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Systemic adenosine given after ischemia protects renal function via A2a adenosine receptor activation
Systemic Adenosine Given After Ischemia Protects Renal Function Via A2a Adenosine Receptor Activation H. Thomas Lee, MD, PhD, and Charles W. Emala, MD ● Ischemia and reperfusion during renal transplant and aortic surgery result in renal ischemic-reperfusion injury. Previously, we showed that preischemic adenosine treatment protects renal function via A1 adenosine receptor (AR) activation. In contrast, in the cardiac and pulmonary systems, postischemic adenosine has potent antiinflammatory attributes and is protective against reperfusion injury via activation of A2a ARs. We questioned whether adenosine given after an ischemic insult protects renal function in rats, and we sought to determine the AR subtype and intracellular second messengers involved. Rats were randomized to a sham operation, 45 minutes of renal ischemia and reperfusion and treatments with systemic adenosine or selective AR agonists and antagonists, or treatments of dibutyryl cyclic adenosine monophosphate (cAMP) after 45 minutes of renal ischemia but before reperfusion. Forty-five minutes of renal ischemia followed by 24 hours of reperfusion led to severe renal dysfunction as indicated by marked rises in creatinine and histologically evident renal tubular damage. Adenosine treatment after ischemia protected renal function and improved tubular histology. This protection was mediated via A2a AR activation because the A2a-selective AR agonist [4-((N-ethyl-5ⴕ-carbamoyadenos-2-yl)-aminoethyl)phenylpropionic acid (CGS-21680)] mimics adenosine-induced renal protection, and the A2a-selective AR antagonist [8-(3-chlorostyryl)caffeine (CSC)] blocks adenosine-induced renal protection. A1 or A3 AR agonists and antagonists did not mimic and block adenosine-induced renal protection. The signaling intermediates of A2a AR–mediated renal protection appear to include cAMP because dibutyryl cAMP mimicked adenosine and CGS21680 mediated renal protection. Rat kidneys can be protected against reperfusion injury via postischemic A2a AR activation or cAMP. These data suggest that A2a adenosine agonists may have clinically beneficial implications when renal ischemia is unavoidable. © 2001 by the National Kidney Foundation, Inc. INDEX WORDS: Acute renal failure; dibutyryl cyclic adenosine monophosphate (cAMP); ischemic-reperfusion injury; kidney.
A
LTHOUGH ISCHEMIA itself can cause injury, significant tissue damage occurs during the reperfusion period.1,2 Renal reperfusion injury resulting in acute renal failure after unavoidable surgical ischemia and reperfusion is a serious clinical concern.3,4 The onset of acute renal failure implies a poor prognosis and frequently is complicated by many other lifethreatening complications, including sepsis and multiorgan failure.4 In high-risk patients undergoing high-risk surgery, the mortality and morbid-
From the Department of Anesthesiology, Columbia University College of Physicians and Surgeons, New York, NY. Received October 16, 2000; accepted in revised form March 30, 2001. Funded in part by intramural grant support from the Department of Anesthesiology, Columbia University College of Physicians and Surgeons. Presented in abstract form at the 1999 American Society of Anesthesiology meeting, Dallas, TX. Address reprint requests to H. Thomas Lee, MD, PhD, Department of Anesthesiology, Columbia Presbyterian Medical Center, P&S Box 46 (PH-5), 630 West 168th Street, New York, NY 10032-3784. E-mail: [email protected] © 2001 by the National Kidney Foundation, Inc. 0272-6386/01/3803-0021$35.00/0 doi:10.1053/ajkd.2001.26888 610
ity rates from perioperative acute renal failure have changed little since the 1970s.3-5 Adenosine has many effects on the kidney, including effects on global renal function (eg, cortical and medullary blood flow) and on endogenous function of specific renal cell types (eg, renin release, electrolyte transport). In the kidney, all four subtypes (A1, A2a, A2b, and A3) of adenosine receptors (ARs) have been identified.6,7 The A1 ARs reduce cortical renal blood flow and renin release and serve as antidiuretic and antinatriuretic receptors. Conversely, A2a ARs increase medullary renal blood flow and renin release and serve as diuretic and natriuretic receptors. The exact roles of the A2b and A3 ARs in the kidney are unclear. In cardiac ischemic-reperfusion (IR) injury, it has been postulated and described that adenosine antagonizes IR injury via two distinct AR subtype modulations. Adenosine given before an ischemic insult protects via A1 AR activation,8-10 whereas adenosine protection is via A2a ARs when given during the postischemic reperfusion period.11-15 In the kidney, we have shown that brief preischemic adenosine treatment (ie, adenosine given for 10 minutes before ischemia and reperfusion) protects rat renal function against IR injury via an A1 AR-Gi-
American Journal of Kidney Diseases, Vol 38, No 3 (September), 2001: pp 610-618
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protein kinase C cascade.16,17 Conversely, neither A2a nor A3 AR activation mimicked the protection of preischemic adenosine. The A2a ARs couple to adenylyl cyclase, which leads to increased intracellular cyclic adenosine monophosphate (cAMP) levels, which in turn activate protein kinase A (PKA). Postischemic adenosine-mediated organ protection against reperfusion injury may involve cAMP and PKA as intracellular signaling intermediates. Exogenous stimulation of the cAMP 3 PKA system with isoproterenol and cAMP analogues has been shown to attenuate renal and pulmonary reperfusion injury in vivo18-20 and in vitro.21 The major aims of this study were to determine whether rat kidneys can be protected against reperfusion injury with postischemic adenosine treatment and to determine the subtype of ARs involved in renal protection against reperfusion injury. Because A2a AR activation classically increases intracellular cAMP, we also wanted to determine whether postischemic administration of a cAMP analogue (dibutyryl cAMP) would protect renal function against reperfusion injury. We hypothesized that postischemic A2a AR activation protects against renal reperfusion injury via intracellular signaling cascades involving cAMP. MATERIALS AND METHODS All protocols were approved by the Institutional Animal Care and Use Committee of Columbia University. Adult male Wistar rats (225 to 275 g, Harlan Sprague-Dawley, Indianapolis, IN) were used. The rats had free access to rodent chow and water. The rats were anesthetized with intraperitoneal pentobarbital (45 mg/kg body weight or to effect). Additional pentobarbital was administered as needed based on response to tail pinch. After 500 U of heparin was given intraperitoneally, rats were placed on an electric heating pad under a warming light. Body temperature was monitored with a rectal probe and maintained at 37°C. The rats were allowed to breathe room air spontaneously. The right femoral vein was cannulated with heparinized (10 U/mL) polyethylene tubing (PE-50) for intravenous drug access and hydration. The right femoral artery was cannulated with heparinized PE-50 tubing for hemodynamic monitoring and blood sampling. After a 10-minute stabilization period, a midline laparotomy was performed. Right nephrectomy was performed, and the left renal artery and vein were isolated. Eleven separate protocols were performed as described subsequently.
