Selective blockade of lysophosphatidic acid LPA3 receptors reduces murine renal ischemia-reperfusion injury
Mark D. Okusa,1
Hong Ye,1
Liping Huang,1
Laura Sigismund,1
Timothy Macdonald,2 and
Kevin R. Lynch3
Departments of 1Medicine,
2Chemistry, and 3Pharmacology,
University of Virginia, Charlottesville, Virginia 22908
Submitted 17 January 2003
; accepted in final form 21 May 2003
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ABSTRACT
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Lysophosphatidic acid (LPA) released during ischemia has diverse
physiological effects via its G protein-coupled receptors, LPA1,
LPA2, and LPA3 (formerly Edg-2, -4, and -7). We tested
the hypothesis that selective blockade of LPA receptors affords protection
from renal ischemia-reperfusion (I/R) injury. By real-time PCR,
LPA1-3 receptor mRNAs were expressed in mouse renal cortex, outer
medulla, and inner medulla with the following rank order LPA3 =
LPA2 > LPA1. In C57BL/6 mice whose kidneys were
subjected to ischemia and reperfusion, treatment with a selective
LPA3 agonist, oleoyl-methoxy phosphothionate (OMPT), enhanced
injury. In contrast, a dual LPA1/LPA3-receptor
antagonist, VPC-12249, reduced I/R injury, but this protective effect was lost
when the antagonist was coadministered with OMPT. Interestingly, delaying
administration of VPC-12249 until 30 min after the start of reperfusion did
not alter its efficacy significantly. We conclude that VPC-12249 reduces renal
I/R injury predominantly by LPA3 receptor blockade and could serve
as a novel compound in the treatment of ischemia acute renal failure.
VPC-12249; oleoyl-methoxy phosphothionate; kidney; acute renal failure
THE SIMPLE PHOSPHOLIPID, lysophosphatidic acid (LPA) is an
autacoid that, like prostaglandins and adenosine, can be generated by many
cell types and binds to a family of G protein-coupled receptors
(LPA1, LPA2, and LPA3)
(4,
7,
16). LPA has heterogeneous
functional effects including cellular proliferation, alterations in
differentiation, cell survival, suppression of apoptosis, and platelet
aggregation (21,
32). In addition to these
effects, LPA is similar to other inflammatory lipid mediators
(platelet-activating factor, prostaglandins, thromboxane) and has the capacity
to evoke an immune response by attracting and activating immune cells and
regulating leukocyte endothelial cell interaction
(9). In the heart, the
precursor to LPA, lysophosphatidyl choline (LPC)
(31), is produced by
activation of phospholipase A2 (PLA2) and yields
deleterious effects on heart muscle
(12,
13). In rat kidneys,
reperfusion leads to an elevation of LPC, free fatty acids, diacyl glycerol,
and phosphatidic acid (20).
Persistent elevation of these levels correlated with injury and, when applied
to primary explants of proximal tubule cells, leads to cell disruption via
damage to the plasma membrane. These multiple divergent responses are likely
mediated by specific LPA receptors that have distinct signaling mechanisms.
The high concentration of LPC in plasma and serum suggests that local
production of LPA could evoke an inflammatory response that may play a
significant role in ischemia-reperfusion (I/R) injury.
We took advantage of recent advances in the pharmacology and molecular
biology of LPA receptors to determine whether LPA receptors are involved I/R
injury. Our results highlight a potential role of LPA3 receptors in
mediating injury and a potential novel target for therapeutic
intervention.
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METHODS
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Renal ischemia surgical protocol. C57BL/6 mice (7-8 wk of age,
Hilltop Laboratory Animals, Scottsdale, PA) were allowed free access to food
and water until the day of surgery. Mice were anesthetized with a regimen that
consisted of ketamine (100 mg/kg ip), xylazine (10 mg/kg ip), and acepromazine
(1 mg/kg im) and were placed on a thermoregulated pad to maintain body
temperature at 37°C. Both renal pedicles were identified and cross-clamped
for 27-32 min. On release of the clamps, the kidneys were observed for
reperfusion. Surgical wounds were closed, and mice were returned to cages for
up to 24 h. At the end of the experimental period, animals were
reanesthetized, blood was obtained by cardiac puncture, and kidneys were
removed for various analyses.
