Am J Physiol Renal Physiol 284: F267–F273, 2003. First published October 15, 2002; 10.1152/ajprenal.00271.2002. A3 adenosine receptor knockout mice are protected against ischemia- and myoglobinuria-induced renal failure H. THOMAS LEE,1 AYUKO OTA-SETLIK,1 HUA XU,1 VIVETTE D. D’AGATI,2 MARLENE A. JACOBSON,3 AND CHARLES W. EMALA1 1 Department of Anesthesiology and 2Department of Pathology, College of Physicians and Surgeons of Columbia University, New York, New York 10032; and 3Department of Pharmacology, Merck Research Laboratories, West Point, Pennsylvania 19486 Submitted 24 July 2002; accepted in final form 8 October 2002 Lee, H. Thomas, Ayuko Ota-Setlik, Hua Xu, Vivette D. D’agati, Marlene A. Jacobson, and Charles W. Emala. A3 adenosine receptor knockout mice are protected against ischemia- and myoglobinuria-induced renal failure. Am J Physiol Renal Physiol 284: F267–F273, 2003. First published October 15, 2002; 10.1152/ajprenal.00271.2002.—A3 adenosine receptor (AR) activation and inhibition worsen and improve, respectively, renal function after ischemiareperfusion (I/R) injury in rats. We sought to further characterize the role of A3 ARs in modulating renal function after either I/R or myoglobinuric renal injury. A3 knockout mice had significantly lower plasma creatinines compared with C57 controls 24 h after I/R or myoglobinuric renal injury. C57 control mice pretreated with the A3 AR antagonist [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6phenyl-1,4-(⫾)-dihydropyridine-3,5 dicarboxylate] or agonist [0.125 mg/kg N6-(3-iodobenzyl)-N-methyl-5⬘-carbamoyladenosine (IB-MECA)] demonstrated improved or worsened renal function, respectively, after I/R or myoglobinuric renal injury. Higher doses of IB-MECA were lethal in C57 mice subjected to renal ischemia. H1 but not H2 histamine receptor antagonist prevented death in mice pretreated with IB-MECA before renal ischemia. Improvement in renal function was associated with significantly improved renal histology. In conclusion, preischemic A3 AR activation (0.125 mg/kg IB-MECA) exacerbated renal I/R injury in mice. Mice lacking A3 ARs or blocking A3 ARs in wild-type mice resulted in significant renal protection from ischemic or myoglobinuric renal failure. after I/R injury in rats (14, 16, 17). All four known AR subtypes (A1, A2a, A2b, and A3) are expressed in the kidney (18, 28), but the function of A3 AR is unknown. We recently showed that preischemic activation of A3 ARs exacerbates renal failure after I/R injury (14). Conversely, preischemic administration of an A3 AR antagonist protected the rat kidney against I/R injury. In this study, we aimed to extend our previous findings of modulation of A3 ARs on renal function after I/R injury utilizing mice deletionally lacking A3 ARs [A3 AR knockout (A3KO)] (23, 26). We hypothesized that A3KO mice would be endogenously protected against I/R injury. We also hypothesized that preischemic activation or inhibition of A3 ARs would worsen and protect, respectively, renal failure after I/R- or myoglobinuria-induced renal failure in mice. A3 AR activation degranulates mast cells and increases plasma histamine in rodents (4, 6, 22, 26). Therefore, we investigated the mechanism of A3 AR modulation of renal failure by using selective blockers of histamine receptors and an effector (compound 48/80) that increases endogenous histamine release. METHODS (ARF) is a major contributor of perioperative mortality and morbidity (7, 20). Ischemic-reperfusion (I/R) injury, toxic nephropathy, and myoglobinuria all can lead to ARF, which is frequently complicated by many other life-threatening complications including sepsis and multiorgan failure (1, 20, 21). The prognosis for patients with ARF is poor (with mortality ⬃50%) and has changed little over the past 40 years (1, 7). We demonstrated previously that