A1 adenosine receptor allosteric enhancer PD-81723 ... · A 1 adenosine receptor allosteric enhancer PD-81723 protects against renal ischemia-reperfusion injury Sang Won Park,1 Joo

doi: 10.1152/ajprenal.00157.2012303:F721-F732, 2012. First published 3 July 2012;Am J Physiol Renal Physiol

D'Agati and H. Thomas LeeSang Won Park, Joo Yun Kim, Ahrom Ham, Kevin M. Brown, Mihwa Kim, Vivette D.protects against renal ischemia-reperfusion injuryA1 adenosine receptor allosteric enhancer PD-81723

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A1 adenosine receptor allosteric enhancer PD-81723 protects against renalischemia-reperfusion injury

Sang Won Park,1 Joo Yun Kim,1 Ahrom Ham,1 Kevin M. Brown,1 Mihwa Kim,1 Vivette D. D’Agati,2

and H. Thomas Lee1

1Department of Anesthesiology, Columbia University, New York, New York; and 2Department of Pathology, ColumbiaUniversity, New York, New York

Submitted 19 March 2012; accepted in final form 29 June 2012

Park SW, Kim JY, Ham A, Brown KM, Kim M, D’Agati VD,Lee HT. A1 adenosine receptor allosteric enhancer PD-81723protects against renal ischemia-reperfusion injury. Am J PhysiolRenal Physiol 303: F721–F732, 2012. First published July 3, 2012;doi:10.1152/ajprenal.00157.2012.—Activation of A1 adenosine re-ceptors (ARs) protects against renal ischemia-reperfusion (I/R) injuryby reducing necrosis, apoptosis, and inflammation. However, extrare-nal side effects (bradycardia, hypotension, and sedation) may limitA1AR agonist therapy for ischemic acute kidney injury. Here, wehypothesized that an allosteric enhancer for A1AR (PD-81723) pro-tects against renal I/R injury without the undesirable side effects ofsystemic A1AR activation by potentiating the cytoprotective effects ofrenal adenosine generated locally by ischemia. Pretreatment withPD-81723 produced dose-dependent protection against renal I/R in-jury in A1AR wild-type mice but not in A1AR-deficient mice. Sig-nificant reductions in renal tubular necrosis, neutrophil infiltration,and inflammation as well as tubular apoptosis were observed in A1ARwild-type mice treated with PD-81723. Furthermore, PD-81723 de-creased apoptotic cell death in human proximal tubule (HK-2) cells inculture, which was attenuated by a specific A1AR antagonist (8-cyclopentyl-1,3-dipropylxanthine). Mechanistically, PD-81723 in-duced sphingosine kinase (SK)1 mRNA and protein expression inHK-2 cells and in the mouse kidney. Supporting a critical role of SK1in A1AR allosteric enhancer-mediated renal protection against renalI/R injury, PD-81723 failed to protect SK1-deficient mice againstrenal I/R injury. Finally, proximal tubule sphingosine-1-phosphatetype 1 receptors (S1P1Rs) are critical for PD-81723-induced renalprotection, as mice selectively deficient in renal proximal tubuleS1P1Rs (S1P1Rflox/flox PEPCKCre/� mice) were not protected againstrenal I/R injury with PD-81723 treatment. Taken together, our exper-iments demonstrate potent renal protection with PD-81723 against I/Rinjury by reducing necrosis, inflammation, and apoptosis through theinduction of renal tubular SK1 and activation of proximal tubuleS1P1Rs. Our findings imply that selectively enhancing A1AR activa-tion by locally produced renal adenosine may be a clinically usefultherapeutic option to attenuate ischemic acute kidney injury withoutsystemic side effects.

acute kidney injury; apoptosis; inflammation; necrosis; sphingosine1-phosphate; sphingosine kinase

ACUTE KIDNEY INJURY (AKI) is a major clinical problem in theUnited States and frequently causes prolonged hospitalization,chronic renal failure, and death (24). Renal ischemia-reperfu-sion (I/R) injury due to surgical renal ischemia or renal hypo-perfusion is a leading cause of AKI (20). Ischemic AKI, inparticular, is a major clinical problem for patients subjected to

major surgical procedures involving the kidney, liver, heart, oraorta, often leading to multiorgan dysfunction and systemicinflammation with extremely high mortality (24). Unfortu-nately, the severity and incidence of AKI have been increasingwithout any improvements in therapy or patient survival overthe past 50 yr (23). Currently, there are no proven therapies toreduce AKI in the perioperative setting.

We (32, 34) have previously demonstrated that systemicadministration of a selective renal A1 adenosine receptor (AR)agonist protected against renal I/R injury in rats and mice withreduced plasma creatinine and dramatically reduced proximaltubular necrosis, inflammation, and apoptosis. Antinecrotic andantiapoptotic effects of renal A1ARs are mediated by activationof ERK, PKB (Akt), and heat shock protein 27 through apertussis toxin-sensitive G protein pathway (25, 27). Unfortu-nately, as A1ARs are ubiquitously expressed in many cell typesand organs, clinical application of A1AR agonist therapy maybe limited by its systemic and undesirable side effects. A1ARagonists produce dose-dependent bradycardia and hypotensionas well as central nervous systemic depression and sedation(5). These nonrenal side effects may not be tolerated in patientssubjected to ischemic AKI or undergoing major surgical pro-cedures.

The potential side effects of selective A1AR agonists may bemitigated by the use of A1AR-selective allosteric enhancers (5,26). Allosteric enhancers for A1ARs bind to a site on thereceptor that is distinct from the adenosine-binding site (knownas the orthosteric site) (5, 7). A1AR allosteric enhancers in-crease the potency of endogenous adenosine-A1AR interac-tions, leading to enhanced receptor activity. Since A1AR allo-steric enhancers selectively increase the efficacy of endoge-nous adenosine in tissues (e.g., the ischemic kidney producingincreased localized adenosine), systemic administration ofA1AR allosteric enhancers may drastically increase renalA1AR activation without producing undesirable systemic sideeffects.

