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1
* Department of Biomedical Engineering and
Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia, USA; and
Klinik und Poliklinik für Anästhesiologie und operative Intensivmedizin, Universitätsklinikum Münster, Münster, Germany
1Correspondence: Department of Biomedical Engineering, Health Sciences Center, Box 800759, Charlottesville, VA 22908, USA. E-mail: klausley{at}virginia.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: neutrophils • ischemia-reperfusion • acute renal failure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
. During reperfusion, neutrophils are thought to mediate tissue damage by two mechanisms (6)
. Together with red blood cells and platelets, neutrophils have been proposed to cause capillary perfusion failure or ‘no-reflow’, which leads to focal tissue hypoxia (7
, 8)
. Second, exacerbated tissue injury (reflow paradox) is induced by postischemic activation of leukocytes, especially neutrophils, which then produce and release cytotoxic substances while adhering to the endothelium and infiltrating into the tissue (9)
. Recruitment of neutrophils into injured tissues is thought to occur in a cascade-like sequence (10
, 11)
. Endothelial and leukocyte adhesion molecules are critically involved in this process. Capturing or tethering of freely flowing leukocytes is the first step and can result in stable rolling of these cells along the endothelium. The selectin class of adhesion molecules (L-, E-, and P-selectin) mediates leukocyte capture and rolling (12
, 13)
. Chemokines or other chemoattractants activate integrins on slowly rolling leukocytes, leading to firm adhesion and transmigration (14)
. Several molecules have been implicated in the transmigration process of neutrophils across the endothelium. These include PECAM-1, ICAM-1, VE-cadherin, LFA-1, and CD47 (15
16
17
18
19)
. The blockade of certain leukocyte endothelial adhesion molecules such as ß2-integrins (20)
, ICAM-1 (1
, 2)
, E-selectin (5)
, or P-selectin (4)
significantly reduces kidney damage after ischemia/reperfusion and thus preserves kidney function.
P-selectin is a 140 kDa type 1 transmembrane glycoprotein and is stored in Weibel-Palade bodies of endothelial cells as well as in 
-granules of platelets (21
, 22)
. From there it can rapidly be brought to the cell surface after exposure to thrombin, histamine, complement 5a, Ca2+ ionophores, or adenosine diphosphate (21
22
23
24)
. In mice, endotoxin injection has also been shown to induce P-selectin expression in liver, heart, lung, and kidney (25)
. Surface P-selectin expression is down-regulated by internalization, degradation, and to a smaller extent by shedding into the plasma (22
, 24)
.
Several in vivo and in vitro studies have provided evidence for P-selectin-dependent interactions between leukocytes and endothelial cells after ischemia/reperfusion (26
, 27)
or hypoxia/reoxygenation (28)
. Evidence for a role of platelet P-selectin is based on reports that neutrophils can roll on platelet P-selectin in flow chamber systems in vitro (29)
. Both the formation of platelet aggregates and rolling of platelets along the endothelium occur after ischemia/reperfusion (30)
. However, little is known about the interaction between neutrophils and platelets after ischemia/reperfusion and its functional relevance in postischemic organ failure. One ex vivo study showed that platelets can further aggravate neutrophil mediated dysfunction in a P-selectin-dependent model of cardiac ischemia/reperfusion (31)
. In a previous study (4)
, we showed that P-selectin is differentially expressed after ischemia/reperfusion in the kidney. In the glomeruli, P-selectin is expressed in both endothelial cells and platelets. However, in peritubular vessels, which provide blood supply to the outer medulla, only platelets stained positive for P-selectin. Postischemic neutrophil accumulation in the kidney is directed into the outer medulla (32
, 33)
, which represents the most vulnerable region within the kidney (34)
. Based on these data, we hypothesized that platelets, in addition to postischemic thrombus formation, may play a role in neutrophil recruitment into the kidney after ischemia/reperfusion.