Protocols To determine the role of postischemic adenosine treatment in renal IR injury, rats were subjected to the following protocols after right nephrectomy:
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1. Control group (sham): Rats were subjected to isolation of left renal artery and vein only. 2. IR group: Rats were subjected to 45 minutes of left renal ischemia. 3. Adenosine treatment group: Rats received systemic intravenous infusion of adenosine (1.75 mg/kg/ min ⫻ 10 minutes) 2 minutes after the termination of 45 minutes of left renal ischemia. To determine the receptor subtypes involved in postischemic adenosine–induced renal protection, rats were subjected to the following protocols after right nephrectomy: 4. A1 AR antagonist before adenosine: Rats received intraperitoneal injections of 2 mg/kg of DPCPX (1,3-dipropyl-8-cyclopentylxanthine), a highly selective A1 AR antagonist, 15 minutes before the termination of 45 minutes of left renal ischemia and 17 minutes before the initiation of a systemic adenosine infusion. 5. A2 AR antagonist before adenosine: Rats received intraperitoneal injections of 2 mg/kg of CSC (8-(3chlorostyryl)caffeine), a highly selective A2 AR antagonist, 15 minutes before the termination of 45 minutes of left renal ischemia and 17 minutes before the initiation of a systemic adenosine infusion. 6. A3 AR antagonist before adenosine: Rats received intraperitoneal injections of 1 mg/kg of MRS-1191 (3-ethyl-5-benzyl-phenylethynyl-6-phenyl-1,4-(⫹)dihydropyridine-3,5-dicarboxylate), a highly selective A3 AR antagonist, 15 minutes before the termination of 45 minutes of left renal ischemia and 17 minutes before the initiation of a systemic adenosine infusion. 7. A1 AR agonist during reperfusion: Rats received intraperitoneal injections of 2 mg/kg of R-PIA (R-N6phenyl-isopropyladenosine), a highly selective A1 AR agonist, 15 minutes before the termination of 45 minutes of left renal ischemia. 8. A2 AR agonist during reperfusion: Rats received intraperitoneal injections of 1 mg/kg of CGS-21680 (4-((N-ethyl-5⬘-carbamoyadenos-2-yl)-aminoethyl)phenylpropionic acid), a highly selective A2 AR agonist, 15 minutes before the termination of 45 minutes of left renal ischemia. 9. A3 AR agonist during reperfusion: Rats received intraperitoneal injections of 1 mg/kg of IB-MECA (N6-(3-iodobenzyl)-N-methyl-5⬘-carbamoyladenosine), a highly selective A3 AR agonist, 15 minutes before the termination of 45 minutes of left renal ischemia. Previously, we showed that the A3 AR antagonist alone given before ischemia and reperfusion protects renal function.16 To determine whether the A3 AR antagonist given alone after renal ischemia but before reperfusion also protects renal function, rats were subjected to the following protocol after right nephrectomy: 10. A3 AR antagonist during reperfusion: Rats received intraperitoneal injections of 1 mg/kg of MRS-1191, a highly selective A3 AR antagonist, 15 minutes before the termination of 45 minutes of left renal ischemia. We hypothesized that postischemic adenosine–mediated renal protection may involve the cAMP 3 PKA pathway. To
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determine whether exogenous cAMP protects renal function, rats were subjected to the following protocol after right nephrectomy: 11. Dibutyryl cAMP after ischemia: Rats received intraperitoneal injections of 5 mg/kg of dibutyryl cAMP, a nonmetabolizable PKA agonist, 5 minutes before the termination of 45 minutes of left renal ischemia. The doses of adenosine, AR agonists and antagonists, and dibutyryl cAMP were selected based on previously published in vivo studies.16,18,22-25 After these treatment protocols, the laparotomy was closed with 2–0 nylon, and the venous and arterial catheters were removed. After the rats regained consciousness, they were returned to the animal care facility and had free access to food and water. At 24 hours postoperatively, the rats were sacrificed with an overdose of intraperitoneal pentobarbital, a plasma sample was obtained for creatinine analysis, and the left kidneys were harvested for histology.
Measurement of Creatinine Plasma creatinine levels were measured spectrophotometrically using a commercially available quantitative colorimetric assay (Sigma, St. Louis, MO).
Histologic Examinations For histologic preparation, explanted kidneys were bisected along the long axis and were cut into three equalsized slices. Kidney slices from sham, IR, adenosine-treated, A2a AR agonist (CGS-21680)–treated, and dibutyryl cAMP– treated groups 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 and eosin. Morphologic assessment was performed by an experienced renal pathologist who was unaware of which treatment the animal had received. A grading scale of 0 to 4, as outlined by Jablonski et al,26 was used for the histopathologic assessment of ischemia and reperfusion–induced damage of the proximal tubules.
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RESULTS
Protective Effects of Systemic Adenosine Infusion After Renal Ischemia The hemodynamic effects of adenosine and selective AR agonists have been reported previously.16 Forty-five minutes of renal ischemia and 24 hours of reperfusion (IR) resulted in significant rises in creatinine (4.2 ⫾ 0.3 mg/dL, n ⫽ 6) compared with the sham-operated group (0.8 ⫾ 0.1 mg/dL, n ⫽ 6; P ⬍ 0.01) (Fig 1). Ten minutes of adenosine infusion given after the termination of 45 minutes of renal ischemia resulted in significant improvements in renal function (creatinine, 1.8 ⫾ 0.3 mg/dL, n ⫽ 8) (Fig 1) compared with animals subjected to IR injury alone. Pilot studies showed that intraperitoneal injections of vehicle (50% dimethyl sulfoxide) has no effect on renal function and morphology (data not shown). A2a Adenosine Receptor Agonist Is Responsible for Renal Protective Effects of Adenosine The A2a AR agonist CGS-21680 mimicked the protective effects of adenosine administration (creatinine, 1.8 ⫾ 0.3 mg/dL, n ⫽ 9) (Fig 1). Figure 1 also shows the effects of the A2a AR antagonist CSC on adenosine-induced renal protection. CSC abolished the renal protective effects of postischemic adenosine (creatinine, 3.7 ⫾ 0.2 mg/dL, n ⫽ 6), indicating that A2a ARs activated by adenosine were responsible for protection from IR-induced damage. Figure 2 shows a representative hypotensive response to a highly
Materials Adenosine and dibutyryl cAMP were dissolved in sterile, isotonic saline. All other drugs were dissolved in 50% dimethyl sulfoxide. Solutions were made daily. Adenosine and DPCPX were obtained from Sigma Chemical Company (St. Louis, MO). Pentobarbital was purchased from Henry Schein Veterinary Company (Indianapolis, IN). All other drugs were obtained from Research Biochemicals Incorporated (Natick, MA).
Statistical Analysis A one-way analysis of variance was used to compare mean values across multiple treatment groups with a Dunnett post-hoc multiple comparison test (eg, sham versus IR group). In all cases, a probability statistic less than 0.05 was taken to indicate significance. All data are expressed throughout the text as mean ⫾ 1 SEM.
Fig 1. Postischemic adenosine treatment protects renal function against reperfusion injury via A2a adenosine receptor (AR) activation. Comparison of mean creatinine (Cr) measured from sham-operated (Sham, n ⴝ 6), ischemic-reperfusion (IR, n ⴝ 6), adenosinetreated (ADO, n ⴝ 8), A2a AR agonist before reperfusion (CGS, n ⴝ 9), A2a AR antagonist before adenosine (CSCⴙADO, n ⴝ 6), and sodium nitroprusside (SNP, n ⴝ 4) treated animals. *P < 0.05 versus sham. #P < 0.05 versus IR. Error bars represent 1 SEM.
POSTISCHEMIC ADENOSINE PROTECTS RENAL FUNCTION
Fig 2. Representative hypotensive response to A2a adenosine receptor (AR) agonist (CGS-21680) and complete reversal by A2a AR antagonist (CSC). Arrows indicate the timing and doses used in the experiments. i.p., intraperitoneal.