In this study, we tested the hypothesis that a selective A1ARallosteric enhancer (PD-81723) produces kidney-specific pro-tection against renal I/R injury without producing undesirablesystemic side effects (e.g., hypotension or bradycardia) in vivo.We determined whether PD-81723 protects against necrosisand apoptosis in human proximal tubule (HK-2) cells in cul-ture. We also examined the mechanisms of PD-81723-medi-ated renal tubular protection in vivo and in vitro.

METHODS

Materials. 2-Amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)phenyl] methanone (PD-81723; a selective allosteric enhancer for theA1AR; Fig. 1), 2-chloro-N6-cyclopentyladenosine (CCPA; a selective

Address for reprint requests and other correspondence: H. T. Lee, Dept. ofAnesthesiology, Anesthesiology Research Laboratories, Columbia Univ., P&SBox 46 (PH-5), 630 W. 168th St., New York, NY 10032-3784 (e-mail: [email protected]).

Am J Physiol Renal Physiol 303: F721–F732, 2012.First published July 3, 2012; doi:10.1152/ajprenal.00157.2012.

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A1AR agonist), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; a selec-tive antagonist for the A1AR) and 4-{[4-(4-chlorophenyl)-2-thiazolyl]amino}phenol [SKI-II, a selective nonlipid inhibitor of sph-ingosine kinase (SK)] were purchased from Tocris (Minneapolis,MN). Unless otherwise specified, all other drugs were purchased fromSigma (St. Louis MO).

Murine model of renal I/R injury. After approval from the Institu-tional Animal Care and Use Committee, we subjected adult maleC57BL/6 (Harlan, Indianapolis, IN) as well as SK1�/� and SK2�/�

mice (kindly provided by Dr. R. L. Proia, National Institutes ofHealth, Bethesda, MD) to 30 min of renal I/R, as previously described(27, 30). To test the renal protective effects of PD-81723, we pre-treated mice with vehicle (1% DMSO) or with PD-81723 (0.3–3mg/kg ip) 15 min before renal ischemia or sham operation. We alsotested whether treatment of mice with PD-81723 after the completionof renal ischemia also provides protection against ischemic AKI.Separate cohorts of mice were treated with vehicle or with PD-81723(3 mg/kg ip) 30 min after reperfusion of the ischemic kidney.

To test whether proximal tubule sphingosine-1-phosphate (S1P)type 1 receptor (S1P1R) activation is critical for PD-81723-mediatedrenal protection in vivo, we generated mice with proximal tubule-specific deletion of S1P1Rs. We crossed mice carrying the floxedS1P1R gene [S1P1Rff mice, provided by Dr. R. L. Proia, NationalInstitutes of Health (2)] with mice expressing Cre recombinase underthe control of phosphoenolpyruvate carboxykinase (PEPCK) pro-moter [PEPCK-Cre recombinase mice, provided by Dr. Volker Haase,Vanderbilt University, (49)]. S1P1R-floxed mice carrying the PEPCK-Cre gene (S1P1R-null or S1P1Rf/f PEPCKCre/� mice) and controlmice [wild-type (WT) or S1P1Rf/f PEPCK�/� mice] were genotypedas previously described (43). We (43) have previously shown thatS1P1Rflox/flox PEPCKCre/� mice display drastically reduced (�10%)S1P1R mRNA in the renal cortex and proximal tubules compared withWT control mice. We also showed that S1P1R mRNA levels in thekidney inner medulla, liver, and lung did not change with proximaltubule S1P1R deletion.

We collected kidneys (cortex and corticomedullary junction) andplasma 6 or 24 h after I/R injury to examine the severity of renaldysfunction (plasma creatinine, renal histology, apoptosis, proinflam-matory mRNA expression, and neutrophil infiltration). To test theeffects of SK inhibition on PD-81723-mediated renal protection,SKI-II (50 mg/kg sc) was administered 15 min before PD-81723treatment. SKI-II is an SK-selective inhibitor with minimal effects onother kinases (17). In some mice, we measured systolic blood pressureas well as heart rate to determine the hemodynamic effects ofPD-81723 or CCPA administration via an indwelling carotid arterycatheter.

HPLC for adenosine. Mouse kidneys were snap frozen in liquidnitrogen at 5-min intervals immediately after renal ischemia and 30min after reperfusion. Kidneys were sonicated in 0.6 M perchloricacid and neutralized by the addition of 0.6 M potassium phosphatetribasic. The supernatant was assayed for adenosine by HPLC. Aden-

osine was quantified on a C18 reverse-phase column with a binarylow-pressure gradient elution system with a UV detector set to 254 nmas previously described (14).

Measurement of renal function. Plasma creatinine was measured aspreviously described with an enzymatic creatinine reagent kit accord-ing to the manufacturer’s instructions (Thermo Fisher Scientific,Waltham, MA) (50). Unlike the Jaffe method, this method of creati-nine measurement largely eliminates the interference from mouseplasma chromagens.

Histological detection of renal necrosis. Morphological assessmentof hematoxylin and eosin staining was performed by an experiencedrenal pathologist (V. D. D’Agati), who was unaware of the treatmentthat each animal had received. An established grading scale ofnecrotic injury (renal injury scores of 0–4) to the proximal tubuleswas used for the histopathological assessment of I/R-induced damageas outlined by Jablonski et al. (21) and as described in our previousstudies (34, 37).

Detection of renal tubular apoptosis. We detected apoptosis afterrenal I/R with TUNEL staining as previously described elsewhere(44) using a commercially available in situ cell death detection kit(Roche, Indianapolis, IN) according to the instructions provided bythe manufacturer.

