A2A adenosine receptor-mediated inhibition of renal injury and neutrophil adhesion

Mark D. Okusa1, Joel Linden1,2, Liping Huang1, Jayson M. Rieger3, Timothy L. Macdonald3, and Long P. Huynh1

Departments of 1 Medicine, 2 Molecular Physiology and Biological Physics, and 3 Chemistry, University of Virginia, Charlottesville, Virginia 22908


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We sought to determine the mechanisms responsible for the reduced renal tissue injury by agonists of A2A adenosine receptors (A2A-ARs) in models of ischemia-reperfusion (I/R) injury. DWH-146e, a selective A2A-AR agonist, was administered subcutaneously to Sprague-Dawley rats and C57BL/6 mice via osmotic minipumps, and animals were subjected to I/R. I/R led to an increase in plasma creatinine and kidney neutrophil infiltration. Infusion of DWH-146e at 10 ng · kg-1 · min-1 produced a 70% reduction in plasma creatinine as well as a decrease in neutrophil density in outer medulla and cortex and myeloperoxidase activity in the reperfused kidney. Myeloperoxidase activity in kidney correlated with the degree of renal injury. P-selectin and intercellular adhesion molecule 1 (ICAM-1) immunoreactivity were most prominent in endothelial cells of peritubular capillaries and interlobular arteries of cortex and outer and inner medulla of vehicle-treated mice whose kidneys were subjected to I/R. DWH-146e treatment led to a pronounced decrease in P-selectin- and ICAM-1-like immunoreactivity. These data are consistent with our hypothesis that A2A-AR agonists limit I/R injury due to an inhibitory effect on neutrophil adhesion.

acute renal failure; neutrophil-endothelial cell interaction; intercellualr adhesion molecule 1; P-selectin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PRESENT STRATEGIES IN THE treatment of acute renal failure have focused on targeting individual mechanisms thought to contribute to ischemia/reperfusion (I/R) injury in kidney (1, 36). This approach is confounded by redundancies in the cascade of I/R injury triggered by multiple cytokines and adhesion molecules. We adopted another strategy to more broadly attenuate inflammatory cascades thought to play a role in I/R injury. Using a novel selective A2A-adenosine receptor (A2A-AR) agonist, DWH-146e, we previously demonstrated that selective activation of A2A-ARs produced a dramatic reduction in renal injury following I/R in rats (26). Renal protection was observed when DWH-146e was started either before or after the ischemic episode and maintained throughout the reperfusion period. Although A2A-AR stimulation can produce vasodilation, the dose that was used in our prior study was far below the threshold for vasodilation. Neither blood pressure nor heart rate was affected when DWH-146e was administered systemically suggesting that other nonhemodynamic factors reduce renal injury.

A physiological role for endogenous adenosine in inflammation became apparent after the demonstration that activated neutrophils or endothelial cells release and respond to adenosine (3, 10, 11, 15). The anti-inflammatory effects of adenosine appear to be mediated by A2A-ARs, one of four subtypes of the G protein-coupled adenosine receptor family that includes A1-, A2A-, A2B-, and A3-ARs (27). Although activation of A2A-ARs expressed on activated neutrophils (12) reduces the release of reactive oxygen metabolites, (10, 12, 31, 33, 34) adenosine may also interfere with neutrophil adherence to endothelial cells (7). Although experimental evidence strongly supports a critical role of adhesion molecules in I/R injury (for review, see 24, 30), the effect of A2A-ARs on the expression of specific adhesion molecules has not been investigated. In this study we sought to determine how activation of A2A-ARs influences neutrophil adherence and affects specific adhesion molecule expression in the setting of renal I/R injury. The results indicate that A2A agonists reduce inflammation in part by reducing adhesion molecule expression and neutrophil adherence to endothelial cells.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Surgery and experimental protocol. C57BL/6 mice (7-8 wk of age, 20-29 g, Hilltop Laboratory Animals, Scottdale, PA) were subjected to bilateral flank incisions (better tolerated than abdominal incisions in mice) under anesthesia with a regimen that consisted of ketamine (100 mg/kg, ip), xylazine (10 mg/kg, ip), and acepromazine (1 mg/kg, im). Both renal pedicles were identified and cross clamped for 27-32 min depending on the experimental protocol. For experiments in rats, surgical details have been described previously (26). The abdomens of adult male Sprague-Dawley rats (200-280 g; Hilltop Laboratory Animals) were opened with a midline incision. The right renal artery and vein were ligated, and the right kidney was removed. After the left renal artery and vein were cross clamped for 45 min, the clamp was released, and the kidney was observed for immediate reperfusion. Alzet osmotic minipumps (model 1003D; Alza, Palo Alto, CA) containing vehicle or DWH-146e were inserted subcutaneously. Surgical wounds were closed, and mice or rats were returned to cages for 24 or 48 h. After reperfusion, animals were reanesthetized, blood was obtained by cardiac puncture, and kidneys were removed for various analyses.

