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,2
* Department of Pharmacology and Toxicology and
Department of Anesthesiology and Intensive Care Medicine and
Department of Pathology, Tübingen University Hospital, Tübingen, Germany;
Pharmaceutical Institute, University of Bonn, Bonn, Germany; and
|| Liver and Transplant Centers, Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
2Correspondence: H.K.E., Department of Anesthesiology and Intensive Care Medicine, Tübingen University Hospital, Zentrum für Medizinische Forschung, Waldhörnle Str. 22, D-72072 Tübingen, Germany; E-mail: heltzschig{at}partners.org or H.O., Department of Pharmacology and Toxicology, Tübingen University Hospital, Wilhelmstr. 56, D-72074 Tübingen, Germany; E-mail: hartmut.osswald{at}uni-tuebingen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: acute renal failure • kidney • preconditioning • NTPDase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Several studies of ischemia and reperfusion injury have revealed profound increases in extracellular nucleotide levels with ischemia (3
4
5
6)
. While nucleotides (particularly ATP) are intracellularly present at a concentration of 
2 to 5 mmol/L, cellular injury but also biological stimuli associated with ischemia or inflammation can result in cellular release of nucleotides into the extracellular space, causing extracellular increases in ATP (5
, 7)
. For example, pharmacological and genetic studies of polymorphonuclear granulocytes (PMN) revealed ATP release from the intracellular to the extracellular space via phosphorylation-controlled connexin-43 hemichannels on inflammatory activation (8)
. In addition, platelets are known to release nucleotides upon activation by ADP or collagen via granular release (9)
. Similarly, endothelia were shown to release ATP into the extracellular space during inflammatory stimulation (10)
or during increased sheer stress via granular release (11)
. Other studies of cellular mechanisms of ATP release found extracellular ATP release through the pore-forming purinergic receptor P2 x 7 (12)
.
In the kidney, extracellular ATP and ADP are rapidly hydrolyzed to adenosine due to the presence of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) (13)
. E-NTPDases represent a recently described family of membrane-bound enzymes (14)
, with E-NTPDases1–3 and 8 facing the extracellular milieu. In contrast, NTPDase4–7 are present at the lumen of intracellular organelles (15)
. E-NTPDases are mainlyinvolved in the enzymatic regulation of extracellular nucleotide levels. As such, E-NTPDase1 (CD39) contributes to blocking platelet aggregation via clearing ADP from the blood (16
, 17)
. Moreover, CD39 protein and function are increased during limited oxygen availability (4)
. This is critically important to the regulation of vascular permeability during conditions of limited oxygen availability; cd39–/– mice develop profound vascular edema, particularly within the kidneys, during whole-body hypoxia exposure (4)
. The critical role of CD39 in vascular function is related at least in part to increased extracellular adenosine generation and signaling due to CD39-dependent conversion of extracellular nucleotides (ATP/ADP) to adenosine (4
, 18
, 19)
. In fact, several studies of tissue protection have suggested an anti-inflammatory and tissue protective role of extracellular adenosine, particularly during hypoxia (20
, 21)
. Moreover, studies using a chimeric approach have found renal protection from ischemia and reperfusion by activation of A2A adenosine receptors (A2AAR) on bone marrow-derived blood cells (22
, 23)
. Similarly, a study using a genetic approach in mice gene targeted for the A1 adenosine receptor (A1AR) found decreased necrosis and inflammation mediated by the A1AR during renal ischemia and reperfusion injury, thus confirming tissue protection by extracellular adenosine during renal ischemia (24)
.
Thus, we hypothesized a role for extracellular nucleotide phosphohydrolysis via E-NTPDases in renal protection from ischemia-reperfusion injury. We pursued this hypothesis, combining genetic and pharmacological approaches in a novel model of in situ ischemia-reperfusion injury and renal protection by ischemic preconditioning (IP). This model uses a hanging weight system for intermittently performing occlusion of the renal artery in mice (25
, 26)
. Systematic evaluation of this technique revealed highly reproducible renal injury and protection by IP, thus minimizing the variability associated with earlier techniques [e.g., by applying a surgical clamp to the renal pedicle (vein and artery)] (26)
. These studies revealed a critical role for CD39-dependent nucleotide phosphohydrolysis in renal protection by IP. Consistent with these findings, treatment of renal ischemia with soluble apyrase recapitulated the renal protective effects of IP.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Age-, gender-, and weight-matched littermates were used as controls. In pharmacological studies, age-, gender-, and weight-matched C57BL/6 x 129Svj were used.