Assessment of renal inflammation. Renal inflammation after I/Rinjury was determined 1) with detection of neutrophil infiltration withimmunohistochemistry 24 h after renal I/R and 2) by measuringmRNA encoding markers of inflammation, including ICAM-1, mono-cyte chemoattractant protein (MCP)-1, macrophage inflammatoryprotein (MIP)-2, and TNF-�. Immunohistochemistry for neutrophilswas performed as previously described (11) with a monoclonalantibody against polymorphonuclear neutrophils (clone 7/4). A pri-mary antibody that recognized IgG2a (MCA1212, Serotec, Raleigh,NC) was used as a negative isotype control in all experiments.Semiquantitative RT-PCR assays for proinflammatory mRNAs wereperformed as previously described (37) (Table 1).

HK-2 cell culture and induction of necrosis and apoptosis. Necroticinjury in HK-2 cells was induced with exposure to 2 mM H2O2 for4 h, and lactate dehydrogenase (LDH) released into cell culture mediawas measured as previously described (33). To induce apoptosis,HK-2 cells were exposed to TNF-� (20 ng/ml) plus cycloheximide(10 �g/ml) for 16 h, as previously described (35). Some HK-2 cellswere pretreated with PD-81723 (1–10 �M) 30 min before the induc-tion of necrosis or apoptosis. Separate cohorts of HK-2 cells weretreated with PD-81723 (1–10 �M) for 6–16 h to test for the inductionof SK1 or SK2 mRNA and protein. Some HK-2 cells were pretreatedwith 100 nM DPCPX, a selective antagonist for A1ARs, 15 minbefore PD-81723 treatment.

RT-PCR and immunoblot analyses for SK. We measured mRNAencoding human SK1 or SK2 6 h after PD-81723 treatment in HK-2cells as previously described (29). Table 1 shows the primer se-quences used in this study. HK-2 cell lysates were also collected forimmunoblot analyses of SK1, SK2, and �-actin (an internal proteinloading control) as previously described 16 h after PD-81723 treat-ment (29).

SK activity assay. SK activity was measured as previously de-scribed (28) using a modified protocol according to Vessey et al. (54).To preferentially measure SK1 activity, we supplemented the assaybuffer with 0.25 M KCl and 0.5% Triton X-100 (46).

Statistical analysis. Data were analyzed with Student’s t-test whenmeans were compared between two groups or one-way ANOVA plusTukey’s post hoc multiple-comparison test when multiple groupswere compared. Two-way ANOVA plus the Bonferroni posttest wasused to test the effects of sham operation or renal I/R injury ondifferent mouse strains, treatment groups, and hemodynamic param-eters. The ordinal values of the renal injury scores were analyzed bya Mann-Whitney nonparametric test. In all cases, P values of �0.05were taken to indicate significance. All data are expressed throughoutthe text as means � SE.

Fig. 1. Chemical structure of the A1 adenosine receptor (AR) allostericenhancer 2-amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)phenyl]metha-none (PD-81723). Previous structure activity relationship studies have deter-mined that chemicals with a 2-amino-3-benzoylthiophene core (outlined) areexcellent A1AR allosteric enhancers.

F722 PD-81723 PROTECTS AGAINST RENAL I/R INJURY

AJP-Renal Physiol • doi:10.1152/ajprenal.00157.2012 • www.ajprenal.org

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RESULTS

Adenosine levels after renal ischemia and reperfusion. Kid-ney adenosine levels (in pmol/�g protein) increased within 5min after renal ischemia compared with preischemic levels(Fig. 2). This increase in renal adenosine levels persistedduring 30 min of ischemia. Interestingly, kidney adenosinelevels decreased to preischemic levels 30 min after reperfusionof the ischemic kidney. In contrast, we found no changes incardiac, hepatic, and lung adenosine levels during or after renalischemia (data not shown).

Differential cardiovascular effects of PD-81723 andCCPA. Next, we compared the effects of a selective A1ARallosteric enhancer (PD-81723) and a selective A1AR agonist(CCPA) on mouse heart rate and blood pressure. The resultsshown in Fig. 3 demonstrate that PD-81723 (3 and 6 mg/kg ip)did not change heart rate or systolic blood pressure in mice. Aselective A1AR agonist [CCPA (0.1 and 0.5 mg/kg ip)], incontrast, caused a dose-dependent decrease in heart rate andsystolic blood pressure in mice. CCPA at 0.1 mg/kg caused an�14% drop in heart rate and an �20% decrease in systolicblood pressure. At a higher dose (0.5 mg/kg), the decrease inheart rate and systolic blood pressure with CCPA were signif-icantly greater (Fig. 3).

Renal protective effects of A1AR allosteric enhancer PD-81723 administration. We initially tested whether PD-81723pretreatment protects against renal I/R injury in mice. Plasmacreatinine values were similar between sham-operated (anes-thesia, laparotomy, right nephrectomy, and recovery) vehicle(1% DMSO)-treated (creatinine: 0.48 � 0.03 mg/dl, n � 3),and PD-81723-treated (creatinine: 0.43 � 0.03 mg/dl, n � 3)mice. Plasma creatinine significantly increased in vehicle-treated mice subjected to 30 min of renal I/R compared withsham-operated mice (Fig. 4). Pretreatment with PD-81723 (15min before renal ischemia) significantly and dose dependently

Fig. 2. Kidney adenosine levels during renal ischemia and after reperfusion.Kidney adenosine levels were measured by HPLC. Kidneys were snap frozenin liquid nitrogen at the indicated times (n � 3–4 kidneys/time point). Kidneyadenosine rapidly increased within 5 min after renal ischemia, and this increasewas maintained during ischemia. However, kidney adenosine decreased topreischemic levels 30 min after reperfusion. *P � 0.01 vs. 0 min.T

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F723PD-81723 PROTECTS AGAINST RENAL I/R INJURY


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attenuated the increases in plasma creatinine in mice (Fig. 4).Further verifying that PD-81723 mediates renal protection viaenhanced activation of A1AR, mice deficient in A1ARs [A1ARknockout (A1KO) mice] were not protected against renal I/Rwith PD-81723 treatment. We also tested whether PD-81723treatment after renal reperfusion (after completion of renalischemia) protected against renal I/R injury. We determinedthat unlike pretreatment, PD-81723 given 30 min after I/R

(creatinine: 2.2 � 0.2 mg/dl, n � 4) failed to protect thekidney.