A solution containing DWH-146e was prepared in phosphate-buffered saline containing <0.01% DMSO and placed in osmotic minipumps that were implanted subcutaneously 5 h before reperfusion under brief vaporized halothane anesthesia (Halothan Vapor 19.1). Our previous studies demonstrated similar degrees of renal protection when DWH-146e was initiated 5 h before ischemia or immediately after the onset of reperfusion (26). In some experiments mice received DWH-146e+ZM-241385, a selective A2A antagonist (7 ng · kg-1 · min-1, an amount that was calculated to be a molar equivalent to delivered amount of DWH-146e). Table 1 summarizes the experimental protocols.

                              
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Table 1.   Control and experimental groups

Plasma creatinine. Plasma creatinine concentration was determined by using a colorimetric assay according to the manufacturer's protocol (Sigma, St. Louis, MO).

Neutrophil infiltration. Neutrophil infiltration was assessed by two independent methods: 1) histological identification of neutrophils and 2) biochemical assay for myeloperoxidase (MPO), an enzyme present in neutrophils. For histological studies, kidneys were embedded in paraffin after fixation with periodate-lysine-paraformaldehyde modified to contain 4% paraformaldehyde (PLP/4%) (17), and 4-µm sections were selectively stained for neutrophils by using napthol AS-D chloroacetate esterase according to the manufacturer's protocol (Sigma) (8, 21, 37). Esterases present on polymorphoneutrophils (PMNs) utilize napthol AS-D chloroacetate as a substrate; the product stains the PMNs red. Other hematopoietic cell lineages contain cell esterases that do not utilize naphthol AS-D as a substrate and therefore will not stain red in tissue sections. This method has been previously utilized successfully in kidney tissue to detect neutrophils (8, 21, 37). To further confirm the specificity of this methodology for staining neutrophils we demonstrated that only PMNs were stained and not monocytes or lymphocytes in peripheral blood smears. The fixation technique was found to be an important determinant of PMN staining. Bouin's solution or high-percentage formaldehyde (10%) reduces staining, and PLP enhances staining. Given these results we have chosen fixation with PLP/4%, a condition that maximizes staining intensity while maintaining tissue preservation.

In some experiments, kidney sections were examined by using a Leitz microscope fitted with a Ludl motor-driven stage and integrated with the Neurolucida software as described in detail (32). The perimeter of the kidney regions was drawn. An optical frame visible through the microscope objective was overlaid on the tissue section, which was viewed at ×250 magnification. The microscope stage was programmed to move one frame at a time without overlapping. To prevent duplicate counting, neutrophils within each frame were marked with a symbol and counted. This software calculates the area within the closed contour and maintains a running tally of PMNs counted. Density of neutrophils was given as the total number of neutrophils in a 4-µm coronal cross section (neutrophils/mm2). A regional map of neutrophil density was generated. Schematic drawings were processed further and printed by using Canvas graphics software (Deneba Software, Miami, FL).