adenosine receptor (AR) modulations significantly affect renal function A3KO mice. Homozygous A3KO (A3⫺/⫺) mice breeding pairs were provided by Merck Research Laboratories (West Point, PA). Generation and initial characterization of the A3KO mice have been described in detail previously (23, 26). The A3KO mice were obtained after they were backcrossed against C57BL/6 mice for 12 generations to obtain a congenic line. C57BL/6 mice (Harlan Laboratories, Indianapolis, IN) served as background A3⫹/⫹ controls (C57). Renal injury protocol. A3KO and C57 mice (25–30 g body wt) were anesthetized with pentobarbital sodium (50 mg/kg or to effect ip) and placed supine on a heating pad under a warming light to maintain body temperature between 36 and 38°C. Additional pentobarbital sodium was given as needed on the basis of response to tail pinch. Bilateral flank incisions were made, and the left kidney was subjected to 30 min of ischemia with microaneurysm clips after right nephrectomy. The duration of ischemia was chosen to maximize reproducibility of renal injury and to minimize mortality in these Address for reprint requests and other correspondence: H. T. Lee, Dept. of Anesthesiology, Anesthesiology Research Laboratories, Columbia Univ., P&S Box 46 (PH-5), 630 West 168th St., New York, NY 10032-3784 (E-mail: tl128@columbia.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. acute renal failure; histamine; ischemic-reperfusion injury ACUTE RENAL FAILURE http://www.ajprenal.org 0363-6127/03 $5.00 Copyright © 2003 the American Physiological Society Downloaded from journals.physiology.org/journal/ajprenal (003.090.035.165) on November 29, 2021. F267 F268 RENAL PROTECTION WITH A3 ADENOSINE RECEPTOR DELETION mice. Separate groups of C57 and A3KO mice were injected with 8.5 mg/kg glycerol intramuscularly after 16 h of water deprivation to induce myoglobinuric renal injury. Some C57 mice were pretreated either with 3-ethyl-5-benzyl-2-methyl4-phenylethynyl-6-phenyl-1,4-(⫾)-dihydropyridine-3,5 dicarboxylate (MRS-1191; 1 mg/kg ip), a selective A3 AR antagonist, or with 0.125 mg/kg N6-(3-iodobenzyl)-N-methyl-5⬘carbamoyladenosine (IB-MECA; 0.0625, 0.125, 0.25, 0.5, 1, and 2 mg/kg ip), a selective A3 AR agonist, 15 min before renal ischemia or glycerol injection. To determine whether histamine release by mast cell degranulation induced by IB-MECA plays any role in lethality and exacerbation of renal injury by A3 AR activation, some C57 mice were pretreated with 10 mg/kg diphenylhydramine or 10 mg/kg ranitidine ip 30 min before 1 mg/kg IB-MECA. Some A3KO and C57 mice were pretreated with 5 mg/kg of compound 48/80 (to degranulate mast cells) 15 min before being subjected to renal I/R injury. Assessment of renal injury. Renal function was assessed by measuring plasma creatinine 24 h after renal ischemia or glycerol injection by using a commercially available colorimetric method (Sigma). For histological preparations, explanted kidneys were bisected along the long axis and were fixed in 10% formalin solution overnight. After automated dehydration through a graded alcohol series, transverse kidney slices were embedded in paraffin, sectioned at 5 ␮m, and stained with hematoxylin-eosin. Morphological assessment was performed by an experienced renal pathologist (V. D. D’Agati) who was blinded to the treatment for each animal. A grading scale of 0–4, as outlined by Jablonski et al. (10), was used for the histopathological assessment of I/R- or myoblobinuria-induced damage of the proximal tubules. Measurement of plasma histamine levels. Plasma histamine levels were measured in A3KO and C57 mice 20 min after IB-MECA or compound 48/80 treatment (5 min after initiation of renal ischemia) by using an ELISA specific for mice (RDI, Flanders, NJ) according to the manufacturer’s instructions. PCR for A3 ARs. We analyzed