In addition to plasma creatinine, we also examined renalhistology after I/R. The results shown in Fig. 5A demonstratesevere necrotic renal injury in vehicle-treated A1AR WT(A1WT) mice subjected to I/R 24 h after injury. Comparedwith sham-operated vehicle-treated A1WT mice (not shown),the kidneys of vehicle-treated A1WT mice subjected to renalI/R showed significant tubular necrosis (proteinaceous castswith increased congestion; Fig. 5A). Consistent with theplasma creatinine data, mice treated with PD-81723 15 minbefore renal ischemia had reduced renal necrosis and tubularinjury (Fig. 5A). In contrast, PD-81723-treated A1KO miceshowed similar degrees of renal tubular necrosis and castformation compared with vehicle-treated A1KO mice. TheJablonski scale (21) renal injury score was used to grade renaltubular necrosis 24 h after renal I/R (Fig. 5B). Thirty minutesof renal ischemia and 24 h of reperfusion resulted in severe

Fig. 5. PD-81723 reduces kidney necrosis (A and B), apoptosis (C), neutrophil infiltration (D), and proinflammatory gene expression (E) in A1WT mice afterrenal I/R. A, C, and D: representative photomicrographs for hematoxylin and eosin (H&E) staining (A; magnification: 200), TUNEL staining (representingapoptotic nuclei; C; magnification: 200), and immunohistochemistry for neutrophil infiltration (D; magnification: 400) of kidney sections of mice. Mice werepretreated with vehicle (1% DMSO) or with 3 mg/kg PD-81723 and subjected to 30 min of renal ischemia and 24 h of reperfusion. Photographs are representativeof 4–5 independent experiments. B: summary of Jablonski scale renal injury scores (graded from H&E staining, scale: 0–4) for mice subjected to renal I/R.Vehicle-treated mice showed severe renal tubular necrosis, apoptosis, and neutrophil infiltration after I/R. Preischemic PD-81723 treatment significantlyattenuated renal I/R injury in A1WT mice but did not protect A1KO mice. Values are means � SE. #P � 0.05 vs. vehicle-treated A1WT mice subjected to renalI/R. E: representative gel images (top) of RT-PCR and densitometric quantification of relative band intensities normalized to GAPDH (bottom) ofproinflammatory markers [ICAM-1, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-2, and TNF-�] from renal cortices ofA1WT mouse kidneys (n � 4–5 mice/group). Mice were treated with vehicle or with PD-81723 (3 mg/kg) and subjected to sham operation or to renal I/R(6 h of reperfusion). PD-81723 selectively reduced expression of TNF-� and MCP-1 without affecting MIP-2 or ICAM-1 expression. Values are means � SE.*P � 0.05 vs. the vehicle-treated sham-operated group; #P � 0.05 vs. the vehicle-treat I/R group. Data were analyzed with one-way ANOVA plus a Tukey posthoc multiple-comparison test.

Fig. 3. Differential cardiovascular effects of PD-81723 and CCPA. Wecompared the effects of a selective A1AR allosteric enhancer (PD-81723) anda selective A1AR agonist [2-chloro-N6-cyclopentyladenosine (CCPA)] onmouse heart rate (A) and systolic blood pressure (B). We measured systolicblood pressure as well as heart rate via an indwelling carotid artery catheter.PD-81723 (3 and 6 mg/kg ip) did not significantly change heart or systolicblood pressure in mice (n � 4). In contrast, CCPA (0.1 and 0.5mg/kg ip, n �4) caused dose-dependent decreases in heart rate and systolic blood pressure inmice. Arrows indicate the time of drug injection. *P � 0.01 vs. baseline;#P � 0.01 vs. mice injected with 6 mg/kg PD-81723.

Fig. 4. Renal protection with PD-81723 pretreatment. Plasma creatinine levelsfrom A1AR wild-type (WT) or A1AR knockout (KO) mice subjected to shamsurgery or to renal ischemia-reperfusion (I/R) are shown. A1WT mice treatedwith 0.3–3 mg/kg PD-81723 15 min before renal ischemia were protectedagainst renal I/R injury (n � 6 mice/group). PD-81723 significantly and dosedependently attenuated the increases in plasma creatinine after renal I/R inA1WT mice. In contrast, PD-81723 failed to protect against renal injury inA1KO mice. Values are means � SE. *P � 0.05 vs. vehicle-treated A1WTor A1KO mice subjected to sham surgery; #P � 0.05 vs. vehicle-treatedA1WT mice subjected to renal I/R.



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acute tubular necrosis in vehicle-treated A1WT or A1KO mice.Consistent with the renal histology data, PD-81723 reduced therenal injury score in A1WT mice but not in A1KO mice.

PD-81723 treatment reduces renal apoptosis after I/R.TUNEL staining detected apoptotic renal cells in the kidneysof mice subjected to renal I/R where proximal tubule cellapoptosis was predominant. Renal ischemia and 24 h of rep-erfusion resulted in significant apoptosis in the kidneys ofvehicle-treated A1WT mice. PD-81723 pretreatment (3 mg/kgip 15 min before renal ischemia) reduced the number ofapoptotic TUNEL-positive cells in the A1WT mouse kidneywithout decreasing apoptosis in the A1KO mouse kidney (Fig.5C; magnification: 200).