Compound administration. A 1 mM stock solution of 1-oleoyl LPA
(18:1 LPA), VPC-12249, or oleoyl-methoxy phosphothionate (OMPT) was prepared
in a 3% fatty acid-free bovine serum albumin/PBS solution (Sigma, St. Louis,
MO). Two protocols were followed for drug administration. For dose-response
experiments (0.01, 0.1, and 1.0
mg·kg-1·dose-1),
vehicle (3% fatty acid-free bovine serum albumin/PBS solution), 18:1 LPA,
VPC-12249, or OMPT was administered every 2 h beginning 2 h before ischemia
and continuing for an additional four doses. A second protocol was followed to
determine whether delayed treatment altered the efficacy of VPC-12249. In this
second protocol, the compound administration was initiated 0.5, 1.0, or 1.5 h
after the onset of reperfusion and was continued every 2 h for four additional
doses.
Tail cuff blood pressure and heart rate measurement. Systolic
blood pressure and heart rate were measured by using a photoelectric sensor
for pulse detection in mouse tail (IITC model 179, IITC/Life Science
Instruments, Woodland Hills, CA). Mice were allowed to rest quietly for 10 min
in a chamber with the temperature controlled at 26°C. Blood pressures were
measured twice and averaged. Measurements were made at baseline before
compound administration, 30 and 60 min after intraperitoneal injection.
Plasma creatinine measurement. Plasma creatinine concentrations
were determined using a colorimetric assay according to the manufacturer's
protocol (Sigma).
Myeloperoxidase activity. Myeloperoxidase (MPO) activity was
determined in kidney homogenates
(25). Kidneys were harvested
from mice subjected to the I/R protocol, and a portion of the kidney was
snap-frozen in liquid N2 until time of assay. 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 of ice-cold 50 mM potassium
phosphate buffer with 0.5% hexadecyltrimethylammonium bromide, and sonicated.
The suspension was subjected to three freeze/thaw cycles, 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 (1).
Histology. Kidneys were fixed in periodate-lysine-paraformaldehyde
(4% paraformaldehyde) and embedded in paraffin, and 4-µm sections were cut.
Sections were subjected to routine staining with hematoxylin and eosin (H and
E) and viewed by light microscopy (Zeiss AxioSkop). We quantified the degree
of tubular necrosis using a semiquantitative index of H and E-stained tissue
sections. Sections were viewed in a masked fashion (MDO) under x400
magnification, and the percentage of tubules showing epithelial necrosis was
assigned the following scoring system: 0 = normal; 1 = <10%; 2 = 10-25%; 3
= 26-75%; 4 = >75%. Five to 10 fields from each of the cortex, outer
medulla, and inner medulla were evaluated, scored, and averaged. Some sections
were stained with napthol-AS-D chloroacetate (Sigma) to localize neutrophils
(19,
25).
Immunohistochemistry. Sections were subjected to
immunohistochemistry using methods previously described
(24). We used a rat monoclonal
antibody to mouse neutrophils (Clone 7/4, Burlingame, CA). Sections were
incubated with primary antibody (2 µg/ml) followed by a biotinylated goat
anti-rat secondary antibody. A peroxidase reaction was performed according to
the manufacturer's protocol (Vectastain ABC Elite kit). Sections were viewed
using a Zeiss AxioSkop microscope, and digital images were taken using a SPOT
RT Camera (software version 3.3; Diagnostic Instruments, Sterling Heights,
MI). Images were imported into Adobe Photoshop (3.0) where brightness/contrast
was adjusted.
RNA isolation and cDNA synthesis. Kidneys were harvested from mice
following reperfusion for 24 h. The capsule was removed, and kidneys were
placed immediately in RNA-later (Ambion, Austin, TX) and stored at -70°C
until RNA preparation. Total RNA was extracted from whole kidney or cortex,
outer medulla, and inner medulla tissue using the Ultraspec RNA Isolation
System according to the manufacturer's protocol (Biotecx Laboratories,
Houston, TX). cDNA was synthesized from the total RNA isolated from the
cortex, outer medulla, and inner medulla tissue using the Thermo-Script RT-PCR
System according to the manufacturer's protocol (Invitrogen, Life
Technologies, Carlsbad, CA).