MPO activity was determined from kidney homogenates. Kidneys were homogenized in 10 vol of ice-cold 50 mM potassium phosphate buffer, pH 7.4, using a Tekmar tissue grinder. The homogenate was centrifuged at 15,000 g for 15 min at 4°C, and the resultant supernatant was discarded. The pellet was washed twice, resuspended in 10 vol ice-cold 50 mM potassium phosphate buffer with 0.5% hexadecyltrimethylammonium bromide, and sonicated. The suspension was subjected to freeze/thaw three times, sonicated for 10 s, and centrifuged at 15,000 g for 15 min at 4°C. The supernatant was added to an equal volume of a solution consisting of o-dianisidine (10 mg/ml), 0.3% H2O2, and 45 mM potassium phosphate, pH 6.0. Absorbance was measured at 460 nm over a period of 5 min (6).

Immunohistochemistry. Kidneys were harvested from mice, fixed in PLP/4%, and embedded in paraffin. Four-micrometer sections were subjected to immunohistochemistry by using methods previously described (17, 25). We used well-characterized monoclonal antibodies to intercellular adhesion molecule 1 (ICAM-1; YN1.1) (28) and P-selectin (RB40.4) (4). In preliminary studies we tested the ability of the antibodies to detect the expression of ICAM-1 and P-selectin in mouse kidneys by using a well-known stimulus of adhesion molecule expression, lipopolysaccharide (LPS) (2). LPS (3 mg/kg ip) was injected, and kidneys were harvested at 2, 4, and 6 h after injection and placed in PLP/4%. Additional studies were performed to detect the expression of ICAM-1 and P-selectin in mice subjected to I/R injury and treated with DWH-146e or vehicle. For I/R injury experiments, kidneys were harvested 6 h post-I/R. Kidneys were embedded in paraffin after fixation with PLP/4%, and 4-µm sections were subjected to antigen retrieval according to the manufacturer's protocol (Vector Laboratories, Burlingame, CA). Sections were incubated with primary antibody (1:1,000 dilution) followed by a biotinylated goat anti-rat secondary antibody. Peroxidase reaction was performed according to the manufacture's protocol (Vectastain ABC Elite kit), and reaction times for sections from control/sham and experimental animals were identical.

Statistical analysis. The randomized block design was used to analyze the data. In this design, we considered the day of the procedure as a block factor. Analysis of variance for the randomized block design and post hoc analysis (Bonferroni or Dunnett's) were performed. In some analyses paired and unpaired t-tests were used. P < 0.05 was used to determine significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DWH-146e reduces plasma creatinine after I/R injury. Previously, we demonstrated that selective A2A activation in rats reduced the rise in plasma creatinine after I/R injury. Taking advantage of the vast array of reagents available for mouse studies, we have now developed a mouse model of I/R injury to begin to determine the mechanisms that participate in the protective role of A2A-AR agonists. Our initial effort was to determine whether a similar degree of protection was seen with DWH-146e after I/R in mice as we have observed previously in rats. We administered vehicle or DWH-146e (10 ng · kg-1 · min-1) via osmotic minipumps beginning 5 h before 27 min of ischemia and continuing through a period of 24 or 48 h of reperfusion (see Table 1 for description of experimental groups). As shown in Fig. 1, a progressive rise in creatinine was observed in vehicle-treated mice after reperfusion for 24 (group 1) and 48 h (group 2). DWH-146e significantly reduced the rise in creatinine in five of five mice at 24 h (group 3) and seven of seven at 48 h (group 4). Plasma creatinine was 0.65 ± 0.09 and 0.48 ± 0.08 mg/dl (n = 5; P < 0.05) at 24 h and 1.16 ± 0.26 and 0.48 ± 0.08 mg/dl (n = 7; P < 0.05) at 48 h for vehicle- and DWH-146e-treated mice, respectively. The percent reduction in plasma creatinine of ~60% was similar to the ~80% reduction observed previously in rats (26).