selective A2a AR agonist (CGS-21680, 1 mg/kg intraperitoneally) and its complete reversal by a selective A2a AR antagonist (CSC, 2 mg/kg intraperitoneally). The elimination half-life of CGS21680 in vivo in plasma is 10 to 15 minutes,27,28 and the hypotension lasted approximately 30 minutes in our studies. Rapid reversal of hypotension after intraperitoneal CSC shows an effective antagonism of the A2a ARs. A1 and A3 Adenosine Receptors Are Not Involved in Adenosine-Induced Renal Protection The highly selective A1 AR antagonist DPCPX (creatinine, 1.8 ⫾ 0.3 mg/dL, n ⫽ 5) (Fig 3) and the highly selective A3 AR antagonist MRS-1191 (creatinine, 1.1 ⫾ 0.1 mg/dL, n ⫽ 6) (Fig 3) failed to block the protection conferred by 10 minutes of systemic adenosine infusion. Similarly, the highly selective A1 AR agonist R-PIA (creatinine, 3.4 ⫾ 0.3mg/dL, n ⫽ 6) (Fig 3) and the highly selective A3 AR agonist IB-MECA (creatinine, 4.0 ⫾ 0.3 mg/dL, n ⫽ 4) (Fig 3) failed to mimic the protection induced by 10 minutes of systemic adenosine infusion. When the A3 adenosine antagonist, MRS-1191, was given after 45 minutes of renal ischemia, it failed to protect renal function (creatinine, 4.8 ⫾ 0.3 mg/dL, n ⫽ 4) (Fig 3). Renal Vasodilation Does Not Induce Renal Protection To determine whether the renal protective effects of systemic adenosine are due to global
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renal vasodilation, sodium nitroprusside was used to induce similar levels of hypotension and vasodilation (40 g/kg/min intravenously for 10 minutes) to mimic the hypotensive effects of adenosine infusion. The rats were subjected to an equivalent duration and degree of hypotension (systolic blood pressure, approximately 75 mm Hg) as occurred with adenosine administration described in our previous study.16 Systemic sodium nitroprusside treatment for 10 minutes after 45 minutes of renal ischemia did not protect the kidneys against IR injury (creatinine, 4.7 ⫾ 0.4 mg/dL, n ⫽ 4) (Fig 1). Postischemic Elevation of Intracellular Cyclic Adenosine Monophosphate Mimics Adenosine-Induced Renal Protection Postischemic dibutyryl cAMP protected renal function after ischemia and reperfusion and mimicked the protective effects of adenosine administration (creatinine, 1.8 ⫾ 0.4 mg/dL, n ⫽ 6) (Fig 4). Postischemic Adenosine, A2a Adenosine Receptor Agonist, and Dibutyryl Cyclic Adenosine Monophosphate Improve Renal Morphology In Fig 5, the renal protective effects of postischemia adenosine are supported further by representative histologic slides of renal medullary tubules. Forty-five minutes of renal ischemia followed by 24 hours of reperfusion resulted in significant renal injury, as evidenced by severe tubular necrosis, medullary congestion and hemorrhage, and development of proteinaceous casts (Fig 5B). Postischemic adenosine, A2a AR agonist (CGS-21680), and dibutyryl cAMP treatments (Fig 5C-E) significantly improved and better preserved near-normal morphology. The Jablonski scale histology grading scores are shown in Fig 6. Forty-five minutes of renal ischemia and 24 hours of reperfusion resulted in severe acute tubular necrosis (grade, 3.5 ⫾ 0.4, n ⫽ 6) when compared with the sham-operated group (0.2 ⫾ 0.2, n ⫽ 6). Postischemic adenosine (grade, 1.9 ⫾ 0.5, n ⫽ 5), A2a AR agonist (CGS 21680, grade, 2.0 ⫾ 0.5, n ⫽ 5), and dibutyryl cAMP (grade, 1.6 ⫾ 0.2, n ⫽ 5) groups showed significant improvements in histologic evaluations when compared with the IR group.
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Fig 3. A1 and A3 adenosine receptors (ARs) are not involved in postischemic adenosine–mediated renal protection. Comparison of mean creatinine (Cr) measured from sham-operated (Sham, n ⴝ 6), ischemicreperfusion (IR, n ⴝ 6), A1 AR antagonist before adenosine (DPCPXⴙADO, n ⴝ 5), A3 AR antagonist before adenosine (MRSⴙADO, n ⴝ 6), A1 AR agonist (PIA, n ⴝ 6), A3 AR agonist (IB-MECA, n ⴝ 4), and A3 AR antagonist before reperfusion (MRSⴙREP, n ⴝ 4) treated animals. *P < 0.05 versus sham. #P < 0.05 versus IR. Error bars represent 1 SEM.
DISCUSSION
The present study shows that acute administration of adenosine or an A2a AR agonist, but not an A1 or A3 AR agonist, mediates renal protection during the reperfusion phase of an IR insult in the rat kidney. Protection was shown by improved serum creatinine levels and improved renal morphology evaluated at 24 hours. Additionally, an A2a AR antagonist, but not an A1 or A3 AR antagonist, blocked adenosine-mediated protection during reperfusion. A cAMP analogue, dibutyryl cAMP, given immediately before reperfusion improved renal function and morphology. Renal IR injury resulting in acute renal failure is a serious and unresolved clinical challenge.3,4 Surgical procedures involving the aorta and renal arteries (eg, suprarenal and juxtarenal abdominal aortic aneurysms and renal transplantation) have significant postoperative renal complications, including acute tubular necrosis and acute renal failure.3,4,29 There are many clinical scenarios in which organ ischemia cannot be predicted in advance, and it is important to understand mechanisms that may mitigate reperfusion injury after an ischemic event. Our findings point to the central role of A2a AR activation in mediating renal protection during the reperfusion phase of an IR insult. These findings complement and extend our previous
study,16 in which a different AR subtype was shown to mediate renal protection when adenosine was given before an ischemic insult. Adenosine or an A1 AR agonist, but not an A2a or A3 AR agonist, given before ischemia and reperfusion improved renal function and morphology evaluated at 24 hours. Taken together, our previous study and the present study suggest that adenosine affords renal protection via A1 AR activation before ischemia and A2a AR activation during reperfusion. These findings agree with studies in the heart in which preischemic A1 and postischemic A2a AR agonists mimic adenosineinduced tissue protection against reperfusion injury.8-12,14,30 This study agrees with the findings of Okusa et al,31 who showed renal protection with long-term (hours) subcutaneous administration of a selective A2a AR agonist during the reperfusion period. In their study, the initiation of drug (a selective A2a AR agonist) delivery via subcutaneous osmotic minipumps 5 hours before an IR insult or initiation of drug delivery during the reperfusion period protected renal morphology and function measured at 24 and 48 hours. Our experimental design differed from the Okusa study in several ways, however. In our study, postischemic adenosine was infused intravenously only for 10 minutes, and the A2a AR agonist was delivered acutely, 15 minutes before reperfusion, via a single intraperitoneal bolus; in the study by Okusa et al,31 the drug delivery continued chronically until animals were sacrificed at 24 or 48 hours. Our current study shows
Fig 4. Postischemic dibutyryl cyclic adenosine monophosphate (DBcAMP) protects renal function against reperfusion injury. Comparison of mean creatinine measured from sham-operated (Sham, n ⴝ 6), ischemic-reperfusion (IR, n ⴝ 6), and DBcAMP treated (DBcAMP, n ⴝ 6) animals. *P < 0.05 versus sham. #P < 0.05 versus IR. Error bars represent 1 SEM.
POSTISCHEMIC ADENOSINE PROTECTS RENAL FUNCTION
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Fig 5. Representative photomicrographs of outer medulla of the kidney. (A) Sham-treated group. (B) Ischemicreperfusion group. (C) Adenosine-treated group. (D) A2a adenosine receptor (AR) agonist (CGS-21680)–treated group. (E) Dibutyryl cyclic adenosine monophosphate (DBcAMP)–treated group. Arrows indicate proteinaceous casts in tubuli. (H&E, original magnification ⴛ200.)
that acute, short-term administration of adenosine during the early postischemic period potently protects renal function and morphology against IR injury. The present study as well as a previous study from our laboratory16 in which an A2a agonist was given before an ischemic insult allows a more complete interpretation of the results of the two drug delivery protocols of Okusa et al.31 Although initiation of an A2a AR agonist infusion
either 5 hours before ischemia or immediately after reperfusion resulted in renal protection, the Okusa study31 does not support A2a AR–mediated protection occurring during the ischemic phase because drug delivery occurred during ischemia and reperfusion. Results from our previous study suggest that A2a AR activation during the ischemic phase does not afford renal protection. Consistent with the findings of the present study and Okusa et al,31 A2a AR agonists mediate
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Fig 6. Jablonski grading scale scores for histologic appearance of acute tubular necrosis from shamoperated (n ⴝ 6), ischemic-reperfusion (IR, n ⴝ 6), adenosine-treated (ADO, n ⴝ 5), A2a adenosine receptor (AR) agonist–treated (CGS, n ⴝ 5), and dibutyryl cyclic adenosine monophosphate (DBcAMP)–treated (DBcAMP, n ⴝ 4) groups. *P < 0.05 versus sham. #P < 0.05 versus IR. Error bars represent 1 SEM.