RT-PCR. The presence of LPA1, LPA2, and
LPA3 receptor mRNAs in the cortex, outer medullary, and inner
medullary tissue was confirmed via RT-PCR. RT-PCR was performed on the cDNA
using the ThermoScript RT-PCR System with Platinum Taq DNA Polymerase
according to the manufacturer's protocol (Invitrogen, Life Technologies). PCR
oligonucleotide primers (Table
1) were designed using the software package Primer Select
(Lasergene, DNASTAR, Madison, WI). The following PCR protocol was used:
initial denaturation (95°C for 3 min), denaturation, annealing, and
elongation program repeated 38 times (95°C for 45 s, 59.5°C for 60 s,
72°C for 60 s), final elongation (72°C for 7 min), and finally a
holding step at 4°C.
Quantitative real-time PCR. Quantitative real-time PCR was
performed on the cDNA from the cortex, outer medullary, and inner medullary
tissue using the QuantiTect SYBR Green PCR Kit according to the manufacturer's
protocol (Qiagen, Valencia, CA). PCR oligonucleotide primers
(Table 1) were designed using
the software package Primer Select (Lasergene, DNASTAR). The PCR master mix
was prepared by combining the following reagents to the indicated end
concentrations and the final volume of 50 µl: 1x QuantiTect SYBR
Green PCR Master Mix, 0.3 µM forward primer, 0.3 µM reverse primer
(Table 1). The PCR master mix
was combined with 2 µl cDNA (1 µg reverse-transcribed total RNA) in a
96-well PCR Plate for iCycler iQ (Bio-Rad Laboratories, Hercules, CA) and
subsequently covered with Optical Quality Sealing Tape for iCycler iQ (Bio-Rad
Laboratories). The PCR plate was centrifuged and placed in the iCycler iQ
Real-Time Detection System (Bio-Rad Laboratories). The following PCR protocol
was used: denaturation program (95°C for 13 min), amplification and
quantification program repeated 45 times (94°C for 30 s, 56°C for 30
s, 72°C for 30 s with a single fluorescence measurement), melting curve
program (55-95°C with a heating rate of 0.5°C per 10 s and a
continuous fluorescence measurement), and finally a cooling step to 20°C.
The data generated were analyzed by iCycler iQ Real-Time Detection System
software (Bio-Rad Laboratories). A threshold value of 10 times the mean
standard deviation of fluorescence in all wells over the baseline cycles was
calculated. This threshold is located in the region of exponential
amplification of all samples. The number of cycles required for a sample to
cross this threshold was compared with the number of cycles required for GAPDH
to cross the threshold. These ratios were plotted and compared. Each sample
was run in duplicate.
Statistical analysis. A randomized block design was used to
analyze the data. In this design, we considered the day of the procedure as a
blocking factor. This is done to minimize variables that may affect our
comparisons. We typically perform experiments in which a small group of mice
(experimental and treatment group) is subjected to I/R injury on the same day.
ANOVA and post hoc analysis (Bonferroni or Dunnett) were performed. In some
analyses, unpaired Student's t-tests were used. P < 0.05
was used to determine significance.
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RESULTS
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LPA and sphingosine 1-phosphate receptor mRNA expression in mouse
kidneys. The family of Edg receptors was first examined to determine the
expression of LPA and sphingosine 1-phosphate (S1P) receptor mRNA in kidneys.
Table 1 lists primers used to
amplify LPA and S1P receptor mRNA from mouse kidneys. LPA1,
LPA2, and LPA3 mRNA were detected in whole mouse kidney
mRNA (Fig. 1). Furthermore,
S1P1, S1P2, and S1P3 mRNA were expressed in
mouse kidney; however, S1P4 and S1P5 mRNA were not found
in mouse kidneys. The results of quantitative real-time PCR of LPA receptor
mRNA in kidney are shown in Table
2. The abundance of LPA receptors relative to GAPDH was in the
following order LPA3 = LPA2 > LPA1 in the
cortex, outer medulla, and inner medulla.