Surgery and perioperative management
Animals were kept on a regular 12 h dark-light cycle with free access to standard chow (Altromin 1320, Altromin, Lage, Germany) and tap water. Animals were anesthetized with sodium pentobarbital (70 mg/kg) and placed on a temperature-controlled heating table (RT, Effenberg, Munich, Germany) to maintain body temperature at 37.0°C in a left lateral decubitus position. Right flank incision was performed with a coagulation electrode (Erbe, ICC50, Tübingen, Germany) to prevent bleeding of muscle and skin vessels. The renal pedicle, including the renal artery and renal vein, were ligated using a 4/0 soie suture, and the right kidney was removed without interfering with the adrenal vessels. The surgical wound was closed using contiguous sutures of the muscle wall and skin. Animals were then placed in a right lateral decubitus position and a left flank incision was performed. The left kidney was carefully removed from connective tissues, avoiding the adrenal gland and vessels. Next, the kidney was turned with its ventral side down into a Lucite cup. The kidney was kept wet and warm with a wet swab soaked with water at 37.0°C. After the experimental procedure (ischemia with or without prior IP), the left kidney was turned and sutured back into its retroperitoneal position using 6/0 nylon sutures. The surgical wound was closed as described above. At the end of surgery, mice received 0.3 ml normal saline i.p. and recovered for 2 h under a heating lamp, then were placed into metabolic cages (Tecniplast Deutschland, Hohenpeissenberg, Germany) to determine renal functional parameters.
Technique of renal artery occlusion
Operations were performed under an upright dissecting microscope (Leica, MZ95, Bensheim, Germany). After exposure of the left kidney and placement into a Lucite cup, the renal artery was easily identified, as it runs on top of the renal vein. The vessel was dissected from adjacent tissues, close to its takeoff from the abdominal aorta, then an 8/0 nylon suture (Ethicon, Norderstedt, Germany) was placed around the artery. This technique allows for interruption of only the arterial blood flow to the kidney without compression of the renal vein compared with clamping of the renal pedicle (26)
. The suture was placed over a small pole and a weight of 1 g was attached to each end. When the weights were unsupported, the renal artery was immediately occluded. In contrast, when the weights were supported, blood flow was immediately restored to the organ (26)
. Successful occlusion was confirmed by a change in color from red to white. During reperfusion, changes of color immediately disappeared when the hanging weights were supported and the kidney was reperfused. All animals survived the surgical procedure and no complications were observed.
Preconditioning protocol
IP was performed with four cycles of 4 min ischemia, followed by 4 min reperfusion, each prior to 45 min of ischemia (26)
. In subsets of experiments, a nonspecific inhibitor of E-NTPDases was used. For this purpose, sodium polyoxotungstate, a water-soluble polyoxometalate (POM-1, Na6[H2W12O40]), was used (28)
. The compound was synthesized at the Pharmaceutical Institute, Pharmaceutical Sciences (Bonn, Germany). Mice were treated with POM-1 (5 mg/kg, i.p.) or sterile saline as vehicle control 30 min before the induction of renal ischemia.
Assessment of renal function
Plasma and urine creatinine were measured 24 h after renal ischemia using a commercially available colorimetric method (LT-SYS, Labor+Technik, Berlin, Germany). Plasma and urine concentrations of Na+ and K+ were determined with a flame emission photometer (ELEX 6361, Eppendorf AG, Hamburg, Germany). Renal excretory and hemodynamic values were calculated using standard formulas. Kidneys were harvested after 24 h and stored at –80°C or embedded in paraffin until further analysis.