protection during the reperfusion phase of the injury process. The present study further extends the understanding of the potential mechanism of A2a AR– mediated renal protection. Classically, renal parenchymal and vascular A2a ARs couple via Gs activation to the stimulation of adenylyl cyclase activity and an elevation of cellular cAMP.6,32 In the present study, administration of a cAMP analogue, dibutyryl cAMP, just before reperfusion mimicked the renal protection afforded by an A2a AR agonist or adenosine. This finding is consistent with an A2a AR 3 Gs protein 3 adenylyl cyclase 3 cAMP pathway mediating renal protection during the reperfusion period. It has been suggested that increased intracellular cAMP protects against renal reoxygenation injury in in vivo and in vitro models, perhaps via modulation of the intracellular rise in calcium and free radical generation during reperfusion.18,21,33 Additionally, it has been proposed that the A2a AR–mediated signaling pathway antagonizes multiple cellular insults responsible for reperfusion injury, including (1) loss of tissue high-energy phosphates during and after the ischemic period; (2) proinflammatory cell (neutrophil and mast cell)–mediated cellular and microvascular injuries via direct cellular toxicity of superoxide free radicals that are generated by these cells during ischemia and subsequent reperfusion; (3) microvascular dysfunction with platelet plugging and endothelial damage, resulting in a no-reflow phenomenon with inadequate tissue
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perfusion during the reperfusion period; and (4) calcium overload–mediated reperfusion injury.2,4,15,29 Tumor necrosis factor-␣, oxygen-mediated free radicals, and neutrophil-mediated cell killing have been implicated in the pathogenesis of acute renal failure.34 Adenosine’s anti-inflammatory actions via A2a AR activation are well characterized.35,36 A2a AR activation is known to inhibit the release and activities of tumor necrosis factor-␣ in cardiomyocytes37,38 and negatively modulates the production of reactive oxygen species and free radicals from activated neutrophils.11,35,36 Adenosine, also via A2a AR activation, attenuates the cytotoxic effects of neutrophils and inhibits platelet-induced vascular dysfunction and occlusions.11,13 Adenosine also protects the endothelial integrity in a reperfused vascular bed.39 At first glance, the renal effects of adenosine infusion appear to be detrimental to renal function because adenosine produces changes that appear to worsen renal function—reduced glomerular filtration rate and afferent cortical blood flow and impaired solute transport.40,41 Several investigators have reported that nonselective adenosine receptor antagonists, such as theophylline, have protective effects against some, but not all, models of ischemic renal failure.41-43 These investigators theorized that AR antagonism would protect against the renal hemodynamic effects of A1 adenosine receptor stimulation (reduction in glomerular filtration rate, solute transport, and renal blood flow via afferent arteriolar vasoconstriction).44 Looking more closely, adenosine has several protective attributes against renal IR injury. Decreases in glomerular filtration rate via reduced cortical blood flow and reduced active solute transport function via tubular glomerular feedback lead to reduced renal oxygen consumption (via A1 AR activation). At the same time, adenosine preferentially increases blood flow and improves oxygenation to the medulla (one of the most susceptible portions of the kidney to hypoxic insult) and reduces the ischemic insult to the tubular cells by vasodilating the vasa recta via A2a AR activation.45,46 Adenosine administration leads to significant improvement in medullary oxygen partial pressure via A2a AR activation.45-47 After renal ischemia, the release and activation of endogenous ARs
POSTISCHEMIC ADENOSINE PROTECTS RENAL FUNCTION
may produce physiologic effects that reduce renal oxygen consumption and improve blood flow to regions that are most susceptible to hypoxic insult (outer border of medulla). In our study, the selective medullary vasodilation of adenosine may have contributed to its renal protective effects. The fact that sodium nitroprusside, a global renal vasodilator, failed to protect renal function after ischemia and reperfusion suggests that increasing total renal blood flow is inadequate for renal protection. The role of A3 ARs in the kidney is not known. We have shown that the selective A3 AR agonist IB-MECA when given before the onset of renal ischemia worsens renal function, and the antagonist MRS-1191 when given before the onset of renal ischemia protects renal function.16 Our previous findings suggest a detrimental role of preischemic A3 AR activation in renal IR injury. In the current study, postischemic treatment with an A3 AR antagonist failed to show protection, indicating that antagonism of A3 ARs must precede the ischemic insult for the protective benefit. In conclusion, we show an in vivo protective effect of postischemic adenosine infusion in the kidney. Treatment with adenosine, an A2a AR agonist, or dibutyryl cAMP protects renal function when given after the renal ischemic period probably via a cAMP-dependent mechanism. These findings may have clinical significance in the protection of the kidney during surgical procedures in which renal ischemia is unavoidable or unpredicted or preischemic optimization of renal function is not possible. ACKNOWLEDGMENT We thank Dr Vivette D’Agati for providing her expertise in renal histopathology.
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Browne DR, Sweeny P, Morehead JF: Prognosis of criticallyill patients with acute renal failure: APACHE II score and other predictive factors. QJM 72:857-866, 1989 6. Spielman WS, Arend LJ: Adenosine receptors and signalling in the kidney. Hypertension 17:117-130, 1991 7. Zou AP, Wu F, Li PL, Cowley AW Jr: Effect of chronic salt loading on adenosine metabolism and receptor expression in renal cortex and medulla in rats. Hypertension 33:511-516, 1999 8. Liu GS, Thornton JD, Van Winkle DM, Stanley AW, Olsson RA, Downey JM: Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation 84:350-356, 1991 9. Auchampach JA, Gross GJ: Adenosine A1 receptors, KATP channels, and ischemic preconditioning in dogs. Am J Physiol 264:H1321-H1336, 1993 10. Liang BT: Direct preconditioning of cardiac ventricular myocytes via adenosine A1 receptor and KATP channel. Am J Physiol 271:H1769-H1777, 1996 11. Jordan JE, Zhao ZQ, Sato H, Taft S, Vinten-Johansen J: Adenosine A2 receptor activation attenuates reperfusion injury by inhibiting neutrophil accumulation, superoxide generation and coronary endothelial adherence. J Pharmacol Exp Ther 280:301-309, 1997 12. Pitarys CJ, Virmani R, Vildibill HD Jr, Jackson EK, Forman MB: Reduction of myocardial reperfusion injury by intravenous adenosine administered during the early reperfusion period. Circulation 83:237-247, 1991 13. Zhao ZQ, Todd JC, Sato H, Ma XL, Vinten-Johansen J: Adenosine inhibition of neutrophil damage during reperfusion does not involve K(ATP)-channel activation. Am J Physiol 273:H1677-H1687, 1997 14. Schlack W, Schafer M, Uebing A, Schafer S, Borchard U, Thamer V: Adenosine A2-receptor activation at reperfusion reduces infarct size and improves myocardial wall function in dog heart. J Cardiovasc Pharmacol 22:8996, 1993 15. Cargnoni A, Ceconi C, Boraso A, Bernocchi P, Monopoli A, Curello S, Ferrari R: Role of A2A receptor in the modulation of myocardial reperfusion damage. J Cardiovasc Pharmacol 33:883-893, 1999 16. Lee HT, Emala CW: Protective effects of renal ischemic preconditioning and adenosine pretreatment: Role of A(1) and A(3) receptors. Am J Physiol Renal Physiol 278: F380-F387, 2000 17. Lee HT, Emala CW: Protein kinase C and Gi/o proteins are involved in adenosine- and ischemic preconditioning-mediated protection of renal ischemic-reperfusion injury. J Am Soc Nephrol 12:233-240, 2000 18. Koiwai T, Oguchi H, Terashima M, Yanagisawa Y, Higuchi M, Furuta S: Beneficial effect of dibutyryl cyclic AMP (DBcAMP) on ischemic acute renal failure in the rat. Ren Fail 14:461-465, 1992 19. Mizus I, Summer W, Farrukh I, Michael JR, Gurtner GH: Isoproterenol or aminophylline attenuate pulmonary edema after acid lung injury. Am Rev Respir Dis 131:256259, 1985 20. Farrukh IS, Gurtner GH, Michael JR: Pharmacological modification of pulmonary vascular injury: Possible role of cAMP. J Appl Physiol 62:47-54, 1987 21. Kawai Y, Gemba M: Amelioration by cAMP of cepha-