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Fig. 1. Lysophingolipid receptor mRNA expression in kidney. Kidney mRNA was
analyzed by the PCR (see METHODS). Briefly, a PCR master mix
consisted of PCR buffer, primer, and each primer and Platinum Taq DNA
Polymerase. The mixture was subjected to initial melting (95°C for 3 min)
followed by 38 successive cycles of denaturation, annealing, and elongation
(95°C for 45 s, 59.5°C for 60 s, 72°C for 60 s), then a final
elongation step (72°C for 7 min) and a holding step at 4°C. Arrows
indicate appropriate size amplification product for lysophosphatidic acid
(LPA1), LPA2, and LPA3 receptor mRNA. Also
shown are amplification products for sphingosine 1-phosphate (S1P) receptor
mRNAs. Primer composition and predicted sizes of amplification products can be
found in Table 1.
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Effect of LPA agonists on I/R. Initially, we sought to determine
the effect of LPA on I/R injury. A nonselective LPA agonist, 18:1 LPA, was
administered 2 h before I/R injury followed by four subsequent doses every 2 h
for four additional doses. As shown in Fig.
2, 18:1 LPA at the lower doses produced a modest degree of
protection. Plasma creatinine values for vehicle, 0.01- and 0.1-mg/kg dosages,
were 2.37 ± 0.81, 1.37 ± 0.17 (58% of vehicle; P <
0.001), and 1.06 ± 0.63 mg/dl (45% of vehicle; P < 0.001),
respectively. This protective effect was lost at the highest dose, i.e., the
plasma creatinine was 1.51 ± 0.17 mg/dl (64% of vehicle; not
significant) at a dosage of 1 mg/kg.

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Fig. 2. Effect of 18:1 LPA, a nonselective LPA agonist, on renal
ischemia-reperfusion (I/R) injury. Mouse kidneys were subjected to 32 min of
ischemia followed by 24-h reperfusion. After reperfusion, mice were killed and
blood was obtained for measurement of plasma creatinine. Mice were treated
with vehicle or 18:1 LPA (0.01, 0.1, and 1
mg·kg-1·dose-1)
beginning 2 h before ischemia followed by additional doses every 2 h for 4
additional doses. Values are means ± SE; n = 4 for each group.
*P < 0.001 compared with vehicle. 18:1 LPA (1.00
mg·kg-1·dose-1) was
not significantly different from vehicle.
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Eighteen-to-one LPA is more potent at the LPA1 and
LPA2 receptors compared with the LPA3 receptor,
suggesting a lower affinity for the latter site
(2). The loss of significant
protection from I/R injury at the highest dose of 18:1 LPA suggested the
possibility that LPA receptors may have different functional effects.
Eighteen-to-one LPA has an apparent lower affinity for LPA3
receptors, thus the highest dosage may have activated this receptor and
negated the beneficial effects observed at lower doses. To test this
hypothesis, we next used OMPT, a highly selective LPA3 receptor
agonist (10). Mouse kidneys
were subjected to 32 min of ischemia followed by 24 h of reperfusion, and OMPT
was administered 2 h before I/R followed by four additional doses every 2 h
beginning at the onset of reperfusion. Unlike 18:1 LPA, OMPT did not exert a
protective effect at any dose tested; indeed, this LPA3
receptor-selective agonist appeared to aggravate injury at the 0.1-mg/kg dose
(Fig. 3A). At this
dose, plasma creatinine following I/R injury was 133% of vehicle treatment;
however, this difference was not significant. We reasoned that the high-grade
injury induced by 32 min of ischemia may have prevented our observing a higher
degree of injury with OMPT. To test this idea, we repeated the experiment,
reducing the degree of ischemia by shortening the time from 32 to 27 min. With
this protocol, we found that OMPT (0.1
mg·kg-1·dose-1)
produced significant injury compared with vehicle
(Fig. 3B). Plasma
creatinine was 0.39 ± 0.05 (n = 8) and 0.92 ± 0.17
mg/dl (n = 6) (P < 0.02) for vehicle and OMPT,
respectively. These results suggest that LPA receptors have multiple
functional effects and that LPA3 receptor activation might enhance
I/R injury.