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Fig. 1.   Pretreatment with DWH-146e improves renal function after ischemia-reperfusion (I/R) injury in mice. Values are means ± SE. Mouse kidneys were subjected to 27-min ischemia and 24 (groups 1 and 3) or 48 h (groups 2 and 4) of reperfusion. DWH-146e (10 ng · kg-1 · min-1) or vehicle was administered continuously via minipumps beginning 5 h before I/R. DWH-146e significantly decreased plasma creatinine in 5/5 mice (P < 0.05) at 24 h and 7/7 mice (P < 0.05) at 48 h. *P < 0.05.

DWH-146e reduces neutrophil infiltration in kidney tissue. Although A2A-agonist infusion reduces renal injury, the mechanism of protection is not known. Because the low doses of A2A agonists used produced no hemodynamic effects (26) and because of the potent anti-inflammatory characteristics of A2A activation, we sought to determine whether A2A agonists reduced renal inflammation. Toward this end we determined whether A2A-agonist infusion reduces neutrophil infiltration in kidney tissue after I/R. By using MPO activity as a measure of neutrophil number in kidney, we found that I/R injury produced an increase in MPO activity in mice after 48 h of reperfusion. DWH-146e reduced kidney MPO activity; MPO activity was 0.84 ± 0.09 and 0.25 ± 0.4 OD460 · g-1 · min-1 at 48 h in vehicle (group 2)- and DWH-146e-treated mice (group 4), respectively (n = 7; P < 0.001); (Fig. 2A), where OD460 is 460-nm optical density. Similar results were noted at 24 h; MPO activity was 1.001 ± 0.091 (n = 4) and 0.498 ± 0.049 OD460 · g-1 · min-1 (n = 5) for vehicle and DWH-146e, respectively (P < 0.001). As shown in Fig. 2B, the degree of injury as assessed by plasma creatinine correlated directly with MPO activity (r2 = 0.73, P < 0.0001).


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Fig. 2.   A2A-adenosine receptor (A2A-AR) activation reduces myeloperoxidase (MPO) activity in mice subjected to I/R. Mice were subjected to 27-min ischemia followed by 24 or 48 h reperfusion. DWH-146e (10 ng · kg-1 · min-1) was administered continuously beginning 5 h before. A: MPO activity (assessed as OD460 · g-1 · min-1, where OD460 is 460-nm optical density) from kidneys of mice treated with vehicle (group 1) or DWH-146e (group 3) (P < 0.005) and perfused for 24 h, or 48 h with vehicle (group 2) or DWH-146e (group 4) (P < 0.001). B: MPO activity was correlated with plasma creatinine (, group 2; black-triangle, group 4; r2 = 0.73, P < 0.0001).

Histological examination of kidney sections from vehicle-treated mice revealed scattered neutrophils throughout the cortex including within glomeruli (Fig. 3A). Dense infiltration of neutrophils was noted in peritubular capillaries primarily in the outer medulla (Fig. 3B). In the inner medulla red cell congestion was apparent (Fig. 3C). It is possible that red cells are trapped in capillaries due to occlusion of postcapillary venules by adherent neutrophils. In DWH-146e-treated mice there was a pronounced decline in neutrophil accumulation throughout the kidney (Fig. 3, D and E), and the inner medulla showed a marked reduction in red cell congestion (Fig. 3F).


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Fig. 3.   DWH-146e reduces dense neutrophil infiltration. Mice were subjected to 27 min of ischemia followed by 48 h of reperfusion. DWH-146e (10 ng · kg-1 · min-1) was administered continuously beginning 5 h before I/R. A-C: vehicle-treated (group 2). D-F: DWH-146e-treated (group 4). A and D, cortex; B and E, outer medulla; C and F, inner medulla. Neutrophils are stained red and appear in peritubular capillaries in A and B (arrowheads). Inner medulla demonstrates vascular congestion (arrowhead) in C. g, Glomerulus. Figure shows a representative example of 5 experiments.