PD-81723 treatment reduces kidney neutrophil infiltrationafter I/R. Figure 5D shows representative images of neutrophilimmunohistochemistry of the kidney (magnification: 400)from mice subjected to 30 min of renal ischemia and 24 h ofreperfusion. There was significant neutrophil infiltration in thekidneys of vehicle-treated A1WT and A1KO mice subjected to24 h of renal I/R. Stained neutrophils appeared as dark brown(Fig. 5D). In sham-operated mice, we were unable to detectany neutrophils in the kidney (data not shown). A1WT micetreated with PD-81723 (3 mg/kg ip) 15 min before renalischemia had reduced neutrophil infiltration in the kidney afterI/R. Again, PD-81723 failed to reduce neutrophil infiltration inA1KO mice.

PD-81723 reduces kidney TNF-� and MCP-1 expressionafter renal I/R. Renal I/R injury resulted in significantly in-creased renal proinflammatory ICAM-1, MCP-1, MIP-2, andTNF-� mRNA expression in the kidney within 6 h comparedwith sham-operated mice (Fig. 5E). PD-81723 pretreatment (3mg/kg ip 15 min before renal ischemia) selectively reduced therenal expression of TNF-� and MCP-1 compared with vehicle-treated A1WT mice without affecting ICAM-1 or MIP-2 ex-pression after renal I/R.

Acute PD-81723 reduces human renal proximal tubule cellapoptosis without affecting necrosis. Acute PD-81723 pretreat-ment protected against HK-2 cell apoptosis. HK-2 cells pre-treated with 10 �M PD-81723 were protected against TNF-�plus cycloheximide-induced apoptosis, as evidenced by re-duced poly(ADP-ribose) polymerase (PARP) and caspase-3fragmentation (Fig. 6, A and B) as well as decreased DNAladdering (Fig. 6C). We also tested whether PD-81723 pre-vented HK-2 cell apoptosis via activation of A1ARs. DPCPX(100 nM) significantly attenuated PARP and caspase-3 frag-mentation (Fig. 6, A and B) and prevented the reduction inDNA laddering (Fig. 6C) in HK-2 cells.

However, acute PD-81723 (1–10 �M) treatment (30 minbefore H2O2 treatment) had no effect on HK-2 cell necrosis(measured with LDH released) induced with 2 mM H2O2 for4 h (LDH in the 10 �M PD-81723-treated group: 25.4 � 2%,n � 4, vs. LDH in the vehicle-treated group: 26.3 � 3%, n �4). Interestingly, when HK-2 cells were pretreated with 10 �MPD-81723 4 h before H2O2 treatment, HK-2 cells were pro-tected against necrosis and released significantly less LDH(LDH in the PD-81723 treated group: 30.6 � 2%, n � 4, vs.LDH in the vehicle-treated group: 43 � 3%, n � 4, P � 0.05).

PD-81723 increases SK1 synthesis and induces SK activityin HK-2 cells. Next, we determined the mechanisms of PD-81723-mediated renal protection. Since we (43) have recentlyshown that a selective A1AR agonist (CCPA) induced SK1 in

HK-2 cells and in the mouse kidney, we tested the hypothesisthat PD-81723 may also induce SK1 in renal proximal tubules.HK-2 cells were treated with 10 �M PD-81723 for SK mRNA(6 h) or protein (16 h) analysis. We determined that PD-81723selectively increased SK1 mRNA and protein expression inHK-2 cells (Fig. 7, A and B). SK2 mRNA or protein expressiondid not change. Furthermore, PD-81723 treatment for 6 hincreased SK activity in HK-2 cells (Fig. 7C). We preferen-tially measured SK1 activity by adding Triton X-100 in our SKactivity assay. A selective A1AR antagonist (100 nM DPPCX)significantly attenuated the PD-81723-induced increases in SKactivity (Fig. 7C).

PD-81723 increases SK1 mRNA and protein synthesis in themouse kidney. PD-81723 also increased SK1 mRNA andprotein expression in the mouse kidney. Figure 8 shows selec-tive SK1 mRNA (A) and protein (B) induction in the kidneysof mice injected with 3 mg/kg ip 6 h after PD-81723 treatment.

Critical role of SK1 in PD-81723-mediated renal protectionagainst I/R. We next tested whether PD-81723 requires theinduction of SK1 for renal protection. PD-81723 (3 mg/kgip)-induced renal protection was abolished in A1WT micepretreated with SKI-II (a selective SK inhibitor, 50 mg/kg, sc;Fig. 9). Furthermore, we determined that SK1�/� mice werenot protected with PD-81723 treatment, whereas SK2�/� micewere significantly protected against renal I/R with PD-81723treatment (Fig. 9).

Critical role of renal proximal tubule S1P1Rs in PD-81723-mediated renal protection. To test whether proximal tubuleS1P1R activation is critical for PD-81723-mediated renal pro-tection, we generated mice with proximal tubule-specific de-letion of S1P1Rs by crossing mice carrying the floxed S1P1Rgene with PEPCKCre recombinase mice (2, 49). Figure 10 showsplasma creatinine from WT (S1P1Rf/f PEPCK�/�) mice andproximal tubule-specific S1P1R-null (S1P1Rf/f PEPCKCre/�)mice subjected to 30 min of renal ischemia and 24 h of reper-fusion. Treatment with PD-81723 (3 mg/kg ip 15 min beforerenal ischemia) protected WT mice but failed to protect prox-imal tubule-specific S1P1R-null mice from renal I/R injury.

