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* Department of Biomedical Engineering and
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA; and
Klinik und Poliklinik für Anästhesiologie und Operative Intensivmedizin, Westfälische Wilhelms-Universität, Münster, Germany
1Correspondence: Department of Biomedical Engineering, Health Sciences Center, Box 377, University of Virginia, Charlottesville, VA 22908, USA. E-mail: kfl3f{at}virginia.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: neutrophils • adhesion molecules • renal function • gene targeting • antibodies
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Postischemic or reperfusion injury is thought to be
mediated mainly by two mechanisms, which can further aggravate tissue
damage: no-reflow and reflow paradox (2)
. No-reflow
describes capillary perfusion failure resulting in focal tissue hypoxia
(3)
. So far, red blood cells, platelets, and leukocytes
have all been proposed to contribute to this phenomenon
(4)
. Reflow paradox refers to exacerbated tissue injury
due to postischemic activation of leukocytes, especially neutrophils,
which then produce and secrete cytotoxic compounds while adhering to
the endothelium and infiltrating into the tissue (5)
.
In many experimental models, neutrophils have been shown to infiltrate
in postischemic kidneys (6
7
8
9
10
11)
. Recruitment of
neutrophils into injured tissues is thought to occur by a sequence of
events leading to firm endothelial adhesion and subsequently to
transmigration (12
, 13)
. Endothelial and leukocyte
adhesion molecules have been proposed as mediators of this cascade-like
process. Capturing or tethering of free-flowing leukocytes represents
the initial step and can be converted into stable rolling of these
cells along the endothelium. The selectin class of adhesion molecules
(L-, P-, and E-selectin) mediates leukocyte capture and rolling
(14
, 15)
. During rolling, leukocytes are believed to get
activated by chemokines or other chemoattractants leading to activation
of integrins, firm adhesion, and finally to transmigration
(16)
.
In ischemia/reperfusion-induced renal injury, only a few adhesion
molecules have been studied so far (6
7
8
9
, 11
, 17
18
19)
.
Whereas inhibiting the function of either
ß2-integrins (17)
or intercellular
adhesion molecule 1 (ICAM-1) (6
, 8
, 11
, 19)
gave a
significant reduction of kidney damage after ischemia-reperfusion
(I/R), blocking L-selectin did not protect from renal failure
(9)
. Although expression of P-selectin has been reported
to be increased in response to renal ischemia-reperfusion
(20)
, the role of P-selectin in the development of acute
renal failure remains unknown.
P-selectin, a 140 kDa type-1 transmembrane glycoprotein, is
constitutively stored in Weibel-Palade bodies of endothelial cells and
in 
-granules of platelets (21
, 22)
. From there it can
be released to the cell surface within a few minutes after exposure to
thrombin, histamine, complement 5a,
Ca2+-ionophores, or adenosine diphosphate
(21
22
23
24)
. In mice, endotoxin injection has been shown to
induce P-selectin expression in liver, heart, lung, and kidney
(25)
. P-selectin expression on the cell surface is
down-regulated by internalization, degradation, and to a smaller extent
by shedding into the plasma (22
, 24)
.
This study seeks to identify the role of P-selectin in I/R-induced severe acute renal failure. To achieve this, we used gene-targeted mice with a null mutation in the P-selectin gene as well as postischemic application of function-blocking monoclonal P-selectin antibody in wild-type mice. To validate our model, we also determined the impact of neutrophils on renal function.
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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 five
generations and maintained in specific pathogen-free conditions at the
Center for Comparative Medicine, University of Virginia.
Chemicals
Unless stated otherwise, chemicals were obtained from Sigma
Chemical Co. (St. Louis, Mo.).
Surgical procedure
Mice were anesthetized with intraperitoneal (i.p.) injections of
ketamine (125 µg/g body weight, Ketalar; Parke-Davis, Morris Plains,
N.J.), xylazine (12.5 µg/g body weight; Phoenix Scientific, Inc., St.