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loridine-induced injury in the porcine kidney cell line LLCPK1. Jpn J Pharmacol 72:67-70, 1996 22. Myers S, Pugsley TA: Decrease in rat striatal dopamine synthesis and metabolism in vivo by metabolically stable adenosine receptor agonists. Brain Res 375:193-197, 1986 23. Von Lubitz DK, Paul IA, Carter M, Jacobson KA: Effects of N6-cyclopentyl adenosine and 8-cyclopentyl-1,3,dipropylxanthine on N-methyl-D-aspartate induced seizures in mice. Eur J Pharmacol 249:265-270, 1993 24. Von Lubitz DK, Carter MF, Deutsch SI, Lin RC, Mastropaolo J, Meshulam Y, Jacobson KA: The effects of adenosine A3 receptor stimulation on seizures in mice. Eur J Pharmacol 275:23-29, 1995 25. Von Lubitz DK, Lin RC, Jacobson KA: Cerebral ischemia in gerbils: Effects of acute and chronic treatment with adenosine A2A receptor agonist and antagonist. Eur J Pharmacol 287:295-302, 1995 26. Jablonski P, Howden BO, Rae DA, Birrel CS, Marshall VC, Tange J: An experimental model for assessment of renal recovery from warm ischemia. Transplantation 35:198204, 1983 27. Chovan JP, Zane PA, Greenberg GE: Automatedextraction, high-performance liquid chromatographic method and pharmacokinetics in rats of a highly A2-selective adenosine agonist, CGS 21680. J Chromatogr 578:77-83, 1992 28. Mathot RA, Cleton A, Soudijn W, IJzerman AP, Danhof M: Pharmacokinetic modelling of the haemodynamic effects of the A2a adenosine receptor agonist CGS 21680C in conscious normotensive rats. Br J Pharmacol 114:761-768, 1995 29. McComs PR, Roberts B: Acute renal failure following resection of abdominal aortic aneurysm. Surg Gynecol Obstet 148:175-178, 1979 30. Ongini E, Adami M, Ferri C, Bertorelli R: Adenosine A2a receptors and neuroprotection. Ann N Y Acad Sci 825:30-48, 1997 31. Okusa MD, Linden J, Macdonald T, Huang L: Selective A2A adenosine receptor activation reduces ischemiareperfusion injury in rat kidney. Am J Physiol 277:F404F412, 1999 32. McCoy DE, Bhattacharya S, Olson BA, Levier DG, Arend LJ, Spielman WS: The renal adenosine system: Structure, function, and regulation. Semin Nephrol 13:31-40, 1993 33. Yonehana T, Gemba M: Ameliorative effect of adenosine on hypoxia-reoxygenation injury in LLC-PK1, a porcine kidney cell line. Jpn J Pharmacol 80:163-167, 1999
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34. Donnahoo KK, Shames BD, Harken AH, Meldrum DR: Review article: the role of tumor necrosis factor in renal ischemia-reperfusion injury. J Urol 162:196-203, 1999 35. Cronstein BN: Adenosine, an endogenous antiinflammatory agent. J Appl Physiol 76:5-13, 1994 36. Engler RL: Adenosine: The signal of life? Circulation 84:951-954, 1991 37. Wagner DR, Combes A, McTiernan C, Sanders VJ, Lemster B, Feldman AM: Adenosine inhibits lipopolysaccharide-induced cardiac expression of tumor necrosis factoralpha. Circ Res 82:47-56, 1998 38. Wagner DR, McTiernan C, Sanders VJ, Feldman AM: Adenosine inhibits lipopolysaccharide-induced secretion of tumor necrosis factor-alpha in the failing human heart. Circulation 97:521-524, 1998 39. Beauchamp P, Richard V, Tamion F, Lallemand F, Lebreton JP, Vaudry H, Daveau M, Thuillez C: Protective effects of preconditioning in cultured rat endothelial cells: Effects on neutrophil adhesion and expression of ICAM-1 after anoxia and reoxygenation. Circulation 100:541-546, 1999 40. Rossi NF, Churchill PC, Jacobson KA, Leahy AE: Further characterization of the renovascular effects of N6cyclohexyladenosine in the isolated perfused rat kidney. J Pharmacol Exp Ther 240:911-915, 1987 41. Rossi N, Churchill P, Ellis V, Amore B: Mechanism of adenosine receptor-induced renal vasoconstriction in rats. Am J Physiol 255:H885-H890, 1988 42. Lin JJ, Churchill PC, Bidani AK: Effect of theophylline on the initiation phase of postischemic acute renal failure in rats. J Lab Clin Med 108:150-154, 1986 43. Lin JJ, Churchill PC, Bidani AK: The effect of dipyridamole on the initiation phase of postischemic acute renal failure in rats. Can J Physiol Pharmacol 65:1491-1495, 1987 44. Bidani AK, Churchill PC, Packer W: Theophyllineinduced protection in myoglobinuric acute renal failure: Further characterization. Can J Physiol Pharmacol 65:42-45, 1987 45. Dinour D, Brezis M: Effects of adenosine on intrarenal oxygenation. Am J Physiol 261:F787-F791, 1991 46. Agmon Y, Dinour D, Brezis M: Disparate effects of adenosine A1- and A2-receptor agonists on intrarenal blood flow. Am J Physiol 265:F802-F806, 1993 47. Dinour D, Agmon Y, Brezis M: Adenosine: An emerging role in the control of renal medullary oxygenation? Exp Nephrol 1:152-157, 1993
A
LTHOUGH ISCHEMIA itself can cause injury, significant tissue damage occurs during the reperfusion period.1,2 Renal reperfusion injury resulting in acute renal failure after unavoidable surgical ischemia and reperfusion is a serious clinical concern.3,4 The onset of acute renal failure implies a poor prognosis and frequently is complicated by many other lifethreatening complications, including sepsis and multiorgan failure.4 In high-risk patients undergoing high-risk surgery, the mortality and morbid-
From the Department of Anesthesiology, Columbia University College of Physicians and Surgeons, New York, NY. Received October 16, 2000; accepted in revised form March 30, 2001. Funded in part by intramural grant support from the Department of Anesthesiology, Columbia University College of Physicians and Surgeons. Presented in abstract form at the 1999 American Society of Anesthesiology meeting, Dallas, TX. Address reprint requests to H. Thomas Lee, MD, PhD, Department of Anesthesiology, Columbia Presbyterian Medical Center, P&S Box 46 (PH-5), 630 West 168th Street, New York, NY 10032-3784. E-mail: [email protected] © 2001 by the National Kidney Foundation, Inc. 0272-6386/01/3803-0021$35.00/0 doi:10.1053/ajkd.2001.26888 610
ity rates from perioperative acute renal failure have changed little since the 1970s.3-5 Adenosine has many effects on the kidney, including effects on global renal function (eg, cortical and medullary blood flow) and on endogenous function of specific renal cell types (eg, renin release, electrolyte transport). In the kidney, all four subtypes (A1, A2a, A2b, and A3) of adenosine receptors (ARs) have been identified.6,7 The A1 ARs reduce cortical renal blood flow and renin release and serve as antidiuretic and antinatriuretic receptors. Conversely, A2a ARs increase medullary renal blood flow and renin release and serve as diuretic and natriuretic receptors. The exact roles of the A2b and A3 ARs in the kidney are unclear. In cardiac ischemic-reperfusion (IR) injury, it has been postulated and described that adenosine antagonizes IR injury via two distinct AR subtype modulations. Adenosine given before an ischemic insult protects via A1 AR activation,8-10 whereas adenosine protection is via A2a ARs when given during the postischemic reperfusion period.11-15 In the kidney, we have shown that brief preischemic adenosine treatment (ie, adenosine given for 10 minutes before ischemia and reperfusion) protects rat renal function against IR injury via an A1 AR-Gi-
American Journal of Kidney Diseases, Vol 38, No 3 (September), 2001: pp 610-618
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protein kinase C cascade.16,17 Conversely, neither A2a nor A3 AR activation mimicked the protection of preischemic adenosine. The A2a ARs couple to adenylyl cyclase, which leads to increased intracellular cyclic adenosine monophosphate (cAMP) levels, which in turn activate protein kinase A (PKA). Postischemic adenosine-mediated organ protection against reperfusion injury may involve cAMP and PKA as intracellular signaling intermediates. Exogenous stimulation of the cAMP 3 PKA system with isoproterenol and cAMP analogues has been shown to attenuate renal and pulmonary reperfusion injury in vivo18-20 and in vitro.21 The major aims of this study were to determine whether rat kidneys can be protected against reperfusion injury with postischemic adenosine treatment and to determine the subtype of ARs involved in renal protection against reperfusion injury. Because A2a AR activation classically increases intracellular cAMP, we also wanted to determine whether postischemic administration of a cAMP analogue (dibutyryl cAMP) would protect renal function against reperfusion injury. We hypothesized that postischemic A2a AR activation protects against renal reperfusion injury via intracellular signaling cascades involving cAMP. MATERIALS AND METHODS All protocols were approved by the Institutional Animal Care and Use Committee of Columbia University. Adult male Wistar rats (225 to 275 g, Harlan Sprague-Dawley, Indianapolis, IN) were used. The rats had free access to rodent chow and water. The rats were anesthetized with intraperitoneal pentobarbital (45 mg/kg body weight or to effect). Additional pentobarbital was administered as needed based on response to tail pinch. After 500 U of heparin was given intraperitoneally, rats were placed on an electric heating pad under a warming light. Body temperature was monitored with a rectal probe and maintained at 37°C. The rats were allowed to breathe room air spontaneously. The right femoral vein was cannulated with heparinized (10 U/mL) polyethylene tubing (PE-50) for intravenous drug access and hydration. The right femoral artery was cannulated with heparinized PE-50 tubing for hemodynamic monitoring and blood sampling. After a 10-minute stabilization period, a midline laparotomy was performed. Right nephrectomy was performed, and the left renal artery and vein were isolated. Eleven separate protocols were performed as described subsequently.