DISCUSSION

The major new findings of our study are that 1) a selectiveallosteric enhancer for A1ARs (PD-81723) dose dependentlyattenuated renal I/R injury in vivo; 2) PD-81723 also attenu-ated HK-2 cell apoptosis in vitro; 3) this in vivo and in vitroprotection afforded by PD-81723 was directly mediated viaactivation of A1ARs; 4) mechanistically, PD-81723 selectivelyinduced SK1 in HK-2 cells and in mouse kidneys; and 5) micedeficient in SK1 were not protected against renal I/R injurywith PD-81723 treatment.

Renal I/R injury creates a cascade of renal tubular necrosis,apoptosis, and inflammation (8). We (25, 32, 34) have previ-ously demonstrated that renal A1AR activation protectedagainst I/R injury in mice and rats. The A1AR-mediated pro-tection against ischemic AKI was characterized by significantreductions in renal necrosis, apoptosis, and inflammation (34).Although A1AR agonist-mediated protection against ischemicAKI has significant therapeutic potential, the nonrenal A1AReffects (e.g., A1AR effects on the heart and brain) may produceundesirable systemic side effects, limiting its clinical applica-tions (5). The present study demonstrates powerful and dose-



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Fig. 6. PD-81723 reduces apoptosis in human renal proximal tubule (HK-2) cells. HK-2 cell apoptosis was induced by TNF-� (20 ng/ml) and cycloheximide(CHX; 10 �g/ml). A: representative poly(ADP-ribose) polymerase (PARP) and caspase-3 fragmentation as indexes of HK-2 cell apoptosis (representative imagesfrom 6 experiments). PD-81723 (10 �M) or vehicle (1% DMSO) was applied 30 min before the induction of apoptosis, and cells were harvested 16 h later. Theselective A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 100 nM) prevented this reduction in HK-2 cell apoptosis. B: densitometric quantificationsof cleaved PARP (n � 6) and caspase-3 (n � 6) in HK-2 cells. Values are means � SE. *P � 0.05 vs. vehicle; #P � 0.05 vs. PD-81723. C: representative gel imagedemonstrating DNA laddering as another index of apoptosis in HK-2 cells. PD-81723 (10 �M) applied 30 min before the induction of apoptosis reduced laddering inHK-2 cells. DPCPX (100 nM) again prevented this reduction in HK-2 cell apoptosis. Photographs are representative of 4 independent experiments.



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dependent protective effects of a selective A1AR allostericenhancer, PD-81723, against renal I/R injury in mice. Specif-ically, we showed that PD-81723 attenuated renal histologicalinjury (Jablonski renal injury scores) and apoptosis (TUNELstaining and PARP/caspase-3 fragmentation). Furthermore, wedemonstrated a significantly reduced influx of proinflammatoryneutrophils after renal I/R in PD-81723-treated mouse kidneys.Therefore, exogenous administration of PD-81723 providespowerful renal protection against ischemic AKI by targeting allthree pathways of cell death in vivo. Few studies have exploredthe tissue protective role of A1AR allosteric enhancers. Mizu-mura et al. (40) also showed that PD-81723 reduced thepreconditioning threshold in the heart. Furthermore, hippocam-pal injury and behavioral recovery were improved by PD-81723 after cerebral ischemia (39). The renal protective effectsof PD-81723 are mediated by A1ARs, as a selective A1ARantagonist (DPCPX) attenuated the induction of SK1 and micedeficient in A1ARs were not protected with PD-81723.

We aimed to test A1AR-based therapeutic strategies toimprove renal function after I/R injury without the potentialsystemic side effects (e.g., bradycardia, hypotension, and se-dation) that may limit A1AR agonist-based therapeutic strate-gies. The selective A1AR allosteric enhancer PD-81723 acts toincrease agonist binding to A1ARs and increases A1AR-medi-ated effects in many cell types (5, 7). PD-81723 increasesradiolabeled adenosine ligand binding and coupling in rat,guinea pig, and human tissues. We postulated that locallyincreased renal adenosine levels after renal ischemia coupledwith an A1AR allosteric enhancer will allow for enhanced renalA1AR activation. Anticipating higher localized renal adenosinelevels after renal I/R, we tested the hypothesis that a selectiveA1AR allosteric enhancer (PD-81723) will produce kidney-specific protection against renal I/R injury without undesirable

systemic side effects in vivo. We demonstrated, in this study,that in vivo renal protection with PD-81723 occurred withoutsignificant effects on heart rate or blood pressure (potentialsystemic effects of A1AR activation in the heart) in mice.

We and others (18, 32, 34, 36, 37, 41, 42) have previouslyshown differential effects of AR subtype activation againstischemic AKI . Selective A1, A2a, or A2bAR activation pro-duces renal protection, whereas A3AR activation in the kidneywas detrimental against ischemic AKI. Therefore, althoughrenal adenosine levels rise rapidly (�3-fold) during ischemia,this increase may not selectively activate cytoprotective ARs toproduce endogenous renal protection. Using an allosteric mod-ulator for A1ARs would use this increase in adenosine forselective activation of cytoprotective renal A1ARs. We alsodetermined that PD-81723 given 30 min after the completionof renal ischemia failed to protect against renal I/R injury. Thisis consistent with our findings showing that renal adenosinelevels fell rapidly after reperfusion to near preischemic levelsafter reperfusion. Therefore, our data confirm that PD-81723-mediated renal protection must be coupled with increasedtissue adenosine in the ischemic organ for protection to occur.

Clinically, pretreatment is an important and valuable optionfor the frequent clinical scenario where patients are subjectedto renal ischemia or hypoperfusion during surgical procedures(e.g., kidney transplantation, open heart surgery, aortovascularsurgery, or liver transplantation). During the perioperativeperiod, renal injury can be frequently anticipated before sur-gery, and a selective A1AR allosteric enhancer can be directlyinfused into the kidney or intravenously. A1AR allostericenhancer-based pretreatment therapies may reduce ischemiaAKI in a wide range of patients subjected to major surgicalprocedures.