Similar results of DWH-146e infusion on neutrophil infiltration were observed in rats. In rats subjected to I/R, the effect of DWH-146e on reducing MPO activity was blocked by the selective A2A antagonist ZM-243185. Compared with MPO activity in vehicle-treated rats (2.72 ± 0.30 OD460 · g-1 · min-1), DWH-146e reduced MPO activity in five of five rats by 33% (1.81 ± 0.21 OD460 · g-1 · min-1; P < 0.5; Dunnett's t-test). Coinfusion of DWH-146e+ZM-243185 led to a 15% reduction in MPO activity, a difference not significantly different from vehicle treatment (n = 5; 2.29 ± 0.087 OD460 · g-1 · min-1). Furthermore, as was the case with mice, the degree of injury correlated with the degree of neutrophil infiltration (r2 = 0.94; P < 0.0001). In three vehicle- and three DWH-146e-treated rats we quantified the degree of neutrophil accumulation (Table 2) and mapped the location of neutrophils in kidney (Fig. 4). As shown in Fig. 4, neutrophil infiltration occurs primarily in the outer medulla and to a lesser degree in the cortex after I/R injury, and DWH-146e treatment results in a profound reduction in neutrophil accumulation. Our findings in rat kidneys are similar to those in mice (Figs. 2 and 3).

                              
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Table 2.   Effect of DWH-146e on neutrophil accumulation in rat kidney after ischemia-reperfusion injury



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Fig. 4.   A2A-AR activation reduces density of neutrophils in outer medulla of rats subjected to I/R. Using Neurolucida the kidney was viewed under ×100 magnification and the entire kidney was drawn. Polymorphoneutrophils (PMNs) were counted by viewing kidney sections under ×250 magnification. Kidney sections were overlaid with optical frames viewed under the microscope, and all PMNs were counted within each frame. Shown is the map of a kidney from a rat subjected to 45-min ischemia and 48-h reperfusion and treated with vehicle (group 8; A) or DWH-146e (4 ng · kg-1 · min-1; group 9; B). The density of neutrophils was 15.65/mm2 for vehicle and 3.02/mm2 for DWH-146e treatment. Map shown is representative example from 3 rats (see Table 2).

DWH-146e reduces ICAM-1 and P-selectin expression in mouse kidneys subjected to I/R injury. Neutrophil adherence to endothelial cells requires the coordinated expression of adhesion molecules for neutrophils to transmigrate into tissues to produce injury. Experiments described above demonstrate that A2A-AR agonists decrease neutrophil accumulation in kidneys subjected to I/R injury. These results suggest the possibility that agonists of A2A-ARs affect adhesion molecule expression or function. Two adhesion molecules known to contribute to neutrophil adhesion and to the pathogenesis of I/R of kidney are P-selectin and ICAM-1 (2). To determine the effect of selective A2A-AR activation on adhesion molecule expression we performed immunohistochemistry to examine the distribution of both P-selectin and ICAM-1 in kidney in response to DWH-146e infusion after I/R injury.

For these experiments we used well-characterized monoclonal antibodies to P-selectin (RB40.4) (4) and ICAM-1 (YN1.1) (28). In preliminary experiments we sought to determine the feasibility of using these antibodies in detecting changes in expression of adhesion molecules in mouse kidneys by using a well-known potent stimulus of adhesion molecule expression, LPS (2). Kidneys were harvested at 2, 4, and 6 h after systemic administration of LPS. LPS led to a time-dependent increase in expression of P-selectin (Fig. 5, A-C). No staining was observed in control, vehicle-injected mice (not shown), and minimal staining was observed in sham-operated mice (Fig. 5D). However, 2 h post-LPS injection, a faint degree of P-selectin-like immunoreactivity was observed in peritubular capillaries in cortex (Fig. 5B) and the endothelial layer of interlobular arteries. Staining was also observed in the outer medulla and inner medulla (data not shown). Kidneys harvested 4 and 6 h post-LPS injection demonstrated a dramatic increase in staining throughout the cortex (Fig. 5, B and C) and outer and inner medulla (not shown). P-selectin expression was limited to the endothelial layer of peritubular capillaries (as shown at higher magnification in Fig. 6) and interlobular arteries. Qualitatively similar results are shown by using monoclonal antibodies to ICAM-1 (Fig. 7, A-C). LPS stimulation showed a gradual increase in staining of the endothelial layer of interlobular arteries, peritubular capillaries, and glomerular capillaries at 2 and 4 h post-LPS treatment. Also apparent is staining in afferent arterioles (Fig. 7C). These results demonstrate the pattern of P-selectin- and ICAM-1-like immunoreactivity by using highly selective and well-characterized antibodies and furthermore that qualitative differences in expression can be observed after treatment that characteristically increases P-selectin and ICAM-1 (2).