Joseph, Mo.), and atropine sulfate (0.025 µg/g body weight;
Elkins-Sinn, Inc., Cherry Hill, N.J.) 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 wild-type mice with a
50% mortality rate at 72 h (6)
. 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 19 of 154 mice, these criteria were not
fulfilled, and these mice were excluded from this study. In 12 mice,
kidneys showed signs of hemorrhagic infarction at the time of
harvesting, indicating venous obstruction rather than arterial
occlusion (27)
. Two mice had polycystic kidneys. 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. Four, 12, 24, or 48 h later mice were killed, blood samples
were taken by heart puncture, and kidneys were harvested. Data from
wild-type mice presented here also served as a control for identifying
the role of E-selectin in ischemia/reperfusion-induced acute renal
failure.
Therapeutic intervention experiments
Two separate groups of wild-type mice received 100 µg of
either function-blocking monoclonal anti-mouse P-selectin antibody
(RB40.34, hybridoma provided by D. Vestweber, Universität
Münster, Germany; described in ref 28
) or
isotype-matched control antibody (PharMingen, San Diego, Calif.) i.p.
10 minutes after reperfusion. Mice were killed at 24 h after
reperfusion and blood was collected.
Neutrophil depletion experiments
In two additional groups of wild-type mice, 2 µl/g body weight
of either rabbit anti-mouse neutrophil serum or conventional rabbit
serum (Inter-Cell Technologies Inc., Hopewell, N.J.) was injected
18 h before ischemia. Blood samples for neutrophil counts were
obtained at the time of ischemia by tail bleeding. Neutrophils were
counted blind using Kimura’s stain. Animals were killed 24 h
later, blood samples were taken, and kidneys were harvested.
Renal function
Whole blood samples were used to determine creatinine and blood
urea nitrogen (BUN) concentrations (NOVA analyzer
16+, NOVA Biomedical, Waltham, Mass.). Creatinine
measurements were based on a three-step enzymatic assay (creatinine
amidohydrolase, creatinine amidininohydrolase, and sarcosine oxidase),
converting creatinine and H2O into formaldehyde,
glycine, and H2O2. BUN was
determined by the urease method converting urea into ammonia and
CO2.
Myeloperoxidase activity (MPO)
MPO, indicating neutrophil infiltration into tissue, was
measured in equal-sized samples of both kidneys. This assay represents
a modified combination of two previously published methods (29
, 30)
. Briefly, samples were homogenized (1:20 w:v) in ice-cold 20
mM KPO4 buffer (pH 7.4). After removing
17,000 x 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)
hexacyltrimethylamonium 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 (17,000xg, 15 min, 4°C),
supernatants were used to measure MPO. Assay buffer (0.2 mg/ml
o-dianisidine and 158 µM
H2O2 in 50 mM
KPO4, pH 6.0) was added in triplicate to
supernatant at a ratio of 4:1. Changes in absorbance were recorded at
460 nm over 5 min. The linear part of the resulting curve was used to
calculate MPO activity. 1 U of activity was defined as change in
absorbance of 1.0 per minute at 25°C. Results were expressed as units
of MPO per gram of protein of supernatant as determined by bichionic
acid assay (Pierce Chemical Co., Rockford, Ill.).
Western blotting
Kidneys were homogenized 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 phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml
aprotinin). After 10 min incubation, homogenate was centrifuged
(10,000xg, 5 min, 4°C). Supernatant was stored at
-80°C. Samples (100 µg protein per lane, bichionic acid assay)
were run under reducing conditions on a sodium dodecyl
sulfate-polyacrylamide gel (5% 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 (31)
. A goat
anti-rabbit antibody conjugated with horseradish peroxidase served as
secondary antibody. Finally, washed blots were impregnated with ECL
chemiluminescence substrate and exposed to X-ray films.
Immunohistochemistry
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 (31)
. This was followed by biotinylated
secondary antibody (1:250, Vector Laboratories Inc., Burlingame,
Calif.) in 10% goat serum to reduce background staining and finally by
avidin-biotin peroxidase (Vector Laboratories).