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Fig. 3. Effect of oleoyl-methoxy phosphothionate (OMPT), a selective
LPA3 agonist on renal I/R injury. A: Mouse kidneys were
subjected to 32 min of ischemia followed by 24-h reperfusion. After
reperfusion, mice were killed and blood was obtained for measurement of plasma
creatinine. Mice were treated with vehicle or OMPT (0.01, 0.1, and 1
mg·kg-1·dose-1)
beginning 2 h before ischemia followed by 4 additional doses every 2 h. Values
are means ± SE; n = 4-5 for each group. B: mouse
kidneys were subjected to 27 min of ischemia followed by 24-h reperfusion.
Mice were treated with vehicle (n = 8) or OMPT (1.0
mg·kg-1·dose-1;
n = 8) beginning 2 h before ischemia followed by 4 additional doses
every 2 h. Values are means ± SE. *P < 0.02.
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Dual LPA1/LPA3
antagonist reduced renal I/R injury. We next performed a series of
experiments to determine the effect of blocking LPA3 receptors on
I/R injury. For these experiments, we used VPC-12249, a dual
LPA1/LPA3 antagonist
(14). Mouse kidneys were
subjected to 32 min of ischemia/24 h reperfusion and treated with vehicle or
VPC-12249 (0.01, 0.1, and 1.0
mg·kg-1·dose-1)
(Fig. 4A). Whereas
vehicle treatment led to a marked increase in plasma creatinine (1.49 ±
0.26 mg/dl), VPC-12249 at 0.01, 0.1, and 1.0
mg·kg-1·dose-1 led
to a dose-dependent decrease in plasma creatinine of 1.06 ± 0.25 (71%
of vehicle; not significant), 0.45 ± 0.125 (45% of vehicle; P
< 0.05), and 0.31 ± 0.04 mg/dl (21% of vehicle; P <
0.01), respectively, compared with control.

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Fig. 4. Effect of dose and timing of administration of VPC-12249 on renal I/R
injury. A: mouse kidneys were subjected to 32-min ischemia and 24-h
reperfusion and treated with vehicle or VPC-12249 (0.01, 0.1, and 1.0
mg·kg-1·dose-1)
beginning 2 h before ischemia followed by 4 additional doses every 2 h. Values
are means ± SE; n = 4 for each group. *P
< 0.05, **P < 0.01 compared with vehicle treatment.
B: administration of VPC-12249 (1
mg·kg-1·dose-1) was
delayed by 0.5, 1.0, and 1.5 h following 32-min ischemia and 24-h reperfusion.
Values are means ± SE; n = 4 for each group.
*P < 0.05 compared with vehicle treatment.
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To determine whether delaying administration until after the start of the
reperfusion period can protect kidneys from injury, we administered vehicle or
VPC-12249 120 min before ischemia as well as 30, 60, and 90 min after the
onset of reperfusion (Fig.
4B). This figure shows that the fractional reduction of
plasma is greatest when VPC-12249 is administered 2 h before ischemia.
Compared with vehicle treatment, plasma creatinine (fractional change) was
reduced by 0.75 ± 0.09. A similar reduction of plasma creatinine was
observed when VPC-12249 was delayed by 30 min. In this case, the fractional
change was 0.56 ± 0.09, which was not significantly different from the
75% reduction observed when VPC-12249 was administered 2 h before ischemia.
However, when VPC-12249 was delayed 1 and 1.5 h, the fractional reduction of
plasma creatinine compared with vehicle treatment was 0.22 ± 0.16
(P < 0.05) and 0.27 ± 0.122 (P < 0.05). These
fractional reductions in plasma creatinine were significantly less than when
VPC-12249 was administered 2 h before ischemia.