Protocols To determine the role of postischemic adenosine treatment in renal IR injury, rats were subjected to the following protocols after right nephrectomy:
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1. Control group (sham): Rats were subjected to isolation of left renal artery and vein only. 2. IR group: Rats were subjected to 45 minutes of left renal ischemia. 3. Adenosine treatment group: Rats received systemic intravenous infusion of adenosine (1.75 mg/kg/ min ⫻ 10 minutes) 2 minutes after the termination of 45 minutes of left renal ischemia. To determine the receptor subtypes involved in postischemic adenosine–induced renal protection, rats were subjected to the following protocols after right nephrectomy: 4. A1 AR antagonist before adenosine: Rats received intraperitoneal injections of 2 mg/kg of DPCPX (1,3-dipropyl-8-cyclopentylxanthine), a highly selective A1 AR antagonist, 15 minutes before the termination of 45 minutes of left renal ischemia and 17 minutes before the initiation of a systemic adenosine infusion. 5. A2 AR antagonist before adenosine: Rats received intraperitoneal injections of 2 mg/kg of CSC (8-(3chlorostyryl)caffeine), a highly selective A2 AR antagonist, 15 minutes before the termination of 45 minutes of left renal ischemia and 17 minutes before the initiation of a systemic adenosine infusion. 6. A3 AR antagonist before adenosine: Rats received intraperitoneal injections of 1 mg/kg of MRS-1191 (3-ethyl-5-benzyl-phenylethynyl-6-phenyl-1,4-(⫹)dihydropyridine-3,5-dicarboxylate), a highly selective A3 AR antagonist, 15 minutes before the termination of 45 minutes of left renal ischemia and 17 minutes before the initiation of a systemic adenosine infusion. 7. A1 AR agonist during reperfusion: Rats received intraperitoneal injections of 2 mg/kg of R-PIA (R-N6phenyl-isopropyladenosine), a highly selective A1 AR agonist, 15 minutes before the termination of 45 minutes of left renal ischemia. 8. A2 AR agonist during reperfusion: Rats received intraperitoneal injections of 1 mg/kg of CGS-21680 (4-((N-ethyl-5⬘-carbamoyadenos-2-yl)-aminoethyl)phenylpropionic acid), a highly selective A2 AR agonist, 15 minutes before the termination of 45 minutes of left renal ischemia. 9. A3 AR agonist during reperfusion: Rats received intraperitoneal injections of 1 mg/kg of IB-MECA (N6-(3-iodobenzyl)-N-methyl-5⬘-carbamoyladenosine), a highly selective A3 AR agonist, 15 minutes before the termination of 45 minutes of left renal ischemia. Previously, we showed that the A3 AR antagonist alone given before ischemia and reperfusion protects renal function.16 To determine whether the A3 AR antagonist given alone after renal ischemia but before reperfusion also protects renal function, rats were subjected to the following protocol after right nephrectomy: 10. A3 AR antagonist during reperfusion: Rats received intraperitoneal injections of 1 mg/kg of MRS-1191, a highly selective A3 AR antagonist, 15 minutes before the termination of 45 minutes of left renal ischemia. We hypothesized that postischemic adenosine–mediated renal protection may involve the cAMP 3 PKA pathway. To
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determine whether exogenous cAMP protects renal function, rats were subjected to the following protocol after right nephrectomy: 11. Dibutyryl cAMP after ischemia: Rats received intraperitoneal injections of 5 mg/kg of dibutyryl cAMP, a nonmetabolizable PKA agonist, 5 minutes before the termination of 45 minutes of left renal ischemia. The doses of adenosine, AR agonists and antagonists, and dibutyryl cAMP were selected based on previously published in vivo studies.16,18,22-25 After these treatment protocols, the laparotomy was closed with 2–0 nylon, and the venous and arterial catheters were removed. After the rats regained consciousness, they were returned to the animal care facility and had free access to food and water. At 24 hours postoperatively, the rats were sacrificed with an overdose of intraperitoneal pentobarbital, a plasma sample was obtained for creatinine analysis, and the left kidneys were harvested for histology.
Measurement of Creatinine Plasma creatinine levels were measured spectrophotometrically using a commercially available quantitative colorimetric assay (Sigma, St. Louis, MO).
Histologic Examinations For histologic preparation, explanted kidneys were bisected along the long axis and were cut into three equalsized slices. Kidney slices from sham, IR, adenosine-treated, A2a AR agonist (CGS-21680)–treated, and dibutyryl cAMP– treated groups 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 and eosin. Morphologic assessment was performed by an experienced renal pathologist who was unaware of which treatment the animal had received. A grading scale of 0 to 4, as outlined by Jablonski et al,26 was used for the histopathologic assessment of ischemia and reperfusion–induced damage of the proximal tubules.
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RESULTS
Protective Effects of Systemic Adenosine Infusion After Renal Ischemia The hemodynamic effects of adenosine and selective AR agonists have been reported previously.16 Forty-five minutes of renal ischemia and 24 hours of reperfusion (IR) resulted in significant rises in creatinine (4.2 ⫾ 0.3 mg/dL, n ⫽ 6) compared with the sham-operated group (0.8 ⫾ 0.1 mg/dL, n ⫽ 6; P ⬍ 0.01) (Fig 1). Ten minutes of adenosine infusion given after the termination of 45 minutes of renal ischemia resulted in significant improvements in renal function (creatinine, 1.8 ⫾ 0.3 mg/dL, n ⫽ 8) (Fig 1) compared with animals subjected to IR injury alone. Pilot studies showed that intraperitoneal injections of vehicle (50% dimethyl sulfoxide) has no effect on renal function and morphology (data not shown). A2a Adenosine Receptor Agonist Is Responsible for Renal Protective Effects of Adenosine The A2a AR agonist CGS-21680 mimicked the protective effects of adenosine administration (creatinine, 1.8 ⫾ 0.3 mg/dL, n ⫽ 9) (Fig 1). Figure 1 also shows the effects of the A2a AR antagonist CSC on adenosine-induced renal protection. CSC abolished the renal protective effects of postischemic adenosine (creatinine, 3.7 ⫾ 0.2 mg/dL, n ⫽ 6), indicating that A2a ARs activated by adenosine were responsible for protection from IR-induced damage. Figure 2 shows a representative hypotensive response to a highly
Materials Adenosine and dibutyryl cAMP were dissolved in sterile, isotonic saline. All other drugs were dissolved in 50% dimethyl sulfoxide. Solutions were made daily. Adenosine and DPCPX were obtained from Sigma Chemical Company (St. Louis, MO). Pentobarbital was purchased from Henry Schein Veterinary Company (Indianapolis, IN). All other drugs were obtained from Research Biochemicals Incorporated (Natick, MA).