the presence of the wildtype or mutant A3 AR DNA in C57 and A3KO mice by PCR. To differentially confirm the genotype of A3KO and C57 mice, primers were designed to recognize the 5⬘- and 3⬘end of the full-length wild-type A3 AR (sense: 5⬘-GA- CTGGCTGAACATCACCTACAT-3⬘ and antisense: 5⬘-ATAGAAGTGCATCTTGACTTGCAG-3⬘) and the PGKneo insert (sense: 5⬘-CTATGACTGGGCACAACAGACAAT-3⬘ and antisense: 5⬘-ATCAGCCATGATGGATACTTTCTC-3⬘) in A3KOs (23). All primers were purchased from Sigma GenoSys (The Woodlands, TX). Genomic DNA was isolated from mouse tails by using the Wizard Genomic DNA extraction kit (Promega, Madison, WI). DNA concentrations were determined by using spectrophotometric absorbance at 260/280. PCR was performed with Taq polymerase (Promega). Genomic DNA (0.2 ␮g) was used as a template and amplified for 30 cycles with a PTC-200 thermal cycler (MJ Research, Waltham, MA) using a primer annealing temperature of 60°C. The products were resolved in a 6% polyacrylmide gel and stained with Syber green (Roche, Indianapolis, IN) for analysis with a FluorS Multi Imager (Bio-Rad, Hercules, CA). Chemicals. Unless otherwise specified, all reagents were purchased from Sigma (St. Louis, MO). Statistics and data analysis. The data were analyzed with Student’s t-test when comparing means between two groups or with one-way analysis of variance plus Dunnett post hoc multiple comparison test to compare mean values across multiple treatment groups. RESULTS Genotyping of wild-type and A3KO C57 mice. To confirm the genotypes of mice used in these studies, genomic DNA extracted from the tail was analyzed by PCR for the expression of the full-length A3 AR (wildtype or C57 mice) or the plasmid insert (PGKneo) originally used to disrupt the A3 AR gene (A3KO mice) (23). The full-length A3 AR (⬃220 bp) was only detected in C57 wild-type mice, whereas the PGKneo insert (⬃290 bp) was only detected in the A3KO mice (Fig. 1). Renal function assessment. C57 control and A3KO mice that underwent sham operations had similar baseline hemodynamic values (heart rate and blood pressure) and renal function [creatinine (Cr) ⫽ 0.3 ⫾ 0.1 mg/dl, n ⫽ 3, for C57, and 0.3 ⫾ 0.1 mg/dl, n ⫽ 3, for A3KO]. However, 24 h after renal I/R injury, A3KO Fig. 1. Representative PCR products using specific primers of mouse A3 adenosine receptor (AR; C57 mice) and PKGneo [A3 AR knockout (A3KO) mice]. Total DNA (0.2 ␮g, extracted from tail) was used in each PCR reaction. DNA encoding A3 AR was only detected in C57 control mice (n ⫽ 2), whereas the PKGneo insert was only detected in A3KO mice (n ⫽ 4). AJP-Renal Physiol • VOL 284 • FEBRUARY 2003 • www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal (003.090.035.165) on November 29, 2021. RENAL PROTECTION WITH A3 ADENOSINE RECEPTOR DELETION mice had significantly lower plasma creatinines (Cr ⫽ 0.7 ⫾ 0.1 mg/dl, n ⫽ 8) compared with C57 controls (Cr ⫽ 3.0 ⫾ 0.3 mg/dl, n⫽ 8, P ⬍ 0.05, Fig. 2). A3KO mice were also protected against myoglobinuric renal injury compared with control mice as demonstrated by reduced creatinines at 24 h (Cr ⫽ 1.8 ⫾ 0.3 mg/dl, n ⫽ 8 for A3KO, vs. 4.1 ⫾ 0.2 mg/dl, n ⫽ 4, for C57, P ⬍ 0.05, Fig. 2). To further illustrate the role of the A3 AR in modulating renal function after injury, control C57 mice were pretreated with an A3 AR antagonist or agonist before I/R or glycerol-induced myoglobinuric injury. C57 mice pretreated with MRS-1191 (a selective A3 AR antagonist) demonstrated improved renal function after either I/R-induced or glycerol-induced renal injury (Cr ⫽ 1.8 ⫾ 0.2 mg/dl, n ⫽ 9, for MRS-1191⫹I/R, or 3.1 ⫾ 0.2 mg/dl, n ⫽ 6, for MRS-1191⫹glycerol, respectively, P ⬍ 0.05; Fig. 2) compared with I/R or glycerol injury alone. In contrast, C57 mice pretreated with IB-MECA, a selective A3 AR agonist, failed to survive the ischemic period at doses of 2 (n ⫽ 6), 