Inulin clearance
Inulin clearance was measured in cd39–/– mice and littermate controls 24 h after renal ischemia (45 min, with or without prior IP treatment) as described previously (29
, 30)
. In contrast to other studies of renal protection, a right nephrectomy was performed immediately before measuring inulin clearance. Briefly, mice were anesthetized using 50 mg/kg i.p. pentobarbital. Animals were then placed on a temperature-controlled operating table to keep rectal temperature at 37°C. Tracheotomy was performed as described previously (31)
and the right jugular vein was cannulated for continuous infusion. Blood samples were taken via a catheter inserted into the left femoral artery. A catheter was placed in the urinary bladder for timed urine collection after removal of the right kidney. After surgery, all mice received a bolus of 0.85% sodium chloride solution in an amount equal to 20% of body weight. Continuous infusion was maintained at a rate of 600 µl/h/30 g body weight and 3H-inulin was added to the infusion to evaluate whole-kidney glomerular filtration rate (GFR). After stabilization of the animals for 20 min, 20 min-timed urine collections were measured to determine urinary flow rate and 3H-inulin. Blood was obtained in the middle of every period to measure 3H-inulin. The concentration of 3H-inulin in plasma and urine was measured by liquid phase scintillation counting, and GFR was calculated by a standard formula.
Histology and immunohistochemistry
Renal tissues were fixed in 4.5% buffered formalin, dehydrated, and embedded in paraffin. Sections (3 µm) were stained with hematoxylin/eosin and periodic acid Schiff. Examination and scoring of three representative sections of each kidney (n=4 to 6 for each condition) were carried out by a board-certified renal pathologist blinded to the experimental group. A grading scale of 0–4, as outlined by Jablonski et al., was used for histopathological assessment of proximal tubular damage (32)
.
Real-time PCR
To assess the influence of IP on renal E-NTPDase1, 2, 3, and 8 transcript levels, we used real-time reverse-transcriptase polymerase chain reaction (RT-PCR, iCycler; Bio-Rad Laboratories Inc., Hercules, CA, USA). For this purpose, total RNA was isolated from kidney tissue using the total RNA isolation NucleoSpin RNA II Kit (Macherey & Nagel, Düren, Germany) as described (26)
. Primer sets (sense sequence, antisense sequence, and transcript size, respectively) for the following genes were E-NTPDase1 (5'-TAC CAC CCC ATC TGG TCA TT-3', 5'-GGA CGT TTT GTT TGG TTG GT-3', 168 bp); E-NTPDase2 (5'-ATG CGC CTA CTC AAC CTG AC-3', 5'-GCC TAC CCA GCC ATA CTT GA-3', 189 bp); E-NTPDase3 (5'-GCT TCT CCC TTA CCT CCT TCA-3', 5' GCT GTT CCC CAC TTC TTT TTC-3', 212 bp); E-NTPDase8 (5'-TGT AAG GGC CAG AAG GAT TG-3', 5'-CAG ACC CGA GGC ACA GTA GT-3', 237 bp). Each target sequence was amplified using increasing numbers of cycles of 94°C for 1 min, 58°C for 0.5 min, and 72°C for 1 min. Murine ß-actin mRNA (sense primer, 5'-CTC TCC CTC ACG CCA TCC TG-3' and antisense primer, 5'-TCA CGC ACG ATT TCC CTC TCA G-3') was amplified in identical reactions to control for the amount of starting template.
Immunoblotting experiments
Kidney tissue was homogenized and lysed for 10 min in ice-cold lysis buffer (150 mM NaCl, 25 mM Tris, pH 8.0, 5 mM EDTA, 2% Triton X-100, and 10% mammalian tissue protease inhibitor cocktail; Sigma-Aldrich). After spinning at 14,000 g to remove cell debris, the pellet was resuspended in reducing Laemmli sample buffer and heated to 90°C for 5 min. Samples were resolved on a 12% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in PBS supplemented with 0.2% Tween 20 and 4% BSA. CD39 was detected with 10 µg/ml rabbit anti-CD39 antibody (Santa Cruz, Santa Cruz, CA, USA) and 1:3000 HRP-goat anti-rabbit Ig (Perbio Science, Bonn, Germany) using standard methods and enhanced chemiluminescence (Applied Biosystems, Foster City, CA, USA). To control for protein loading, blots were stripped and reprobed for ß-actin using murine anti-human ß-actin antibody (Abcam Inc., Cambridge, UK).