Statistical Analysis A one-way analysis of variance was used to compare mean values across multiple treatment groups with a Dunnett post-hoc multiple comparison test (eg, sham versus IR group). In all cases, a probability statistic less than 0.05 was taken to indicate significance. All data are expressed throughout the text as mean ⫾ 1 SEM.
Fig 1. Postischemic adenosine treatment protects renal function against reperfusion injury via A2a adenosine receptor (AR) activation. Comparison of mean creatinine (Cr) measured from sham-operated (Sham, n ⴝ 6), ischemic-reperfusion (IR, n ⴝ 6), adenosinetreated (ADO, n ⴝ 8), A2a AR agonist before reperfusion (CGS, n ⴝ 9), A2a AR antagonist before adenosine (CSCⴙADO, n ⴝ 6), and sodium nitroprusside (SNP, n ⴝ 4) treated animals. *P < 0.05 versus sham. #P < 0.05 versus IR. Error bars represent 1 SEM.
POSTISCHEMIC ADENOSINE PROTECTS RENAL FUNCTION
Fig 2. Representative hypotensive response to A2a adenosine receptor (AR) agonist (CGS-21680) and complete reversal by A2a AR antagonist (CSC). Arrows indicate the timing and doses used in the experiments. i.p., intraperitoneal.
selective A2a AR agonist (CGS-21680, 1 mg/kg intraperitoneally) and its complete reversal by a selective A2a AR antagonist (CSC, 2 mg/kg intraperitoneally). The elimination half-life of CGS21680 in vivo in plasma is 10 to 15 minutes,27,28 and the hypotension lasted approximately 30 minutes in our studies. Rapid reversal of hypotension after intraperitoneal CSC shows an effective antagonism of the A2a ARs. A1 and A3 Adenosine Receptors Are Not Involved in Adenosine-Induced Renal Protection The highly selective A1 AR antagonist DPCPX (creatinine, 1.8 ⫾ 0.3 mg/dL, n ⫽ 5) (Fig 3) and the highly selective A3 AR antagonist MRS-1191 (creatinine, 1.1 ⫾ 0.1 mg/dL, n ⫽ 6) (Fig 3) failed to block the protection conferred by 10 minutes of systemic adenosine infusion. Similarly, the highly selective A1 AR agonist R-PIA (creatinine, 3.4 ⫾ 0.3mg/dL, n ⫽ 6) (Fig 3) and the highly selective A3 AR agonist IB-MECA (creatinine, 4.0 ⫾ 0.3 mg/dL, n ⫽ 4) (Fig 3) failed to mimic the protection induced by 10 minutes of systemic adenosine infusion. When the A3 adenosine antagonist, MRS-1191, was given after 45 minutes of renal ischemia, it failed to protect renal function (creatinine, 4.8 ⫾ 0.3 mg/dL, n ⫽ 4) (Fig 3). Renal Vasodilation Does Not Induce Renal Protection To determine whether the renal protective effects of systemic adenosine are due to global
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renal vasodilation, sodium nitroprusside was used to induce similar levels of hypotension and vasodilation (40 g/kg/min intravenously for 10 minutes) to mimic the hypotensive effects of adenosine infusion. The rats were subjected to an equivalent duration and degree of hypotension (systolic blood pressure, approximately 75 mm Hg) as occurred with adenosine administration described in our previous study.16 Systemic sodium nitroprusside treatment for 10 minutes after 45 minutes of renal ischemia did not protect the kidneys against IR injury (creatinine, 4.7 ⫾ 0.4 mg/dL, n ⫽ 4) (Fig 1). Postischemic Elevation of Intracellular Cyclic Adenosine Monophosphate Mimics Adenosine-Induced Renal Protection Postischemic dibutyryl cAMP protected renal function after ischemia and reperfusion and mimicked the protective effects of adenosine administration (creatinine, 1.8 ⫾ 0.4 mg/dL, n ⫽ 6) (Fig 4). Postischemic Adenosine, A2a Adenosine Receptor Agonist, and Dibutyryl Cyclic Adenosine Monophosphate Improve Renal Morphology In Fig 5, the renal protective effects of postischemia adenosine are supported further by representative histologic slides of renal medullary tubules. Forty-five minutes of renal ischemia followed by 24 hours of reperfusion resulted in significant renal injury, as evidenced by severe tubular necrosis, medullary congestion and hemorrhage, and development of proteinaceous casts (Fig 5B). Postischemic adenosine, A2a AR agonist (CGS-21680), and dibutyryl cAMP treatments (Fig 5C-E) significantly improved and better preserved near-normal morphology. The Jablonski scale histology grading scores are shown in Fig 6. Forty-five minutes of renal ischemia and 24 hours of reperfusion resulted in severe acute tubular necrosis (grade, 3.5 ⫾ 0.4, n ⫽ 6) when compared with the sham-operated group (0.2 ⫾ 0.2, n ⫽ 6). Postischemic adenosine (grade, 1.9 ⫾ 0.5, n ⫽ 5), A2a AR agonist (CGS 21680, grade, 2.0 ⫾ 0.5, n ⫽ 5), and dibutyryl cAMP (grade, 1.6 ⫾ 0.2, n ⫽ 5) groups showed significant improvements in histologic evaluations when compared with the IR group.
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Fig 3. A1 and A3 adenosine receptors (ARs) are not involved in postischemic adenosine–mediated renal protection. Comparison of mean creatinine (Cr) measured from sham-operated (Sham, n ⴝ 6), ischemicreperfusion (IR, n ⴝ 6), A1 AR antagonist before adenosine (DPCPXⴙADO, n ⴝ 5), A3 AR antagonist before adenosine (MRSⴙADO, n ⴝ 6), A1 AR agonist (PIA, n ⴝ 6), A3 AR agonist (IB-MECA, n ⴝ 4), and A3 AR antagonist before reperfusion (MRSⴙREP, n ⴝ 4) treated animals. *P < 0.05 versus sham. #P < 0.05 versus IR. Error bars represent 1 SEM.