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Fig. 5.   DWH-146e decreases P-selectin immunoreactivity in kidney after I/R. We performed immunohistochemical studies using a well-characterized monoclonal antibody to P-selectin, RB40.34. Lipopolysaccharide (LPS; 3 mg/kg ip, mouse) was injected, and kidneys were harvested at 2, 4, and 6 h after injection (a-c). For I/R injury experiments (d-h), kidneys were harvested 6 h post-I/R. a-c, Cortex 2, 4, and 6 h post-LPS treatment, respectively; d-f, cortex; g-i, outer medulla; d and g, sham; e and h, vehicle; f and g, DWH-146e. *, Endothelial cells of interlobular arteries; arrows, peritubular capillaries. Magnification: ×400 (a-f); ×200 mag (g-i).



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Fig. 6.   Endothelial cells of peritubular capillaries express P-selectin. P-selectin immunoreactivity is shown in kidneys harvested 6 h after LPS administration (ip). Note that immunoreactivity was limited to endothelial cells outlying entrapped red blood cells. Magnification: ×650 (oil immersion).



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Fig. 7.   DWH-146e decreases intracellular adhesion molecule 1 (ICAM-1) immunoreactivity in kidney after I/R injury. We performed immunohistochemical studies using a well-characterized monoclonal antibody to ICAM-1 (YN1.1). LPS (3 mg/kg ip, mouse) was injected, and kidneys were harvested at 2 and 4 h after injection (a-c). For I/R injury experiments (d-h), kidneys were harvested 6 h post-I/R. a-c, Cortex control, 2 h, 4 h, and post-LPS treatment, respectively; d-f, cortex; g-i, outer medulla; d and g, sham; e and h, vehicle; f and g, DWH-146e. *, Endothelial cells of interlobular arteries; arrowheads, afferent arterioles; arrows, peritubular capillaries. Magnification: ×400 (b-i); ×200 (a).

Having established the utility of this immunochemical approach we then subjected mouse kidneys to I/R (Fig. 5, D-I). Sham-operated mice demonstrated very little staining in cortex (Fig. 5D) and outer medulla (Fig. 5G). However, I/R led to an increase in P-selectin immunoreactivity in endothelial layers of peritubular capillaries and interlobular arteries of kidneys from vehicle-treated mice (Fig. 5, E and H). A significant reduction in P-selectin-like immunoreactivity was observed in kidneys from DWH-146e-treated mice (Fig. 5, F and I). I/R also produced a modest increase in ICAM-1 immunoreactivity in the same structures in the cortex (Fig. 7E). Dense immunoreactivity was present in outer medulla (Fig. 7H). Under higher resolution magnification ICAM-1-like immunoreactivity can be seen in the endothelial layers of peritubular capillaries (Fig. 8). Treatment with DWH-146e led to a pronounced reduction in ICAM-1 immunoreactivity (Fig. 7, F and I). The effect of DWH-146e on P-selectin (Fig. 9A) and ICAM-1 (Fig. 9B) expression was blocked with ZM-243185.


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Fig. 8.   ICAM-1 expression in endothelial cells of peritubular capillaries after I/R. ICAM-1 immunoreactivity is shown in kidneys harvested 6 h after I/R. Using differential interference contrast optics, immunoreactivity can be seen in endothelial cells of peritubular capillaries. Magnification: ×630 (oil immersion).