Fig. 7. PD-81723 induces sphingosine kinase (SK)1 expression and activity in HK-2 cells. A and B: representative images (top) and relative normalized bandintensities (bottom) for SK mRNA (by RT-PCR; A) and protein (by immunoblot analysis; B) expression in HK-2 cells treated with vehicle (1% DMSO) or 10�M PD-81723 (n � 4 per group). HK-2 cells were treated for 6 h (mRNA analysis) or 16 h (protein analysis). Note that PD-81723 increased SK1 mRNA andprotein expression in HK-2 cells but did not change SK2 expression. C: SK activity in HK-2 cells treated with vehicle or with 1–10 �M PD-81723 6 h (n �4 per group). PD-81723 caused a dose-dependent increase in SK activity in HK-2 cells. We preferentially measured SK1 activity by the addition of Triton X-100in our SK activity assay. A selective A1AR antagonist (100 nM DPPCX) significantly attenuated the PD-81723-induced increase in SK activity. Values aremeans � SE. *P � 0.05 vs. vehicle; #P � 0.05 vs. PD-81723.



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Upregulation of proinflammatory cytokines and adhesionmolecules after renal I/R contributes to the pathophysiology ofkidney injury (12). Therefore, we determined whether PD-81723 attenuates ischemic AKI by modulating renal proinflam-matory mRNA expression. We showed that, as expected, all ofthe proinflammatory mRNAs examined (TNF-�, MCP-1,MIP-2, and ICAM-1) showed enhanced expression in thekidney after I/R. We found that PD-81723 selectively reducedthe expression of TNF-� and MCP-1 without changing

ICAM-1 or MIP-2 expression. TNF-� is a well-known medi-ator of kidney injury after I/R and upregulates early to mediateneutrophil infiltration after renal I/R (15, 16). TNF-� promotesthe migration of inflammatory cells into the kidney by upregu-lating additional cytokines and adhesion molecules (e.g.,MCP-1, ICAM-1, and MIP-2) (15, 16, 48). MCP-1 is also animportant mediator in the pathogenesis of AKI (47, 51). Up-regulation of MCP-1 leads to neutrophil recruitment duringAKI induced by renal I/R or nephrotoxic agents. Indeed, ourstudy showed that renal I/R-induced neutrophil infiltration wassignificantly attenuated by PD-81723 pretreatment, consistentwith targeting of these proinflammatory and neutrophil attract-

Fig. 8. PD-81723 induces renal SK1 in mice. A and B: representative images(top) and relative normalized band intensities (bottom) for kidney SK mRNA(by RT-PCR; A) and protein (by immunoblot analysis; B) expression aftervehicle (1% DMSO) or PD-81723 (3 mg/kg ip) treatment (n � 4 per group).Kidneys were isolated 6 h after treatment. PD-81723 increased SK1 mRNAand protein expression in HK-2 cells but did not change SK2 expression.Values are means � SE. *P � 0.05 vs. vehicle.

Fig. 9. Critical role for SK1 in PD-81723-mediated renal protection againstI/R. Plasma creatinine levels in vehicle (1% DMSO)-treated or PD-81723(3 mg/kg)-treated mice subjected to sham surgery (n � 4 mice/group) or torenal I/R (n � 6 mice/group) are shown. WT mice treated with a potent SKinhibitor (SKI-II; 50 mg/kg sc 15 min before ischemia) were not protectedagainst renal I/R injury with PD-81723 treatment. Moreover, mice deficient inSK1 were not protected against renal I/R injury with PD-87123. In contrast,SK2-deficient mice were protected against renal I/R with PD-81723 treatment.Values are means � SE. *P � 0.05 vs. vehicle-treated mice to subjected torenal I/R.

Fig. 10. Critical role of renal proximal tubule sphingosine-1-phosphate type 1receptors (S1P1Rs) in PD-81723-mediated protection against renal I/R injury.Plasma creatinine from WT (S1P1Rf/f PEPCK�/�) mice or from proximaltubule-specific S1P1R-null (S1P1Rf/f PEPCKCre/�) mice subjected to 30 min ofrenal ischemia and 24 h of reperfusion are shown. Treatment with an A1ARagonist (PD-81723, 3 mg/kg ip 15 min before renal ischemia) protectedS1P1Rf/f PEPCK�/� mice but failed to reduce plasma creatinine levels inS1P1Rf/f PEPCKCre/� mice subjected to renal I/R. *P � 0.05 vs. vehicle-treated S1P1Rf/f PEPCK�/� mice; #P � 0.05 vs. PD-81723-treated S1P1Rf/f

PEPCK�/� mice.



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ing cytokines. The role of neutrophils in the pathophysiologyof organ injury (including in the liver and kidney) is wellrecognized (13). MCP-1 also acts as a chemoattractant formonocytes and lymphocytes to areas of injury (47, 51). Mono-cytes and lymphocytes play critical roles in the pathogenesis ofrenal I/R (3, 12).

Our data showed that PD-81723 produces renal protectionby direct induction of renal tubular SK1 via enhanced activa-tion of A1ARs as 1) PD-81723 induced SK1 in HK-2 cells andin mouse kidneys, 2) the PD-81723-mediated induction inHK-2 cell SK activity was blocked with a selective A1ARantagonist (DPCPX), and 3) mice deficient in SK1 were notprotected against renal I/R injury with PD-81723. PD-81723-mediated induction of SK1 has never been previously de-scribed, and our data may provide a novel mechanistic cyto-protective mechanism triggered by PD-81723 administration.However, our present data cannot rule out the induction of SK1in other nephron segments with PD-81723 treatment.