1 (n ⫽ 6), 0.5 (n ⫽ 6), or 0.25 mg/kg (n ⫽ 6). At 0.125 mg/kg IBMECA, C57 mice survived renal I/R injury and demonstrated significantly worsened renal function (Cr ⫽ 4.5 ⫾ 0.2 mg/dl, n ⫽ 6, P ⬍ 0.05, compared with I/R injury alone; Fig. 2). C57 mice pretreated with 0.0625 mg/kg IB-MECA before renal I/R did not exhibit significantly worsened renal function (Cr ⫽ 3.4 ⫾ 0.2 mg/dl, n ⫽ 4). Selective A3 AR agonist (Cr ⫽ 0.6 ⫾ 0.1 mg/dl, n ⫽ 5, after 0.125 mg/kg IB-MECA) or antagonist (Cr ⫽ 0.6 ⫾ 0.1 mg/dl, n ⫽ 4, after 1 mg/kg MRS-1191) has no impact on renal protection observed in A3KO mice subjected to I/R injury. IB-MECA (0.5 mg/kg) also exacerbated renal function in C57 mice after myoglobinuric renal injury (Cr ⫽ 4.8 ⫾ 0.1 mg/dl, n ⫽ 4, P ⬍ 0.05, compared with myoglobinuric injury alone). C57 mice injected with F269 IB-MECA (2 mg/kg) alone without I/R or myoglobinuric injury survived and showed normal renal function. When C57 (n ⫽ 4) and A3KO (n ⫽ 4) mice were pretreated with compound 48/80 (5 mg/kg) and subjected to renal I/R injury, all the mice died during renal ischemia. Increase in plasma histamine by IB-MECA or compound 48/80 is lethal when associated with renal ischemia. A3 AR activation causes mast cell degranulation and histamine release in rodents (6, 22, 26). We questioned whether the systemic elevations in histamine played a role in the lethality and impaired renal function of A3 AR activation coupled with renal ischemia and reperfusion. We measured plasma histamine levels in A3KO and C57 mice after IB-MECA or compound 48/80 (induces mast cell degranulation) pretreatment by using a mouse-specific ELISA. A3KO and control C57 mice that underwent sham operations had similar baseline plasma histamine levels (4.6 ⫾ 1.0 and 5.0 ⫾ 1.6 ng/ml, respectively, n ⫽ 3). IB-MECA (1 mg/kg) increased plasma histamine concentration to 226 ⫾ 24.8 ng/ml in C57 mice (n ⫽ 6) and to 31.0 ⫾ 4.3 ng/ml in A3KO mice (n ⫽ 4, P ⬍ 0.01 vs. C57) 20 min after injection (5 min after initiation of renal ischemia). Plasma histamine concentrations increased in C57 and A3KO mice pretreated with compound 48/80 (320 ⫾ 8.8 ng/ml, n ⫽ 4, and 249 ⫾ 29.3 ng/ml, n ⫽ 5, respectively). Because all control C57 mice pretreated with compound 48/80 or 0.25–2.0 mg/kg IB-MECA and all A3KO mice pretreated with compound 48/80 died during ischemia, we questioned whether inhibition of histamine receptor subtypes would alter lethality or renal function. Thirty minutes of pretreatment with H1 (diphenylhydramine, 10 mg/kg ip) but not with H2 (ranitidine, 10 mg/kg ip) histamine receptor antagonist prevented death in C57 mice treated with IB-MECA Fig. 2. Comparison of mean creatinine measured from sham-operated C57 mice (C57 sham; n ⫽ 3), sham-operated A3KO mice (A3KO sham; n ⫽ 3), C57 mice subjected to ischemia and reperfusion (C57 IR; n ⫽ 8), A3KO mice subjected to ischemia and reperfusion (A3KO IR; n ⫽ 8), C57 mice pretreated with IB-MECA before ischemia and reperfusion (IBMECA IR; n ⫽ 6), C57 mice pretreated with MRS1191 before ischemia and reperfusion (MRS IR; n ⫽ 9), C57 mice subjected to glycerol injury (C57 Glyc; n ⫽ 4), A3KO mice subjected to glycerol injury (A3KO Glyc; n ⫽ 8), C57 mice pretreated with IBMECA before glycerol injury (IB-MECA Glyc; n ⫽ 4), and C57 mice pretreated with MRS-1191 before glycerol injury (MRS Glyc; n ⫽ 6). *P ⬍ 0.05 vs. sham-operated C57 mice. #P ⬍ 0.05 vs. C57 mice subjected to ischemia and reperfusion. %P ⬍ 0.05 vs. C57 mice subjected to glycerol injury. Error bars, 1 SE. AJP-Renal Physiol • VOL 284 • FEBRUARY 2003 • www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal (003.090.035.165) on November 29, 2021. F270 RENAL PROTECTION WITH A3 ADENOSINE RECEPTOR DELETION and in C57 or A3KO mice treated with compound 48/80. However, pretreatment with diphenylhydramine did not protect renal