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Fig. 9.   A2A-agonist reduction of ICAM-1 and P-selectin expression in mouse kidneys subjected to I/R is receptor mediated. Shown is the outer medulla of mouse kidneys subjected to I/R and coinfusion of DWH-146e (10 ng · kg-1 · min-1)+ZM-243185 (7 ng · kg-1 · min-1, an amount that was calculated to be a molar equivalent to delivered amount of DWH-146e). A: effect of DWH-146e+ZM-243185 on P-selectin expression. B: effect of DWH-146e+ZM-243185 on ICAM-1 expression. Magnification: ×400.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Agonists of A2A-ARs produce a dramatic reduction in renal tissue injury when administered to animals subjected to I/R (26). The results of the present study demonstrate that the decrease in injury produced by A2A-AR agonists is associated with a reduction in neutrophil accumulation, particularly in peritubular capillaries of the outer medulla where neutrophils are most prone to accumulate during I/R. Because adhesion molecules play a critical role in the pathogenesis of neutrophil-mediated renal injury in acute renal failure, we examined the expression of ICAM-1 and P-selectin expression in kidney after I/R. We observed intense staining of both P-selectin and ICAM-1 at 6 h after I/R in the outer medulla. Lesser degrees of adhesion molecule immunoreactivity were observed in the cortex and inner medulla. To further examine the mechanism underlying the protective effects of A2A-AR agonists in I/R injury, we administered DWH-146e to mice before injury. Treatment with DWH-146e led to a decrease in adhesion molecule immunoreactivity. The data are consistent with the conclusion that A2A-AR agonists limit I/R injury due to an inhibitory effect on neutrophil adhesion.

During the early reperfusion phase of I/R injury, neutrophils become adherent to endothelial cells of postcapillary venules and may migrate into renal tissue. Neutrophil-endothelial cell adhesion in the vasa recta in the outer stripe of the outer medulla leads to capillary plugging and vascular congestion. Furthermore, neutrophils release additional reactive oxygen species, proteolytic enzymes, and cytokines that incite cell death. Accumulation of neutrophils in kidney has been clearly identified at various times in models of I/R injury in rats and mice and has been associated with renal injury (8, 20, 23, 30, 37). Injury is exacerbated after maneuvers that increase neutrophil infiltration (22). Conversely, therapeutic interventions to reduce neutrophil infiltration have been shown to protect kidneys from I/R injury (14, 16, 18-20, 23, 29).

Our studies, as assessed by two independent measures of neutrophil function, demonstrate a dramatic decrease in neutrophil infiltration after I/R that was reduced by infusion with DWH-146e. MPO, an enzyme present in neutrophils, has been used as a biochemical marker for neutrophil infiltration in I/R studies of the kidney. In both rats and mice, MPO activity was reduced with infusion of DWH-146e, and furthermore the degree of activity correlated with injury. Napthol AS-D choroacetate has been used as a reliable marker for neutrophils in tissue sections (8, 21, 37). We carefully examined the specificity of this stain in peripheral blood smears and found staining limited to neutrophils and not monocytes or lymphocytes. Using this staining method the localization of neutrophils primarily in the outer medulla is consistent with previous studies of renal I/R injury (8). MPO is also present in monocyte/macrophages and, because we did not stain for these cells, the contribution of these cell types cannot be assessed.

An early step leading to neutrophil-mediated tissue injury is the adherence of these leukocytes to endothelial cells through adhesion molecules (14, 19, 29, 30). Adhesion of neutrophils to endothelial cells occurs through a complex series of events that may involve several classes of adhesion molecules including selectins, mucin and other selectin ligands, integrins, and the Ig superfamily (30). In particular the role of ICAM-1 has been well studied. ICAM-1 (CD54) is expressed on endothelial cells and binds to counterreceptors on neutrophils, lymphocyte function-antigen (LFA-1; CD11alpha /CD18), and Mac-1 (CD11beta /CD18). Abundant data have accumulated that demonstrate convincingly that CD11/CD18beta 2-integrins and ICAM-1 are important in the pathogenesis of ischemic renal injury (9, 13, 16, 18, 19, 29, 30). P-selectin and E-selectin, adhesion molecules expressed on endothelial cells thought to be responsible for leukocyte rolling, have also been found to mediate I/R injury (35). Thus the adherence of neutrophils to endothelium mediated by adhesion molecules plays a critical role in I/R injury.