E-NTPDase1/CD39 immunohistochemistry
Mice were euthanized at the indicated time points, and the kidneys were removed and fixed in Tissue-Tek (Sakura, Japan) for 24 h. Cryostat sections were mounted on glass slides, air dried, and postfixed in acetone/methanol (1:1) for 10 min at room temperature. Sections were blocked by incubation in 5% powdered skimmed milk and 0.1% Triton-X-100 (Sigma, Munich, Germany) in Tris-buffered saline (TBS) for 30 min. Sections were incubated for 1 h at room temperature in rabbit anti-CD39 antibody (1:200; Santa Cruz). After three washes with TBS, sections were incubated for 45 min with Cy3 goat anti-rabbit Ig (Amersham Pharmacia, Freiburg, Germany) at room temperature. Fluorescence was visualized with a confocal laser scanning microscope (Leica, Wetzlar, Germany).
Adenosine measurements
Whole kidneys from cd39–/– mice or age-, gender-, and weight-matched littermates were removed and immediately snap-frozen with clamps precooled to the temperature of liquid nitrogen with or without prior IP treatment (4 cycles consisting of 4 min of ischemia and 4 min of reperfusion). The frozen kidneys were pulverized under liquid nitrogen and tissue protein was precipitated with 0.6 N ice-cold perchloric acid. Tissue adenosine levels were determined via HPLC as described previously (33)
.
Data analysis
Renal injury score data are given as median (range); all other data are presented as mean ± SD from 6–8 animals per condition. Renal hemodynamics in cd39–/– mice and controls with or without drug application were analyzed by 2-way ANOVA, followed by Bonferroni correction for multiple comparisons. Renal mRNA induction after IP was evaluated by 1-way ANOVA. Renal injury was analyzed with the Kruskal-Wallis rank test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For this purpose, mice on the C57BL/6 x 129Svj strain were subjected to 45 min of renal artery occlusion with or without prior IP (4 cycles, 4 min ischemia, 4 min reperfusion), followed by 24 h of reperfusion, and assessed for renal hemodynamics using a recently described model of in situ preconditioning via a hanging weight system for renal artery occlusion (26)
. Consistent with earlier studies of renal IP (26)
, plasma creatinine (Fig. 1
A) and potassium (Fig. 1B
), creatinine clearance (Fig. 1C
), urinary flow rate (Fig. 1D
), urinary excretion of sodium (Fig. 1E
), and potassium (Fig. 1F
) were improved by IP. To determine the contribution of E-NTPDases to renal protection by IP, mice were treated with POM-1 (5 mg/kg, i.p.) or vehicle control. Consistent with our hypothesis, POM-1 treatment abolished the renal protective effects of IP (Fig. 1A-F
). Plasma creatinine (Fig. 1A
) and potassium (Fig. 1B
), creatinine clearance (Fig. 1C
), urinary flow rate (Fig. 1D
), urinary excretion of sodium (Fig. 1E
), potassium (Fig. 1F
), and GFR (Fig. 1G
) were not improved by IP in POM-1-treated mice. Taken together, these studies reveal for the first time an inhibition of renal protection by IP associated with E-NTPDase inhibition, suggesting a critical role for extracellular nucleotide phosphohydrolysis in renal protection from ischemia and reperfusion injury.
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Increases in renal adenosine content with IP are attenuated following POM-1
After showing inhibition of renal protection by IP after nonspecific inhibition of E-NTPDases, we were curious to see whether POM-1 treatment affects renal adenosine generation during IP. For this purpose, we determined renal adenosine tissue levels in kidneys shock frozen immediately after IP treatment (4 cycles, 4 min of ischemia, and 4 min reperfusion). As shown in Fig. 2
, renal adenosine levels were 9-fold higher after renal IP. To demonstrate an effect of ENTPDases for increasing tissue adenosine levels during IP, we repeated this experiment in mice that were treated with POM-1 (5 mg/kg, i.p.) prior to IP. While basal adenosine levels were lower in POM-1-treated mice, increases in extracellular adenosine with IP were attenuated. Taken together, these studies demonstrate that E-NTPDases are critical for increasing renal adenosine levels during IP.