function after I/R injury in C57 mice treated with IB-MECA (Cr ⫽ 4.0 ⫾ 0.2 mg/dl, n ⫽ 4). Similarly, when C57 mice were pretreated with diphenyhydramine before compound 48/80 and subjected to renal I/R, they survived renal ischemia but failed to show improved renal function compared with the I/R injury alone group (Cr ⫽ 2.9 ⫾ 0.2 mg/dl, n ⫽ 3). Therefore, although elevated plasma histamine and activation of the H1 histamine receptor coupled with renal ischemia are lethal in mice, H1 receptor activation alone cannot account for A3 AR activation-mediated exacerbation of renal function. Injured A3KO mice maintained improved renal histology. In Fig. 3, enhanced renal protection of A3KO mice against I/R- and glycerol-mediated injury are further supported by representative histological slides. C57 control mice subjected to 30 min of renal ischemia (Fig. 3B) or glycerol injection (Fig. 3C) followed by 24 h of reperfusion resulted in significant renal injury, as evidenced by severe tubular necrosis, medullary congestion and hemorrhage, and the development of proteinaceous casts. A3KO mice (Fig. 3, D and E) or C57 mice (Fig. 3, F and G) pretreated with the A3 AR antagonist MRS-1191 before I/R- or glycerol-induced injury showed improved renal morphology. The Jablonski (10) scale histology grading scores are shown in Fig. 4. Twenty-four hours after 30 min of renal ischemia or glycerol treatment in C57 control mice resulted in severe acute tubular necrosis (grade: 3.0 ⫾ 0.3, n ⫽ 6, and 3.7 ⫾ 0.4, n ⫽ 4, respectively). A3KO mice subjected to I/R or glycerol injury showed significant improvements in histological scoring compared with C57 mice subjected to I/R (grade: 0.7 ⫾ 0.5, n ⫽ 4, and 1.7 ⫾ 0.3, n ⫽ 4, respectively). Additionally, C57 mice pretreated with MRS-1191 before I/R or glycerol injury showed significant improvements in histological scoring compared with C57 mice subjected to I/R injury (grade: 2.1 ⫾ 0.2, n ⫽ 5, and 2.3 ⫾ 0.3, n ⫽ 4, respectively). IB-MECA-pretreated C57 mice subjected to I/R or glycerol injury failed to show statistically significant worsening histological injury compared with C57 mice (grade: 3.7 ⫾ 0.4, n ⫽ 4, and 3.8 ⫾ 0.4, n ⫽ 3, respectively). DISCUSSION The major findings of our study are the following: 1) mice deletionally lacking A3 ARs are protected against both ischemic and myoglobinuric forms of renal injury, 2) blocking A3 ARs protected and activating A3 ARs worsened renal failure after I/R- and myoglobinuriainduced injury in C57 control mice, 3) high doses of the A3 AR agonist (IB-MECA) coupled with renal ischemia are lethal in C57 mice presumably by degranulating resident mast cells and releasing histamine, and 4) blocking H1 histamine receptors prevented A3 AR agonist- or mast cell degranulation-induced mortality during renal ischemia in C57 control or A3KO mice. In previous studies by our laboratory, we showed that preischemic A1 or postischemic A2a AR activation protects against renal I/R injury in rats (14, 16, 17). In addition, we demonstrated in rats that A3 AR activation with IB-MECA and inhibition with MRS-1191 worsened and protected, respectively, against I/R-induced renal failure (14). In the present study, we utilized mice that lack A3 ARs to further probe the role of A3 ARs in renal I/R and myoglobinuric injury. The A3 AR subtype is the most recently characterized member of the AR family (5, 11). Although the expression of the A3 AR subtype in the kidney has been demonstrated (18, 28), its function in the kidney is unknown. The present studies in mice agree with our previous studies in rats in that pretreatment with the highly selective A3 AR agonist IB-MECA worsened renal I/R injury, whereas pretreatment with the highly selective A3 adenosine antagonist MRS-1191 protected renal function after I/R injury (14). Although MRS1191 is a potent and