Little is known regarding the regulation of adhesion molecules by adenosine. In vitro studies were used to examine the effects of adenosine on adhesion molecule expression (5). Adenosine reduced expression of vascular cell adhesion molecule 1 and E-selectin but not ICAM-1 in activated human umbilical vein endothelial cells. Regulation of adhesion molecule expression by adenosine receptor subtypes is not well characterized. In the present study, I/R injury led to a dramatic increase in expression of P-selectin and I-CAM-1 that was inhibited by infusion of the selective A2A-AR agonist DWH-146e. We demonstrated by pharmacological means, using selective A2A-AR agonists and antagonists (26), that the protective effect of DWH-146e in animal models of I/R injury is due to activation of A2A-ARs. Therefore, it is likely that the observed regulation of adhesion molecule expression in the present study involves the A2A-AR subtype of adenosine receptors. The data suggest that the reduced expression of adhesion molecules after A2A-AR activation could contribute to a decease in neutrophil accumulation and contribute to the renal tissue protection from I/R injury as a consequence of activating these receptors.

In addition to the regulation of adhesion molecules, A2A agonists also appear to directly influence the function of inflammatory cells. Adenosine modulates the release of cytokines from inflammatory cells and endothelial cells. It is notable that adenosine decreases the release of proinflammatory cytokines and increases release of anti-inflammatory cytokines by endothelial cells (5). Determining the extent to which these factors contribute to the protective effects of A2A agonists will require additional investigation.

The therapeutic possibilities for the use of DWH-146e and other highly selective agonists of A2A-ARs are quite apparent from the foregoing discussion. First, DWH-146e is capable of maximally reducing renal injury at extremely low concentrations that are not known to produce systemic hemodynamic effects (26). In rats with an infusion rate of 4 ng · kg-1 · min-1, a plasma level of <1 nM was observed. The low dose is likely to minimize any potential clinical side effect. Activation of A2A-ARs likely inhibits inflammation by multiple mechanisms involving several different cell types. This therapeutic approach may prove to be more effective at limiting inflammation than maneuvers that target a single adhesion molecule or cytokine. It is likely that disabling one proinflammatory protein may be compensated for by another protein. By broad abrogation of the effects of inflammatory factors at multiple sites and levels within this cascade, inflammatory-mediated renal injury may be minimized. A2A-ARs may be a good target for therapy because they broadly attenuate the inflammatory cascade. Thus the use of A2A-AR agonists holds promise for future clinical trials as a novel approach in the preservation of renal tissue and function from I/R injury in kidney as well as other organs.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge Dr. Diane Rosin (Dept. of Pharmacology, Univ. of Virginia) for helpful discussions and editorial assistance, Dr. Kai Singbartl (Dept. of Biomedical Engineering) for assistance in establishing the ischemia-reperfusion mouse model in our laboratory, Dr. Klaus Ley (Dept. of Biomedical Engineering) for the gift of P-selectin and ICAM-1 antibodies, and Dr. Ruth Stornetta (Dept. of Pharmacology, Univ. of Virginia) for assistance with the use of the imaging system for quantitative neutrophil mapping.


    FOOTNOTES

This work was supported in part by funds to M. D. Okusa from the American Heart Association (0050329N), National Kidney Foundation, Virginia Affiliate, and the University of Virginia (Research and Development Committee), and to J. Linden from the National Heart, Lung, and Blood Institute (2RO1 HL-37942). M. D. Okusa was a recipient of the Clinical Scientist Award from the National Kidney Foundation (CSA-16). A portion of this work has been published in abstract form (J Am Soc Nephrol 10: 638A, 1999).

Address for reprint requests and other correspondence: M. D. Okusa, Div. of Nephrology, Box 133, Univ. of Virginia Health System, Charlottesville, VA 22908 (E-mail: mdo7y{at}virginia.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 February 2000; accepted in final form 3 July 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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