Protective effect of VPC-12249 on mice subjected to renal I/R injury is
mediated by LPA3 receptors. To determine
whether the protective effect of VPC-12249 was due to blockade of
LPA3 and/or LPA1 receptors, we coadministered OMPT and
VPC-12249 (Fig. 5). We
performed renal I/R in mice treated with vehicle, VPC-12249 (0.1
mg·kg-1·dose-1), or
the combination of OMPT (1.0
mg·kg-1·dose-1) +
VPC-12249. Plasma creatinine following 32 min of ischemia/24 h reperfusion was
1.51 ± 0.21 (n = 4), 0.36 ± 0.11 (24% of vehicle;
n = 4; P < 0.01), and 0.99 ± 0.17 mg/dl (66% of
vehicle; n = 4; not significant) for vehicle, VPC-12249, and OMPT +
VPC12249. These results suggest that the protective effect of VPC-12249 was
mediated, in large part, by blocking LPA3 receptors.

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Fig. 5. Effect of VPC-12249 on renal injury is mediated by LPA3
receptors. Mouse kidneys were subjected to 32-min ischemia and 24-h
reperfusion and treated with vehicle, VPC-12249 (0.1
mg·kg-1·dose-1), or
VPC-12249 (0.1
mg·kg-1·dose-1) +
OMPT (1.0
mg·kg-1·dose-1).
Values are means ± SE. NS, not significant.
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VPC-12249 reduces tubular injury and ischemic necrosis following I/R
injury in mice. Light microscopic analysis of the outer medulla following
I/R injury revealed a loss of brush-border villi, tubular necrosis, and
obstruction of proximal tubule cells in outer medulla of vehicle-treated mice
(Fig. 6C). The
severity of the injury was reduced markedly in kidneys from mice treated with
VPC-12249 (Fig. 6D).
Similar reduction of tissue injury was evident from the cortex and outer
medullary (Fig. 6, A and
B, vehicle and VPC-12249, respectively) and inner
medullary (Fig. 6, E and
F, vehicle and VPC-12249, respectively) sections.
Table 3 demonstrates injury
quantitatively in the cortex, outer medulla, and inner medulla of kidneys.
VPC-12249 reduced injury significantly in the outer medulla.

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Fig. 6. Effect of VPC-12249 on renal morphology following I/R. Mouse kidneys were
subjected to 32-min ischemia and 24-h reperfusion and were treated with
vehicle (A, C, D) or VPC-12249 (1
mg·kg-1·dose-1;
B, D, F) beginning 2 h before ischemia followed by 4 additional doses
every 2 h. Kidneys were fixed and paraffin embedded, and 4-µm sections were
cut and stained with hematoxylin and eosin. A and B: cortex;
C and D: outer medulla; and E and F: inner
medulla. Magnification x200.
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VPC-12249 reduces leukocyte infiltration in kidneys subjected to I/R
injury in rats. Leukocytes are thought to contribute to renal injury
following I/R, therefore we examined the degree of leukocyte infiltration by
using MPO activity. We found that I/R injury produced an increase in MPO
activity in mice following 24 h of reperfusion. VPC-12249 reduced kidney MPO
activity; MPO activity was 1.50 ± 0.10 (n = 15) and 0.84
± 0.14
OD460·g-1·min-1
at 24 h (n = 15) in vehicle- and VPC-12249-treated mice, respectively
(P < 0.005) (Fig.
7). With the use of a stain for neutrophils, napthol AS-D
choloracetate, or an anti-neutrophil antibody, we observed that neutrophils in
vehicle-treated mice accumulated primarily in the peritubular capillaries of
the outer medulla (Fig. 8, A and
C). VPC-12249 decreased neutrophil accumulation in this
region (Fig. 8, B and
D).

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Fig. 7. Effect of VPC-12249 on myeloperoxidase (MPO) activity of kidneys subjected
to I/R. Mouse kidneys were subjected to 32-min ischemia followed by 24-h
reperfusion. VPC-12249 (1
mg·kg-1·dose-1) was
administered intraperitoneally beginning 2 h before I/R followed by 4
additional doses every 2 h. MPO activity
( OD460·g-1·min-1)
for vehicle (filled bar; n = 15) or VPC-12249 treated (hatched bar;
n = 15). Values are means ± SE. *P <
0.005.