Statistics
Analysis of variance, followed by an unpaired t
test, with Bonferroni correction when appropriate, was used for
statistical analysis. Most data are shown as fold change vs.
corresponding sham group. All results are given as mean ±
SE for groups of six mice each. Statistical
significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Moreover, in neutropenic mice renal MPO was only 3.0 ± 0.6 U/g
protein at that time, whereas mice that had received control serum
revealed renal MPO levels of 10.9 ± 3.8 U/g protein
(P<0.05).
|
In wild-type mice, 32 min bilateral renal pedicle clamping, followed by
reperfusion, led to dramatically elevated creatinine concentrations
after 24 (13-fold over sham) and 48 h (19-fold over sham)
(Fig. 2a
). A significant increase in BUN (1)
concentrations (Fig. 2b
) could also be seen after 24
(10-fold over sham) and 48 h (10-fold over sham). Concomitant with
this drastic impairment of renal function, untreated wild-type mice
revealed a continuous increase in MPO, reaching 17.9 ± 3.6 U/g
protein at 24 h after reperfusion (19-fold increase above sham
control, Fig. 2c
). Thereafter, MPO declined, reaching
10.3 ± 3.5 U/g protein (ninefold above sham) 48 h after
reperfusion. Creatinine and BUN concentrations trailed MPO by ~24 h,
reaching a maximum at 48 h after reperfusion.
|
In contrast to the dramatic rise in both creatinine and BUN
concentrations seen in wild-type mice, P-selectin-deficient mice had
significantly smaller elevations in creatinine (Fig. 2a
) and
BUN (Fig. 2b
) concentrations after 24 and 48 h of
reperfusion. In P-selectin-deficient mice, renal MPO was only 4.5-fold
elevated above corresponding sham values 24 h after I/R (Fig. 2c
). This indicates a significant reduction of neutrophil
influx by ~85% compared to wild-type mice.
Protein extracts from kidneys of representative sham-operated and I/R
wild-type mice were analyzed for P-selectin expression using Western
blot (Fig. 3a
). P-selectin was highly up-regulated soon after
reperfusion, peaked at 12 h, and declined thereafter. In
sham-operated animals, a similar but far less intense expression
pattern was found. Western blot analysis of kidney samples from
P-selectin-deficient mice confirmed that no P-selectin was expressed
(Fig. 3b
) in these mice.
|
Immunostaining localized P-selectin expression at 12 h after
ischemia-reperfusion to glomeruli, peritubular vessels, and to a lesser
extent to arteries and veins. In glomeruli, P-selectin expression was
found in platelet as well as in endothelial cells (Fig. 4a
), whereas in peritubular vessels only platelets stained
positive for P-selectin (Fig. 4b
). Sham-operated animals
also revealed P-selectin expression (Fig. 3)
, but this was considerably
smaller and could only be found in glomeruli (data not shown).
|
To identify any therapeutic benefit from blocking P-selectin, another
group of wild-type mice that had undergone 32 min bilateral ischemia,
received either function blocking monoclonal anti-P-selectin antibody
(RB40.34) or isotype- matched control antibody
(IgG1) i.p. 10 minutes after clamp removal. This
treatment resulted in a significant attenuation of creatinine increase
at 24 h after reperfusion, similar to that seen in mice gene
deficient for P-selectin (Fig. 5
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several studies have shown neutrophil infiltration into the kidney
after ischemia-reperfusion (6
7
8
9
10
11)
. However, the effect of
neutrophils on renal function after ischemia-reperfusion remains
unsettled. In some studies preischemic neutrophil depletion provided
protection from renal failure (6
, 32
33
34)
, whereas other
studies could not demonstrate a protective role of neutrophil depletion
(18
, 35)
. In addition to variations in model design,
different levels of neutrophil depletion achieved may have contributed
to these conflicting results. We achieved a significant reduction in
neutrophil counts of ~85% at the time of ischemia and show that
neutrophils play a crucial role in this model of acute renal failure.
This demonstrates that our model is suitable to study the role of
adhesion molecules such as P-selectin, relevant to neutrophil
infiltration in the postischemic kidney.
