’s Trichrome–Stained SectionsTen consecutive nonoverlapping fields of a mouse kidney stained

with the Trichrome were analyzed. The fibrotic areas stained in light

blue were picked up on the digital images using a computerized den-

sitometry (ImagePro Plus, version 6.0; Media Cybernetics, Silver

Spring, MD) coupled to a microscope equipped with a digital camera

as described.50,51 The percentage of the fibrotic area relative to the

whole area of the field was calculated (percent fibrosis area). The

average densities of 10 areas per kidney were averaged, the SEM is

indicated, and n � 4 to 6 kidneys for each category.

Collagen QuantificationSoluble collagen levels were quantified in kidney tissues using the

Sircol collagen assay (Biocolor, Belfast, Ireland) according to the

manufacturer’s instructions.

Total RNA Isolation and Real-Time RT-PCR AnalysisTotal RNA was isolated using TRIzol reagent (Invitrogen). RNase-

free DNase (Invitrogen) was used to eliminate genomic DNA con-

tamination. Transcript levels were quantified using real-time quanti-

tative RT-PCR. Cyber green was used for analysis of �2 (I)

procollagen, IL-6, plasminogen activator inhibitor-1 (Pai-1), and

�-actin using the following primers: �2 (I) procollagen, forward, 5�-

AGACATGCTCAGCTTTGTGGATAC-3� and reverse, 5�-CGTACT-

GATCCCGATTGCAAAT-3�; Pai-1, forward, 5�-AGTGATGGAGC-

CTTGACAG-3� and reverse, 5�-AGGAGGAGTTGCCTTCTCTT-3�;

�-actin, forward, 5�- GCTCTGGCTCCTAGCACCAT-3� and reverse,

5�-CCACCGATCCACACAGAGTAC-3�. Adenosine receptor tran-

scripts were analyzed using Taqman probes, with primer sequences

and conditions as described previously.45,46 The sequence for four

adenosine receptor were described previously.33

Isolation of Kidneys and Organ Culture ConditionsKidneys were surgically isolated from WT mice and four adenosine

receptor– deficient mice. The isolated kidneys were washed in PBS,

minced into 2- to 3-mm3 pieces, suspended in DMEM containing

10% FBS, and cultured in a humidified atmosphere of 5% CO2 at 37

°C. After 24 hours in culture, tissues were serum-starved in DMEM

without FBS and treated with various drugs. Specifically, NECA (20

�M), a potent nonmetabolized adenosine analog, and MRS1754 (20

�M), a specific A2BR antagonist, were used. IL-6 neutralizing anti-

body or isotype control antibody (R&D Systems) at a final concentra-

tion of 0.5 �g/ml was used. After 24-hour treatment, collagen content

in the supernatants and total RNA from tissues were isolated as de-

scribed above.

Statistical AnalysisAll data are expressed as the mean � SEM. Unpaired t tests were

applied in two-group analysis. Statistical significance of the difference

of multiple groups of mice was assessed by two-way repeated-mea-

sures ANOVA, followed by Bonferroni post tests. Data were analyzed

for statistical significance using GraphPad Prism 4 software (Graph-

Pad Software, San Diego, CA).48,49 A value of P � 0.05 was considered

significant.



ACKNOWLEDGMENTS

This work was supported by National Institute of Health Grants

DK077748 (to Y.X.) and DK083559 (to Y.X.) and China Scholarship

Council Grant 2008637068 (to J.W.).

DISCLOSURESNone.

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