This study was designed to evaluate the role of platelet vs. endothelial P-selectin in postischemic neutrophil recruitment. To selectively address the role of endothelial vs. platelet P-selectin, we generated chimeric mice by bone marrow transplantation using wild-type (wt) and P-selectin deficient mice. Chimeric mice expressed P-selectin on endothelial cells, platelets, both, or neither. Bone marrow-transplanted mice then underwent bilateral renal ischemia/reperfusion and were evaluated for neutrophil infiltration, creatinine concentration, and P-selectin expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Mutant mice were back-crossed into a C57BL/6 background for at least six generations and maintained under specific pathogen-free conditions at the Center for Comparative Medicine, University of Virginia.
Bone marrow transplantation
Bone marrow was harvested from donor mice and transplanted into recipient mice as described previously (36)
. Recipient mice were lethally irradiated in two doses of 600 rads, each 
4 h apart. Donor mice were killed by lethal injection of sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL). Bone marrow cells from both femurs and tibias were harvested under sterile conditions. Approximately 50 million nucleated cells were obtained from each donor mouse. Bones were flushed with RPMI (Life Technologies, Grand Island, NY) (without phenol red) containing 10% fetal calf serum (Atlanta Biologicals, Norcross, GA). Suspended bone marrow cells were washed and erythrocytes were lysed in 0.15 M NH4Cl lysing solution. Approximately 4–5 million unfractionated bone marrow cells in 200 µl of media were injected intravenously through the tail vein of each recipient mouse. Recipient mice were housed in a barrier facility (individually ventilated cages, HEPA-filtered air, sterile bedding, autoclaved food) under pathogen-free conditions before and after bone marrow transplantation. After bone marrow transplantation, mice were maintained on autoclaved water supplemented with antibiotics (5 mM sulfamethoxazole, 0.86 mM trimethoprim; Sigma, St. Louis, MO). These conditions were maintained for 9 wk, after which mice were tail bled in order to obtain systemic and differential blood counts. Leukocytes were counted using Kimura’s stain; platelet counts were obtained by ammonium oxalate staining. Pilot studies had shown that in our neutrophil-dependent model (4
, 5)
, a neutrophil concentration of 900/µl or more was necessary to reproducibly induce severe postischemic renal failure (data not shown). Thus, only mice with a neutrophil concentration of at least 900/µl were included (n=76). Using wt and P-selectin deficient mice, we generated chimeric mice (bone marrow transplantation between mice of different genotypes, Table 1
) and control mice (bone marrow transplantation between mice of the same genotype). Bone marrow transplantation was validated by Western blot and immunostaining (see below).
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Surgical procedure
Mice were anesthetized with intraperitoneal injections of ketamine (125 µg/g body weight, Ketalar; Parke-Davis, Morris Plains, NJ), xylazine (12.5 µg/g body weight; Phoenix Scientific, St. Joseph, MO), and atropine sulfate (0.025 µg/g body weight; Elkins-Sinn, Cherry Hill, NJ) and placed on a heating pad to maintain body temperature. Both renal pedicles were prepared using a median dorsal skin incision and bilateral paramedian opening of the retroperitoneal space. In animals undergoing I/R, both pedicles were clamped off for 32 min with hemostatic microclips. This model is known to induce severe acute renal failure in untreated wt mice, with a 50% mortality rate at 72 h (1)
. Kidneys were inspected for immediate color change indicating successful clamping. After clamp removal, kidneys were checked for a change in color within 3 min to ensure reperfusion. In four of 76 mice these criteria were not fulfilled, and these mice were excluded from the study. In five mice, kidneys showed signs of hemorrhagic infarction at time of harvesting, indicating venous obstruction rather than arterial occlusion (37)
. These mice were also excluded. In animals subjected to sham operation, the surgical procedure was identical except that no clamps were applied. After surgery, the wounds were covered with saline-soaked gauze. Incisions were closed in two layers and animals were allowed to recover. Postoperative analgesia was provided by subcutaneous injections of buprenorphine (2 µg/g body weight) diluted with isotonic saline to provide sufficient volume substitution for 12 h. Twenty-four and 48 h later, mice were killed, blood samples were taken by heart puncture, and kidneys were harvested.
Renal function
Serum samples were used to determine creatinine concentrations, using a commercially available enzymatic test kit (Sigma).