selective antagonist of human A3 receptors, it has only limited potency and selectivity at the mouse A3 AR receptor (Ki ⬃ 1.42 ␮M) (12, 13). For this reason, we cannot rule out the possibility that MRS-1191 might act in part by blocking other AR subtypes. For this reason, the data derived from the use of A3KO mice are important to bolster the conclusion on the basis of the use of MRS-1191. IB-MECA also exacerbated glycerol-induced myoglobinuric renal failure. Furthermore, mice lacking the A3 AR exhibited endogenous protection against both I/Rinduced and myoglobinuric-induced renal injury. Because the A3 AR agonist alone had no effect on renal function in both rats (14) and mice, it can be concluded that A3 AR activation must be coupled with an I/R or myoglobinuric renal insult for the receptor to have a detrimental effect on renal function. The detrimental effects of preischemic A3 AR agonist treatment on the kidney differs from the heart, where significant cardiac protection with A3 AR agonist pretreatment has been reported (19). The mechanism(s) by which A3 AR activation or inhibition exacerbates or protects against, respectively, renal injury remains to be determined. A3 adenosine agonists cause apoptosis in multiple cell lines, including cardiomyocytes, human leukemia cell lines, and human proximal tubule (HK-2) cells, via incompletely characterized mechanisms (11, 15, 24). Chronic A3 AR activation or overexpression is detrimental to cell survival (11). Moreover, overexpression of A3 AR is Fig. 3. Representative light microscopic photographs of outer medulla of the kidney. A: sham-operated C57 mice. B: C57 mice subjected to ischemia and reperfusion. C: C57 mice subjected to glycerol injury. D: A3KO mice subjected to ischemia and reperfusion. E: A3KO mice subjected to glycerol injury. F: C57 mice pretreated with MRS-1191 before ischemia and reperfusion. G: C57 mice pretreated with MRS-1191 before glycerol injury. Magnification, ⫻200. AJP-Renal Physiol • VOL 284 • FEBRUARY 2003 • www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal (003.090.035.165) on November 29, 2021. RENAL PROTECTION WITH A3 ADENOSINE RECEPTOR DELETION AJP-Renal Physiol • VOL 284 • FEBRUARY 2003 • www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal (003.090.035.165) on November 29, 2021. F271 F272 RENAL PROTECTION WITH A3 ADENOSINE RECEPTOR DELETION Fig. 4. Jablonski grading scale scores for histological appearance of acute tubular necrosis from sham-operated C57 mice (n ⫽ 3), shamoperated A3KO mice (n ⫽ 3), C57 mice subjected to ischemia and reperfusion (n ⫽ 6), A3KO mice subjected to ischemia and reperfusion (n ⫽ 4), C57 mice pretreated with IB-MECA before ischemia and reperfusion (n ⫽ 4), C57 mice pretreated with MRS-1191 before ischemia and reperfusion (n ⫽ 5), C57 mice subjected to glycerol injury (n ⫽ 4), A3KO mice subjected to glycerol injury (n ⫽ 4), C57 mice pretreated with IB-MECA before glycerol injury (n ⫽ 3), and C57 mice pretreated with MRS-1191 before glycerol injury (n ⫽ 4). *P ⬍ 0.05 vs. sham-operated C57 mice. #P ⬍ 0.05 vs. C57 mice subjected to ischemia and reperfusion. %P ⬍ 0.05 vs. C57 mice subjected to glycerol injury. Error bars, 1 SE. embryologically lethal in mice with prominent fragmentation of DNA (27). Additionally, mice deletionally lacking A3 ARs are protected against cardiac I/R injury by unclear mechanisms (3, 8, 9). The A3 AR activation degranulates resident mast cells, which results in the release of stored inflammatory mediators, including histamine and proteolytic enzymes, as well as other proinflammatory mediators (e.g., IL-␤ and TNF-␣) (6, 22). We examined the role of mast cell degranulation and histamine release after A3 AR activation utilizing histamine receptor blockers and by measuring plasma histamine concentrations. We demonstrate that the A3 AR agonist