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Departments of 1 Medicine, 2 Molecular Physiology and Biological Physics, and 3 Chemistry, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 · min1 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
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 · kg1 · min1, 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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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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 · kg1 · min1) 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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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 · g1 · min1
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 · g1 · min1
(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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1 · min1),
DWH-146e reduced MPO activity in five of five rats by 33% (1.81 ± 0.21 OD460 · g1 · min1;
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 · g1 · min1).
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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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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;
CD11
/CD18), and Mac-1 (CD11
/CD18). Abundant data have accumulated
that demonstrate convincingly that CD11/CD182-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 · kg1 · min1, 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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TOP
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METHODS
RESULTS
DISCUSSION
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, group 2; 
, group
4; r2 = 0.73, P < 0.0001).