DISCUSSION
The present study shows that acute administration of adenosine or an A2a AR agonist, but not an A1 or A3 AR agonist, mediates renal protection during the reperfusion phase of an IR insult in the rat kidney. Protection was shown by improved serum creatinine levels and improved renal morphology evaluated at 24 hours. Additionally, an A2a AR antagonist, but not an A1 or A3 AR antagonist, blocked adenosine-mediated protection during reperfusion. A cAMP analogue, dibutyryl cAMP, given immediately before reperfusion improved renal function and morphology. Renal IR injury resulting in acute renal failure is a serious and unresolved clinical challenge.3,4 Surgical procedures involving the aorta and renal arteries (eg, suprarenal and juxtarenal abdominal aortic aneurysms and renal transplantation) have significant postoperative renal complications, including acute tubular necrosis and acute renal failure.3,4,29 There are many clinical scenarios in which organ ischemia cannot be predicted in advance, and it is important to understand mechanisms that may mitigate reperfusion injury after an ischemic event. Our findings point to the central role of A2a AR activation in mediating renal protection during the reperfusion phase of an IR insult. These findings complement and extend our previous
study,16 in which a different AR subtype was shown to mediate renal protection when adenosine was given before an ischemic insult. Adenosine or an A1 AR agonist, but not an A2a or A3 AR agonist, given before ischemia and reperfusion improved renal function and morphology evaluated at 24 hours. Taken together, our previous study and the present study suggest that adenosine affords renal protection via A1 AR activation before ischemia and A2a AR activation during reperfusion. These findings agree with studies in the heart in which preischemic A1 and postischemic A2a AR agonists mimic adenosineinduced tissue protection against reperfusion injury.8-12,14,30 This study agrees with the findings of Okusa et al,31 who showed renal protection with long-term (hours) subcutaneous administration of a selective A2a AR agonist during the reperfusion period. In their study, the initiation of drug (a selective A2a AR agonist) delivery via subcutaneous osmotic minipumps 5 hours before an IR insult or initiation of drug delivery during the reperfusion period protected renal morphology and function measured at 24 and 48 hours. Our experimental design differed from the Okusa study in several ways, however. In our study, postischemic adenosine was infused intravenously only for 10 minutes, and the A2a AR agonist was delivered acutely, 15 minutes before reperfusion, via a single intraperitoneal bolus; in the study by Okusa et al,31 the drug delivery continued chronically until animals were sacrificed at 24 or 48 hours. Our current study shows
Fig 4. Postischemic dibutyryl cyclic adenosine monophosphate (DBcAMP) protects renal function against reperfusion injury. Comparison of mean creatinine measured from sham-operated (Sham, n ⴝ 6), ischemic-reperfusion (IR, n ⴝ 6), and DBcAMP treated (DBcAMP, n ⴝ 6) animals. *P < 0.05 versus sham. #P < 0.05 versus IR. Error bars represent 1 SEM.
POSTISCHEMIC ADENOSINE PROTECTS RENAL FUNCTION
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Fig 5. Representative photomicrographs of outer medulla of the kidney. (A) Sham-treated group. (B) Ischemicreperfusion group. (C) Adenosine-treated group. (D) A2a adenosine receptor (AR) agonist (CGS-21680)–treated group. (E) Dibutyryl cyclic adenosine monophosphate (DBcAMP)–treated group. Arrows indicate proteinaceous casts in tubuli. (H&E, original magnification ⴛ200.)
that acute, short-term administration of adenosine during the early postischemic period potently protects renal function and morphology against IR injury. The present study as well as a previous study from our laboratory16 in which an A2a agonist was given before an ischemic insult allows a more complete interpretation of the results of the two drug delivery protocols of Okusa et al.31 Although initiation of an A2a AR agonist infusion
either 5 hours before ischemia or immediately after reperfusion resulted in renal protection, the Okusa study31 does not support A2a AR–mediated protection occurring during the ischemic phase because drug delivery occurred during ischemia and reperfusion. Results from our previous study suggest that A2a AR activation during the ischemic phase does not afford renal protection. Consistent with the findings of the present study and Okusa et al,31 A2a AR agonists mediate
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Fig 6. Jablonski grading scale scores for histologic appearance of acute tubular necrosis from shamoperated (n ⴝ 6), ischemic-reperfusion (IR, n ⴝ 6), adenosine-treated (ADO, n ⴝ 5), A2a adenosine receptor (AR) agonist–treated (CGS, n ⴝ 5), and dibutyryl cyclic adenosine monophosphate (DBcAMP)–treated (DBcAMP, n ⴝ 4) groups. *P < 0.05 versus sham. #P < 0.05 versus IR. Error bars represent 1 SEM.
protection during the reperfusion phase of the injury process. The present study further extends the understanding of the potential mechanism of A2a AR– mediated renal protection. Classically, renal parenchymal and vascular A2a ARs couple via Gs activation to the stimulation of adenylyl cyclase activity and an elevation of cellular cAMP.6,32 In the present study, administration of a cAMP analogue, dibutyryl cAMP, just before reperfusion mimicked the renal protection afforded by an A2a AR agonist or adenosine. This finding is consistent with an A2a AR 3 Gs protein 3 adenylyl cyclase 3 cAMP pathway mediating renal protection during the reperfusion period. It has been suggested that increased intracellular cAMP protects against renal reoxygenation injury in in vivo and in vitro models, perhaps via modulation of the intracellular rise in calcium and free radical generation during reperfusion.18,21,33 Additionally, it has been proposed that the A2a AR–mediated signaling pathway antagonizes multiple cellular insults responsible for reperfusion injury, including (1) loss of tissue high-energy phosphates during and after the ischemic period; (2) proinflammatory cell (neutrophil and mast cell)–mediated cellular and microvascular injuries via direct cellular toxicity of superoxide free radicals that are generated by these cells during ischemia and subsequent reperfusion; (3) microvascular dysfunction with platelet plugging and endothelial damage, resulting in a no-reflow phenomenon with inadequate tissue
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perfusion during the reperfusion period; and (4) calcium overload–mediated reperfusion injury.2,4,15,29 Tumor necrosis factor-␣, oxygen-mediated free radicals, and neutrophil-mediated cell killing have been implicated in the pathogenesis of acute renal failure.34 Adenosine’s anti-inflammatory actions via A2a AR activation are well characterized.35,36 A2a AR activation is known to inhibit the release and activities of tumor necrosis factor-␣ in cardiomyocytes37,38 and negatively modulates the production of reactive oxygen species and free radicals from activated neutrophils.11,35,36 Adenosine, also via A2a AR activation, attenuates the cytotoxic effects of neutrophils and inhibits platelet-induced vascular dysfunction and occlusions.11,13 Adenosine also protects the endothelial integrity in a reperfused vascular bed.39 At first glance, the renal effects of adenosine infusion appear to be detrimental to renal function because adenosine produces changes that appear to worsen renal function—reduced glomerular filtration rate and afferent cortical blood flow and impaired solute transport.40,41 Several investigators have reported that nonselective adenosine receptor antagonists, such as theophylline, have protective effects against some, but not all, models of ischemic renal failure.41-43 These investigators theorized that AR antagonism would protect against the renal hemodynamic effects of A1 adenosine receptor stimulation (reduction in glomerular filtration rate, solute transport, and renal blood flow via afferent arteriolar vasoconstriction).44 Looking more closely, adenosine has several protective attributes against renal IR injury. Decreases in glomerular filtration rate via reduced cortical blood flow and reduced active solute transport function via tubular glomerular feedback lead to reduced renal oxygen consumption (via A1 AR activation). At the same time, adenosine preferentially increases blood flow and improves oxygenation to the medulla (one of the most susceptible portions of the kidney to hypoxic insult) and reduces the ischemic insult to the tubular cells by vasodilating the vasa recta via A2a AR activation.45,46 Adenosine administration leads to significant improvement in medullary oxygen partial pressure via A2a AR activation.45-47 After renal ischemia, the release and activation of endogenous ARs
POSTISCHEMIC ADENOSINE PROTECTS RENAL FUNCTION
may produce physiologic effects that reduce renal oxygen consumption and improve blood flow to regions that are most susceptible to hypoxic insult (outer border of medulla). In our study, the selective medullary vasodilation of adenosine may have contributed to its renal protective effects. The fact that sodium nitroprusside, a global renal vasodilator, failed to protect renal function after ischemia and reperfusion suggests that increasing total renal blood flow is inadequate for renal protection. The role of A3 ARs in the kidney is not known. We have shown that the selective A3 AR agonist IB-MECA when given before the onset of renal ischemia worsens renal function, and the antagonist MRS-1191 when given before the onset of renal ischemia protects renal function.16 Our previous findings suggest a detrimental role of preischemic A3 AR activation in renal IR injury. In the current study, postischemic treatment with an A3 AR antagonist failed to show protection, indicating that antagonism of A3 ARs must precede the ischemic insult for the protective benefit. In conclusion, we show an in vivo protective effect of postischemic adenosine infusion in the kidney. Treatment with adenosine, an A2a AR agonist, or dibutyryl cAMP protects renal function when given after the renal ischemic period probably via a cAMP-dependent mechanism. These findings may have clinical significance in the protection of the kidney during surgical procedures in which renal ischemia is unavoidable or unpredicted or preischemic optimization of renal function is not possible. ACKNOWLEDGMENT We thank Dr Vivette D’Agati for providing her expertise in renal histopathology.
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