IB-MECA profoundly increases plasma histamine levels in C57 mice (⬃45-fold increase). In contrast, compound 48/80 increased histamine levels in both C57 and A3KO mice. It is interesting to note that IB-MECA increased plasma histamine levels even in A3KO mice by approximately sevenfold, although this increase is significantly less than that observed in C57 mice. It may be that IB-MECA has some additional mechanism of action to increase plasma histamine levels independently of A3 AR activation. We show that 0.125 mg/kg IB-MECA, a selective A3 AR agonist, worsened renal function after I/R renal injury. Moreover 0.5 mg/kg IB-MECA exacerbated renal injury after glycerol-mediated myoglobinuric renal injury. Surprisingly, C57 mice were very sensitive to A3 AR agonist (IB-MECA) injection when subjected to renal ischemia, because doses ⱖ0.25 mg/kg were lethal. Moreover, degranulation of mast cells with compound 48/80 coupled with renal ischemia was also lethal in both C57 and A3KO mice. IB-MECA and compound 48/80 injections without renal ischemia were well tolerated in both A3KO and C57 mice. The AJP-Renal Physiol • VOL mechanism of mortality after higher doses of IB-MECA (0.25–2 mg/kg) coupled with renal ischemia is likely related to H1 histamine receptor activation. Systemic effects of histamine release on the cardiac and vascular systems likely account for mortality after high doses of IB-MECA. Death associated with elevated systemic histamine complicates the interpretation of A3 ARmediated exacerbation of renal function after renal ischemia and is the major limitation of the present, in vivo approach. Pretreatment with an H1 histamine receptor blocker (diphenylhydramine) prevented death during renal ischemia in C57 mice treated with 1 mg/kg IB-MECA, whereas pretreatment with an H2 histamine receptor blocker (ranitidine) failed to rescue C57 mice from death. Although C57 mice survived renal ischemia after H1 histamine receptor antagonist and IB-MECA, their renal function was not improved. This indicates that an increase in plasma histamine concurrent with renal ischemia is lethal in mice because of H1 histamine receptor activation. However, H1 histamine receptor activation cannot account for exacerbation of renal function after ischemia- or glycerol-induced renal injury, because pretreatment with H1 histamine blocker failed to improve plasma creatinine or histology. It remains to be determined whether other histamine receptor subtype blockades (H2, H3, or H4) with or without concomitant H1 histamine receptor blockade are required to protect against renal I/R injury. We recognize that the background strain of mice is an important determinant of renal injury (2). In this study, we utilized commercially available C57BL/6 mice as controls because the homozygous A3KO mice were backcrossed against C57BL/6 mice for 12 generations to obtain a congenic line. Backcrossing to this 284 • FEBRUARY 2003 • www.ajprenal.org Downloaded from journals.physiology.org/journal/ajprenal (003.090.035.165) on November 29, 2021. RENAL PROTECTION WITH A3 ADENOSINE RECEPTOR DELETION extent makes a congenic line that is 99.99% genetically C57BL/6. In conclusion, we demonstrate that mice lacking A3 AR expression are protected against both I/R-induced and myoglobinuria-induced renal failure. Moreover, A3 AR agonist and antagonist worsened and protected, respectively, I/R- and myoglobinuric-mediated renal injury in C57 control mice. The mechanism(s) of A3 AR-mediated exacerbation of renal function after I/R or myoglobinuric injury and the signaling pathway(s) responsible for potent renal protection in A3KO mice remain to be determined. These findings further support the potential role of a selective A3 AR antagonist for protection against perioperative renal failure. 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