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Renal E-NTPDase1 (CD39) is selectively induced by renal IP
Having shown that pharmacological inhibition of NTPDases results in attenuated adenosine production during IP and abolished renal protection, we sought to determine the contribution of individual E-NTDPases (E-NTPDase1–3 and 8) to renal protection. For this purpose, we studied transcriptional responses of renal E-NTPDase expression patterns to IP. We used a previously described IP model with four cycles of intermittent renal artery occlusion and reperfusion (4 min ischemia, 4 min reperfusion) prior to 45 min of ischemia by using a hanging weight system for isolated renal artery occlusion (Fig. 3
A) (26)
; preconditioned renal tissues were snap-frozen at indicated time points and investigated by real-time RT-PCR for renal E-NTPDase expression. Baseline expressional levels relative to ß-actin mRNA revealed the lowest expressional rates of E-NTPDase3, followed by 8 and 2, with highest expression of E-NTPDase1 (data are not shown). In addition, we found a robust and selective induction of E-NTPDase1 (CD39) mRNA (e.g., 90 min after renal IP, 2.9±0.5-fold, P<0.01, Fig. 3B
). Furthermore, Western blot analysis (Fig. 3C
) and immunohistological staining and imaging via confocal laser scanning microscopy (Fig. 3D
) demonstrated an induction of CD39 protein 60 and 120 min after IP. Staining of wild-type tissue with a secondary antibody alone revealed no signal (data not shown). These findings indicate a selective and robust induction of E-NTPDase1/CD39 by renal IP.
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Renal protection by IP is abolished in cd39–/– mice
After having shown that nonselective inhibition of E-NTPDases abolishes renal protection by IP whereas E-NTPDase1 is selectively induced by IP, we next considered a functional contribution of CD39 to renal protection by IP. Although there is no selective CD39 inhibitor available at this time, we pursued this question using mice gene targeted for cd39 (27)
. We performed IP in cd39–/– mice and corresponding littermate controls matched in age, gender, and weight using 45 min of renal artery occlusion with or without prior IP (4 cycles, 4 min ischemia, 4 min reperfusion), followed by 24 h of reperfusion. While measurements of plasma creatinine (Fig. 4
A) and potassium (Fig. 4B
), creatinine clearance (Fig. 4C
), urinary flow rate (Fig. 4D
), urinary excretion of sodium (Fig. 4E
) and potassium (Fig. 4F
), and GFR (Fig. 4G
) were dramatically improved by IP in littermate controls, no renal protection by IP was noted in cd39–/– mice. Taken together, these studies reveal for the first time a lack of renal protection by IP in cd39–/– mice.
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Histological attenuation of acute tubular necrosis by IP before ischemia is abolished in cd39–/– mice
To evaluate the consequences of renal ischemia and preconditioning on a morphological level, we next performed a histological examination of renal tissues from cd39–/– and littermate controls with or without IP prior to 45 min of ischemia. Forty-five minutes of renal ischemia in littermate controls without IP (Fig. 5
A) was associated with severe acute tubular necrosis, including loss of tubular cell nuclei in the cortex and outer medullary stripe, with almost complete destruction of the proximal tubular brush border. Casts containing brush border blebs, hyaline cast formation, and intraluminal necrotic cellular debris were observed in the outer medulla. As predicted, kidneys of littermate controls subjected to four cycles of IP prior to 45 min of renal ischemia showed only mild histological signs of acute tubular necrosis (Fig. 5B
). The proximal tubular brush border damage occurs sporadically and was quantitatively mild; only small numbers of hyaline casts were apparent. Histological examination of renal tissues from cd39–/– mice after 45 min of ischemia represented a histological picture similar to that of littermate controls (see above, Fig. 5C
). In striking contrast to littermates, however, renal IP in cd39–/– mice was not associated with an attenuation of histological damage. In fact, we again observed histological signs of severe acute tubular necrosis, including loss of tubular cell nuclei in the cortex and outer medullary stripe, with complete destruction of the proximal tubular brush border and casts containing brush border blebs, hyaline cast formation, and intraluminal necrotic cellular debris in the outer medulla (Fig. 5D
). To quantify these findings, a board-certified renal pathologist who was blinded to the experimental conditions performed semiquantitative histological analysis of renal tissues. Consistent with the above findings, histological scoring confirmed severe acute tubular necrosis with 45 min of renal ischemia alone (Jablonski index of 3, range 2 to 4). IP treatment was associated with a significant attenuation of tubular destruction (Jablonski index of 1.5, range 1 to 3, P<0.05, Fig. 5E
). Taken together, these data provide genetic evidence for CD39-dependent renal protection by IP on a functional and histological level.