Myeloperoxidase activity (MPO)
MPO, indicating neutrophil infiltration into tissue, was measured in equal-sized samples of both kidneys according to our previously published protocol (4
, 5)
. Samples were homogenized (1:20 w:v) in ice-cold 20 mM KPO4 buffer (pH 7.4). After removing 17,000 g supernatants (4°C, 30 min), pellets were again resuspended in ice-cold 20 mM KPO4 buffer (pH 7.4), followed by two additional spins. Then 0.5% (w/v) hexacyltrimethylammonium bromide-10 mM EDTA in 50 mM KPO4 (pH 6.0) was added to the remaining pellet (buffer:pellet 6:1). Suspensions were sonicated for 5 x 1 s on ice, freeze-thawed 3x, and incubated for 20 min at 4°C. After final centrifugation (17,000 g, 15 min, 4°C), supernatants were used to measure MPO. In triplicate, assay buffer (0.2 mg/ml o-dianisidine and 158 µM H2O2 in 50 mM KPO4, pH 6.0) was added to supernatant at a ratio of 4:1. Changes in absorbance were recorded at 460 nm over 3.5 min. The linear part of the resulting curve was used for calculating MPO activity. 1 U of activity was defined as change in absorbance of 1.0/min at 25°C. Results were expressed as units of MPO/g of protein of supernatant as determined by bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL).
Western-blotting
Whole blood was mixed (1:2) in ice-cold protein extraction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10 µg/ml phenyl-methane-sulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin). Protein extracts were stored at -80°C. Samples (25 µl/lane) were run under reducing conditions on an SDS-polyacrylamide gel (4% stacking gel, 7.5% separating gel). Thereafter, gels were electroblotted on nitrocellulose membranes. After blocking, blots were incubated for 2 h with a rabbit polyclonal antibody (1:5000) against a peptide corresponding to the 25 COOH-terminal amino acids of P-selectin (38)
. A goat anti-rabbit antibody conjugated with horseradish-peroxidase served as secondary antibody (Immun-Star, Bio-Rad, Hercules, CA). Washed blots were finally impregnated with the chemiluminescence substrate and exposed to X-ray films.
Immunohistochemistry: P-selectin
After antigen retrieval, paraffin-embedded kidney sections (5 µm) were incubated with a rabbit polyclonal antibody (1:4000) against a peptide corresponding to the 25 COOH-terminal amino acids of P-selectin (38)
. This was followed by biotinylated secondary antibody (1:250, Vector Laboratories, Burlingame, CA) in 10% goat serum to reduce background staining, and finally by avidin-biotin-peroxidase (Vector Laboratories).
Immunohistochemistry: neutrophils
Paraffin embedded kidney sections (5 µm) were incubated (1:4000) with a rat anti-mouse monoclonal antibody (clone 7/4; Serotec, Raleigh, NC) against a polymorphic 40 kDa antigen expressed by neutrophils (39)
. This was followed by a biotinylated secondary antibody (1:250, Vector Laboratories) in 10% goat serum to reduce background staining, and finally by avidin-biotin-peroxidase (Vector Laboratories). Neutrophils were then located and counted in representative kidney sections (n=5).
Statistics
For statistical analysis, one-way ANOVA, Dunnett’s test, Student-Newman-Keuls test, and paired t test were used. All results are given as mean ± SE. Statistical significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, we generated chimeric and control mice (Table 1)
. Successful bone marrow transplantation was confirmed by Western blot analysis of whole blood and immunostaining on paraffin-embedded kidney sections. Lethally irradiated wt mice that had received bone marrow from P-selectin-deficient mice exclusively expressed endothelial P-selectin (EC-P mice). Lethally irradiated P-selectin-deficient mice injected with bone marrow from wt mice expressed only platelet P-selectin (Plt-P mice). Wild-type mice receiving bone marrow from wt mice (Wt-BMT) and P-selectin-deficient mice obtaining bone marrow from P-selectin-deficient mice (P-/-BMT) served as positive and negative controls, respectively. Only whole blood from Wt-BMT and Plt-P mice, but not from EC-P or P-/-BMT, stained positive for P-selectin in Western blots using whole blood protein extracts (Fig. 1
a). Moreover, immunostaining on kidney sections shows that in Wt
Wt mice P-selectin expression was found in both platelets and endothelium (Fig. 1b
). However, in Plt-P mice, P-selectin expression was restricted to platelets (Fig. 1c
) whereas in EC-P it was seen only in endothelial cells (Fig. 1d
). Leukocyte and platelet counts were not significantly different between the groups of mice (Table 2
).