SK is a multifunctional lipid kinase that phosphorylatessphingosine to form S1P. Of the two forms of SK, SK1 is acytosolic enzyme that migrates to the plasma membrane or tothe nucleus upon activation (19, 31). SK1 is a well-knownmediator of tissue protection (including protection against I/Rinjury), growth, and survival (38). SK1 overexpression protectsagainst acute lung and kidney injury (45, 56). Furthermore,SK1 activation plays a potent cytoprotective role, as SK1-deficient cardiomyocytes had increased injury after ischemia(55). We and others (4, 22, 28, 29) have previously demon-strated a renal protective role of SK1 as well as S1P1 receptoractivation. Overall, activation of SK1 has been shown toproduce antinecrotic, anti-inflammatory, and antiapoptotic ef-fects. Our finding that PD-81723 induced SK1 is consistentwith our recent finding that a selective A1AR agonist (CCPA)induced SK1 in the mouse kidney and in HK-2 cells (43). Itremains to be tested whether PD-81723 induces SK1 in othercell types.

Our experiments also showed that PD-81723 treatment notonly induces SK1 but that its renal protective effects aredependent on SK1, as mice deficient in the SK1 enzyme werenot protected against renal I/R injury with PD-81723 treatment.In addition, renal proximal tubule S1P1Rs are required forPD-81723-mediated renal protection. S1P is a potent lipidsignaling molecule that activates five known S1P receptors toregulate diverse biological effects, including cell growth, sur-vival, and modulation of inflammation (1, 10, 53). S1P1Ractivation, in particular, has been shown to produce cytopro-tective effects, including reducing T lymphocyte-mediated in-flammation, strengthening endothelial vascular permeabilityand producing direct renal tubular protection (2, 4). Indeed,direct S1P1R activation with a selective agonist (4) or en-hanced activation of S1P1Rs secondary to increased SK1activity (28) produces protection against renal I/R injury inmice. Collectively, our data suggest that PD-81723-mediatedSK1 induction enhances the synthesis of endogenous S1P inthe kidney to activate renal tubular S1P1Rs.

Although protective against HK-2 cell apoptosis, acute PD-81723 treatment failed to attenuate HK-2 cell necrosis in vitro.This is in contrast to the in vivo protective effects of acutePD-81723 administration, where we observed reduced necrosisas well as apoptosis against ischemic AKI. Differences inmodels of cell death (H2O2 vs. renal I/R) may explain the

discrepancies between our in vitro and in vivo effects ofPD-81723 against necrosis. However, since 4 h of pretreatmentwith PD-81723 did produce protection against necrosis, a morelikely explanation is that PD-81723-mediated protection againstcell death requires the induction of a downstream cytoprotectivefactor (such as SK1).

One of the limitations of this study is that we used anenzymatic creatinine assay method that overestimates plasmacreatinine values, especially when the values are low (e.g., insham-operated mice). Previous studies by Yuen et al. (57) andTakahashi et al. (52) have shown that enzymatic creatinineassays overestimate plasma creatinine. Indeed, the plasmacreatinine values obtained from our sham-operated mice (�0.4mg/dl) were higher than the values obtained with either tandemmass spectrometry (�0.07 mg/dl) or HPLC (�0.1–0.2 mg/dl)(52, 57). However, mice subjected to renal I/R and treated withvehicle or with an A1AR allosteric enhancer had plasmacreatinine values of �2.3 and �1.2 mg/dl, respectively. Thedegree of overestimation by enzymatic creatinine assay isreduced at plasma creatinine values above 0.5 mg/dl (57).Furthermore, the degree of overestimation would be similarfor both vehicle- and PD-81723-treated mice subjected torenal I/R.

Detailed structure activity relationship studies (6, 9) todevelop a potent A1AR allosteric enhancer have been previ-ously reported. The common structural theme that has emergedfrom these studies is that chemicals with a 2-amino-3-benzo-ylthiophene core are excellent A1AR allosteric enhancers.Furthermore, Bruns et al. (9) demonstrated that an unsubsti-tuted 2-amino group and an adjacent ketone group are impor-tant for the potency of the allosteric enhancers. Subsequentstructure activity relationship studies revealed that modifica-tion of the benzoyl moiety at the 3-position of the thiopentenering with chlorine or trifluromethyl chlorine moiety resulted inenhanced allosteric actions.

In summary, our findings of PD-81723-mediated SK1 in-duction leading to reduced renal injury represent a potentialnew direction in A1AR allosteric enhancer therapy for isch-emia AKI. Our experiments may lead to new therapeuticapproaches with a drug that can reduce all three pathways ofrenal cell death (necrosis, apoptosis, and inflammation) tolessen the clinical perils from AKI and have implications inorgan protection strategies beyond the kidney. These therapeu-tic approaches may be able to protect the kidney againstimpending but anticipated renal I/R injury during the periop-erative period.

ACKNOWLEDGMENTS

The authors thank R. L. Proia (National Institute of Diabetes and Digestiveand Kidney Diseases) for providing the SK1�/� and SK2�/� mice.

GRANTS

This work was supported by National Institutes of Health Grants R01-DK-058547 and GM-06708.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: S.W.P. and H.T.L. conception and design of research;S.W.P., J.Y.K., A.H., K.M.B., M.K., and H.T.L. performed experiments; S.W.P.,J.Y.K., A.H., K.M.B., M.K., V.D.D., and H.T.L. analyzed data; S.W.P., J.Y.K.,



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A.H., K.M.B., M.K., V.D.D., and H.T.L. interpreted results of experiments;S.W.P., J.Y.K., A.H., K.M.B., M.K., and H.T.L. prepared figures; S.W.P. andH.T.L. edited and revised manuscript; S.W.P., J.Y.K., A.H., K.M.B., M.K.,V.D.D., and H.T.L. approved final version of manuscript; H.T.L. draftedmanuscript.

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A1 adenosine receptor allosteric enhancer PD-81723 ... · A 1 adenosine receptor allosteric enhancer PD-81723 protects against renal ischemia-reperfusion injury Sang Won Park,1 Joo