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Increases in renal adenosine content with IP are attenuated in cd39–/– mice
Based on the above findings of abolished renal protection by IP after targeted gene deletion of E-NTPDase1/CD39 and on previous studies showing a critical role of extracellular adenosine in renal protection from ischemia and reperfusion (22
23
24)
, we hypothesized that increases in renal adenosine with IP are attenuated in cd39–/– mice. For this purpose, we determined renal adenosine tissue levels in kidneys shock frozen immediately after IP treatment (4 cycles, 4 min of ischemia, and 4 min reperfusion). As shown in Fig. 6
, renal adenosine levels were 9-fold higher after renal IP compared with baseline values. To demonstrate that CD39 is important for increasing tissue adenosine levels during IP, we repeated this experiment in cd39–/– mice. While basal adenosine levels were lower in cd39–/– mice, increases in extracellular adenosine with IP were dramatically attenuated compared with littermate controls. Taken together, these studies demonstrate that E-NTPDase1/CD39 is critical for increasing renal adenosine levels during IP.
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Improvement of renal function in cd39–/– mice is reconstituted by apyrase treatment
As proof of principle and to demonstrate that the absence of renal protection by IP in cd39–/– mice is in fact related to a lack of extracellular nucleotide phosphohydrolysis, we next reconstituted cd39–/– mice using soluble apyrase derived from potatoes (5 U of apyrase, i.p.). As shown in Fig. 7
A, B, increases in serum creatinine and serum potassium were reduced after apyrase treatment even without IP. Similarly, mice with apyrase treatment showed an increase in creatinine clearance (Fig. 7C
), urinary flow rate (Fig. 7D
), and urinary excretion of sodium (Fig. 7E
) and potassium (Fig. 7F
) in cd39–/– mice with and without IP. Taken together, these results confirm our genetic studies that CD39 plays a crucial role in increasing renal resistance to ischemia after IP.
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Apyrase treatment mimics the protective IP effect in wild-type mice
After having shown that renal protection by IP can be restored in cd39–/– mice by treatment with apyrase, we next pursued application of apyrase in wild-type mice. As shown in Fig. 8
A–F, apyrase treatment provided a degree of renal protection similar to that of IP. Taken together, these data demonstrate for the first time a therapeutic effect of treatment with apyrase in renal ischemia and suggest it as a novel therapeutic tool during acute renal ischemia.
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Apyrase treatment increases renal adenosine
After having shown that treatment of renal ischemia with apyrase offers a degree of renal protection similar to that from IP treatment, we hypothesized that such treatment results in adenosine increases similar to those in renal IP. Therefore, we determined renal adenosine tissue levels in kidneys shock frozen immediately after IP treatment (4 cycles, 4 min of ischemia, and 4 min reperfusion) with or without prior apyrase therapy (5 µ of apyrase). As shown in Fig. 9
, apyrase treatment resulted in a similar elevation of renal adenosine as IP treatment itself. Taken together, these studies demonstrate increases in renal adenosine by apyrase treatment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These studies revealed complete inhibition of renal protection from ischemia by preconditioning. To gain insight into the contribution of individual E-NTPDases, we next performed transcriptional profiling of preconditioned renal tissue. These studies revealed a selective and prominent induction of E-NTPDase1 (CD39) mRNA. Western blot analysis and confocal laser scanning confirmed these findings on a protein level. Consistent with these studies, targeted gene deletion of cd39 abolished the renal protective effects of in situ IP; renal increases of adenosine concentrations were attenuated in these mice. Moreover, soluble apyrase treatment successfully reconstituted cd39–/– mice and mimicked renal protection by IP in wild-type. In fact, this pharmacological regimen was associated with an increase in renal resistance to ischemia similar to that from renal IP treatment. Taken together, these studies reveal E-NTPDase1/CD39 in innate protection from renal ischemia, and suggest pharmacological strategies of increasing extracellular nucleotide phospho-hydrolysis in treating acute ischemic injury of the kidneys.