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APRF
After a 32-min ischemia, the creatinine concentration reached its maximum at 48 h after reperfusion (1
, 4
, 5)
. Here Wt-BMT mice revealed the highest creatinine concentration 48 h after ischemia/reperfusion, which was 11-fold higher than in corresponding sham-operated animals (2.96±0.96 mg/dl vs. 0.22±0.02 mg/dl) (Fig. 2
). By contrast, in P-/-BMT mice the creatinine concentration was significantly lower and only twofold over that of matching sham-operated mice (0.61±0.19 mg/dl vs. 0.27±0.03 mg/dl). This corresponded to an 80% protection from APRF in P-/-BMT mice, similar to the protection observed in P-selectin-deficient mice (4)
. Thus, bone marrow transplantation does not interfere with our model of neutrophil-dependent APRF.
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Mice that expressed endothelial but not EC-P had only an twofold increase in creatinine concentration 48 h after ischemia/reperfusion (0.58±0.34 mg/dl vs. 0.25±0.02 mg/dl), which was significantly smaller than that in Wt-BMT mice (Fig. 2)
. The protection from APRF seen in EC-P mice equaled that found in P-/-BMT mice. This rise was significantly lower than that observed in mice in which P-selectin expression was restricted to only platelets (Plt-P); here, the creatinine concentration increased eightfold over sham after I/R (2.13±0.46 mg/dl vs. 0.28±0.02 mg/dl) (Fig. 2)
.
Postischemic neutrophil recruitment
We have previously shown (4
, 5)
that in wt mice, renal myeloperoxidase activity reaches its maximum at 24 h after ischemia/reperfusion. After I/R, Plt-P mice and EC-P mice had similar, statistically significant increases in absolute as well as relative renal myeloperoxidase activities vs. sham controls (Fig. 3
a). MPO activity was elevated sixfold over matching sham-operated animals (Fig. 3b
), indicating a comparable overall neutrophil infiltration.
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Several studies (1
, 33
, 40)
have shown that in wt mice postischemic neutrophil infiltration into the kidney is concentrated in the outer medulla, which represents the most vulnerable region in the kidney (34)
. To a lesser extent, neutrophils also infiltrate into the inner medulla and cortical region. Using immunostaining on paraffin-embedded kidney sections with a monoclonal antibody against a neutrophil antigen, we localized postischemic neutrophil infiltration into kidneys from Plt-P mice and EC-P mice. In Plt-P mice, we found a massive, highly concentrated influx of neutrophils into the outer medulla (Fig. 4
a) and some infiltration into the inner medulla (Fig. 4c
). In the outer medulla, both intra- and extravascular neutrophils were found (Fig. 4a
), whereas in the inner medulla neutrophils were seen mainly inside the peritubular vessels (Fig. 4c
). By contrast, EC-P mice demonstrated a different pattern of postischemic neutrophil recruitment (Fig. 4b, d
). Here the influx of neutrophils into the outer medulla was drastically reduced (Fig. 4b
) and almost completely abolished in the inner medulla (Fig. 4d
). Instead, more neutrophils were found in sections of the renal cortex of EC-P mice (Fig. 4f
) than in Plt-P mice (Fig. 4e
). Thus, the overall neutrophil infiltration in EC-P mice was similar in extent, but more diffuse than in Plt-P mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, the dominant role of platelet P-selectin was surprising, because after ischemia/reperfusion P-selectin is also expressed on glomerular endothelial cells (4)
. Even though P-selectin -/- mice reconstituted with wt bone marrow (Plt-P) and wt mice reconstituted with P-selectin-/- bone marrow (EC-P) demonstrated equal total neutrophil recruitment into the kidney, only EC-P were protected from renal failure. In these mice, neutrophil accumulation was redirected from a highly concentrated infiltrate in the outer medulla to a more diffuse pattern. The dissociation between global neutrophil recruitment and outcome suggests that neutrophils recruited into certain vulnerable postischemic regions mediate renal damage at a clinically relevant level. These findings also call into question the value of global measures of neutrophil infiltration without histological studies to localize the infiltrating cells.