In our experiments, we found a sharp increase in total, i.e., surface
and intracellular P-selectin expression as early as 4 h after
reperfusion, indicating ischemia/reperfusion-induced biosynthesis.
P-selectin expression was maximal at 12 h after reperfusion. In a
30 min model of murine renal ischemia, followed by reperfusion, Zizzi
et al. (20)
showed that P-selectin surface expression in
perfused vessels, as measured by injection of radio-labeled P-selectin
antibody, peaked within 5 h of reperfusion and declined
thereafter. However, postischemic kidneys are known to have a disturbed
regional perfusion pattern, which can last for up to 48 h
(36)
. Blood flow in the outer medulla is reduced and blood
flow in the inner medulla is increased (36)
. Thus, the
observed difference in P-selectin expression may be explained at least
partially by P-selectin expressed on cell surfaces in blood vessels
that were not perfused.
We found that sham operation could also induce considerable P-selectin
expression, which was restricted to glomeruli. By contrast, after
ischemia-reperfusion, P-selectin was found in glomeruli and peritubular
vessels. In glomeruli, both endothelial and platelet P-selectin were
detected whereas in peritubular vessels, only platelets stained
positive for P-selectin. In a recent biopsy study of human kidney
transplants, P-selectin expression was also found in platelets in both
glomeruli and peritubular vessels (37)
. Activated and
adherent platelets have been shown to support leukocyte rolling,
adhesion, and transmigration via P-selectin and
ß2-integrins in a way similar to that of
activated endothelium (38)
.
Development of acute renal failure in our model is strongly neutrophil
dependent; blocking P-selectin dramatically reduced renal
myeloperoxidase activity, indicating attenuated neutrophil
infiltration. Therefore, we suggest that inhibition of P-selectin
dependent neutrophil recruitment may be the mechanism responsible for
the protection seen in P-selectin-deficient mice and in wild-type mice
after antibody treatment. As the peritubular vessels provide blood
supply to the outer medulla representing the most
ischemia/hypoxia-sensitive zone of the kidney (39)
,
neutrophils adherent to either endothelium or platelets could damage
the surrounding tissue by two mechanism. First, adherent neutrophils
can impair or obstruct blood flow in small vessels leading to local
tissue hypoxia in downstream regions (no-reflow phenomenon). Second,
while sticking to and later infiltrating through the endothelium,
neutrophils can release cytotoxic substances, destroying neighboring
cells and tissue matrix (reflow-paradox). Both mechanisms have been
postulated in ischemia/reperfusion-induced acute renal failure
(3)
.
Blocking P-selectin to protect from ischemia/reperfusion-induced organ
failure has been shown in the heart (40
, 41)
, brain
(42
, 43)
, and liver (44)
. In a rat model of
cold renal ischemia-reperfusion, Takada et al. (45)
showed
a protective effect of soluble P-selectin glycoprotein ligand 1
(PSGL-1), suggesting a role for P-selectin under these circumstances.
However, PSGL-1 may bind to and block E- and L-selectin in addition to
P-selectin (14)
.
Our study is the first showing a clear role for P-selectin in
ischemia/reperfusion-induced acute renal failure. Similar results have
been obtained for ICAM-1 (6)
and CD11a/CD11b
(17)
. It is not possible to compare our results with that
of blocking CD11a/CD11b (17)
, as the study protocol
differs from ours with respect to animals (uninephrectomized rats) and
duration of ischemia. ICAM-1-deficient mice showing decreased renal
myeloperoxidase levels were protected against I/R-induced renal failure
(6)
in the same model of renal ischemia presented here.
Since no data from sham-operated, ICAM-1-deficient mice were reported
in that study, the efficiency of blocking P-selectin cannot rigorously
be compared with that of blocking ICAM-1. However, in ICAM-1-deficient
mice, absolute creatinine concentrations at 24 and 48 h were
reduced by 60 and 90%, respectively.
In conclusion, in our neutrophil-dependent model of severe ischemia/reperfusion-induced acute renal failure, blocking P-selectin reduces neutrophil infiltration into postischemic kidneys and protects from the development of renal failure, even after the onset of reperfusion.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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