The induction of renal CD39 by IP is consistent with earlier studies showing that exposure of endothelia to ambient hypoxia (2% oxygen) resulted in a robust induction of CD39 transcript, protein, and function (4)
. Similarly, studies in cd39–/– mice demonstrated an increased vascular leak syndrome upon hypoxia exposure (8% oxygen over 4 h) (4)
. Conceptually, increases in extracellular nucleotide phosphohydrolysis during hypoxia result in a decrease of extracellular ATP/ADP levels in conjunction with increased extracellular AMP. Because the E-NTPDase end product AMP serves as metabolic substrate for ecto-5'-nucleotidase (CD73) -dependent generation of extracellular adenosine, it is not surprising that increases of renal adenosine with IP are attenuated in cd39–/– mice. Moreover, cd73–/– mice develop profound vascular leakage, acute pulmonary edema, and increased neutrophil tissue accumulation when exposed to ambient hypoxia, similar to cd39–/– mice (18
, 34)
. In view of these findings and the fact that other studies have provided genetic evidence for renal protection from ischemia by extracellular adenosine signaling (22
23
24)
, it seems more likely that the renal protective effects observed in the present study with E-NTPDase1/CD39 are related to increases in extracellular adenosine generation rather than to decreased extracellular nucleotide signaling effects. In addition, a recent study using an in vitro model of renal ischemia and reperfusion found that ATP or hydrolysis-resistant ATP analog (ATP-gamma-S or 2-methylthio-ATP) appears to be protective during ischemic injury-induced activation of nuclear factor NF-
B activation (35)
. In fact, this study suggests a protective effect of ATP signaling in renal ischemia related to inhibition of NF-B activation via P2Y activation. Thus, a CD39-dependent reduction of extracellular ATP levels would not explain a protective effect of extracellular nucleotide metabolism in renal ischemia. Other studies have found a protective role of CD39 in attenuating platelet aggregation. For example, a carefully executed study on the role of CD39 in ischemic brain found increased cerebral infarct volumes and reduced postischemic perfusion in cd39–/– mice (16)
. In addition, pharmacological treatment with soluble CD39 reconstituted these mice, restoring postischemic cerebral perfusion and rescuing them from cerebral injury, revealing a protective thromboregulatory role of CD39 in stroke. In contrast, the cd39–/– mice used in the present study have a prolonged bleeding time and dysregulation of platelet function. These findings are most likely related to a desensitization of the purinergic platelet P2Y1 receptor (27)
. Taken together, these studies and results from the present study strongly suggest that abolished renal protection by IP in cd39–/– mice is most likely related to attenuated renal adenosine generation and signaling.
Although extracellular adenosine generation and signaling have been shown to modulate tubulo-glomerular feedback mechanism in the kidney via the A1AR (3
, 30
, 36
37
38
39)
, it remains unclear which ARs mediate renal protection by IP. Studies in other organs have found tissue protection during hypoxia or inflammation through signaling pathways involving the A2AAR (18
, 21
, 40)
. For example, a thorough study found extensive tissue damage, prolonged and higher levels of proinflammatory cytokines, and death in A2AAR–/– mice exposed to an inflammatory stimulus that caused minimal tissue damage in control mice. Similar observations were made using other models of inflammation, liver damage, or bacterial endotoxin-induced septic shock (41)
. Moreover, elegantly performed studies of renal ischemia and reperfusion in chimeric mice found that bone marrow-derived cells play an important role in A2AAR-mediated tissue protection (22
, 23
, 42)
. Further work using adoptive transfers into Rag-1–/– mice revealed that IFN
produced by CD4+ T cells appears to be an important mediator for this complex interplay (22)
. Other studies have indicated a critical role of signaling through the A2BAR in the resealing of endothelia during transendothelial migration of neutrophils (43)
, particularly during conditions of limited oxygen availability (4)
. Moreover, pharmacological inhibition of the A2BAR during hypoxia exposure was associated with increased pulmonary edema and vascular leakage (18)
.
In summary, the present study demonstrates protection during renal ischemia via CD39-dependent nucleotide metabolism. Gene-targeted mice lacking the major extracellular pathway of ATP/ADP phosphohydrolysis in the kidney (CD39) are not protected from renal ischemia by IP. The significant attenuation of renal injury after ischemia by apyrase treatment suggests possible new strategies to ameliorate the consequences of renal hypoxia. Future challenges include the identification of cell types responsible for extracellular nucleotide phosphohydrolysis (e.g., by tissue-specific deletion of cd39), thus allowing the delivery of adenosine-precursor molecules (AMP) to specific anatomic sites.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication February 17, 2007. Accepted for publication March 15, 2007.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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