Several studies have shown that neutrophil infiltration into the postischemic kidney is important for the development of renal failure (1
, 41
42
43
44)
. However, none of these studies investigated the role of platelets for neutrophil recruitment. Our study is the first to specifically show a role for platelet-induced neutrophil recruitment in postischemic renal injury. Moreover, it is the first in vivo study to demonstrate the relevance of platelet-induced neutrophil recruitment for the development of any postischemic organ failure. So far, only one ex vivo (31)
study has shown a role for platelets in postischemic organ dysfunction. Lefer et al. (31)
showed that postischemic cardiac reperfusion with platelets and neutrophils resulted in a far greater myocardial dysfunction than reperfusion with either cell type alone. Dual-cell reperfusion also leads to a much higher postischemic neutrophil infiltration than reperfusion with neutrophils alone. Adding either a sialyl Lewisx-oligosaccharide or a recombinant soluble P-selectin ligand to the reperfusion solution drastically reduced both cardiodepressant effects and neutrophil infiltration. Kogaki et al. (45)
examined neutrophil adhesion after hypoxia-reoxygenation in a static endothelial cell/cardiomyocyte coculture system in vitro. The coincubation with platelets resulted in significantly higher neutrophil adhesion and transmigration than the incubation with endothelial cells/cardiomyocytes alone. This increase was inhibited by a monoclonal anti-P-selectin antibody, suggesting a critical role for P-selectin in platelet-mediated neutrophil adhesion in this model.
After ischemia/reperfusion, neutrophils accumulate in the outer medulla (32
, 33)
, which has long been known to undergo extensive postischemic damage (46)
. These findings suggest an association between neutrophil infiltration in this area and subsequent renal damage. Our present data provide a potential mechanistic link between platelet-mediated neutrophil infiltration into the outer medulla and acute renal failure. In mice lacking platelet P-selectin, neutrophil recruitment into the outer medulla was reduced, and these mice were protected from APRF. Although we show here that neutrophil recruitment to the outer medulla requires platelet P-selectin, the mechanism of recruitment remains unknown. It is possible that platelets may adhere to endothelial cells and subsequently cause secondary attachment of neutrophils. This has been shown in vitro (47
48
49)
and in vivo, especially after arterial injury by angioplasty (48
49
50)
. However, platelets could also adhere to circulating neutrophils after ischemia/reperfusion, which could then become trapped in narrow peritubular capillaries. The latter possibility is supported by a recent intravital microscopy study (30)
. In a mouse model, Massberg et al. (30)
showed that after intestinal ischemia/reperfusion, platelet–endothelial cell-interactions were almost completely blocked by eliminating endothelial P-selectin, but platelet–leukocyte interactions were platelet P-selectin dependent (30)
. Since the development of APRF in our model depends on platelet but not on endothelial P-selectin, it appears more likely that circulating platelets and neutrophils form aggregates, which then adhere or become trapped in the renal microcirculation.
In addition to P-selectin (4)
, blockade or elimination of E-selectin (5)
, ß2-integrins (20)
, or ICAM-1 (1
, 2)
has been shown to reduce kidney damage after ischemia and reperfusion. By contrast, L-selectin blockade had no effect (51)
. Our study not only adds to this list of protective interventions in postischemic renal failure, it also provides evidence that selective elimination of platelet P-selectin is protective. This novel finding suggests that targeted therapy aimed at platelets or, more specifically, platelet–leukocyte interaction may be beneficial in patient management to prevent postischemic renal failure. Importantly, such interventions may impair host defense or coagulation much less than globally blocking leukocyte adhesion molecules.
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ACKNOWLEDGMENTS |
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Received for publication April 19, 2001.
Revision received July 13, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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