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Fig. 8. Effect of VPC-12249 on neutrophil accumulation in the outer medulla of
kidneys subjected to I/R. Mouse kidneys were subjected to 32-min ischemia
followed by 24-h reperfusion. Vehicle (A and
C) or VPC-12249 (1
mg·kg-1·dose-1;
B and D) was administered intraperitoneally beginning 2 h
before I/R followed by 4 additional doses every 2 h. Kidneys were harvested,
fixed, and embedded in paraffin. Four-micrometer sections were cut.
Neutrophils were detected using napthol-AS-D chloroacetate (A and
B) or an antineutrophil antibody (C and D).
Arrowheads indicate neutrophils in peritubular capillaries of the outer
medulla. Magnification x400.
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Effect of VPC-12249 and OMPT on blood pressure and heart rate.
Alterations in systemic hemodynamics could contribute to the observed effect
of VPC-12249 and OMPT; therefore, we measured blood pressure and heart rate at
baseline and 30 and 60 min after intraperitoneal administration of vehicle,
VPC-12249 (0.1
mg·kg-1·dose-1),
OMPT (1.0
mg·kg-1·dose-1), or
VPC-12249 + OMPT. Blood pressure (Fig.
9A) and heart rate
(Fig. 9B) were not
affected by any of the treatment regimens at any of the time points.

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Fig. 9. Effect of VPC-12249 and OMPT on blood pressure and heart rate. Mice were
administered intraperitoneal vehicle, VPC-12249 (0.1
mg·kg-1·dose-1),
OMPT (1.0
mg·kg-1·dose-1), or
VPC-12249 + OMPT every 2 h, and blood pressure (A) and heart rate
(B) were measured at baseline before injection, 30 and 60 min after
each injection. There was no significant difference between groups at each of
the time points. SE were omitted for clarity; n = 4 for each
group.
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DISCUSSION
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We found that LPA had a paradoxical effect in our model of kidney I/R
injury. That is, at lower doses, 18:1 LPA was protective, but this protection
was lost at a higher dosage. However, when a LPA3
receptor-selective agonist was substituted for 18:1 LPA, there was no
protective effect at any dose; indeed, administration of this agonist
exacerbated moderate injury. Interestingly, a LPA3-receptor
antagonist reduced injury in a dose-dependent fashion and this protection was
lessened by collision with the LPA3 receptor agonist. Because two
LPA receptor types predominate in the kidney and one of these
(LPA2) appears to have a distinctly higher affinity than the other
(LPA3), we explain our results by ascribing a protective effect to
agonist occupation of the LPA2 receptor while agonist occupation of
the other (LPA3) receptor facilitates I/R injury. There is a
biphasic effect of a nonselective agonist (i.e., 18:1 LPA) because protection
occurs when the "protective" LPA receptor (LPA2) is
occupied, but this is reversed at increased doses as the LPA3
receptor is occupied. The beneficial action of the antagonist is due to
blockade of the "injury-promoting" receptor (LPA3).
This explanation predicts that a selective LPA2-receptor
antagonist, were it available, would not be protective and might worsen
injury.
Our conclusions are based on the binding characteristics of LPA, VPC-12249,
and OMPT for LPA receptors. Several groups working in multiple systems (e.g.,
Sf9/baculovirus, Xenopus oocyte, HEK293 cells, broken cell
[35S]GTP
S binding, etc.) showed consistently that the
EC50 values for LPA at LPA3 are one to two log orders
higher than those measured at the LPA1 and LPA2
receptors.1 We
interpret these data as meaning that the LPA3 receptor is a
low-affinity LPA binding site compared with the LPA1 and
LPA2 receptors. However, some of the difference might be due to
poorer coupling of the LPA3 receptor to G proteins.
VPC-12249 behaves as a competitive antagonist, thus its binding affinity
can be determined by Schild analyses; i.e., a radioligand binding assay is not
required. The Ki values so determined are LPA1
= 125 nM, LPA2 > 5,000 nM, and LPA3 = 425 nM
(22). Note that although the
affinity of LPA3 for VPC-12249 is lower than that of
LPA1, VPC-12249 might be a "better" blocker at
LPA3, if we are correct in our assumption that this receptor has a
lower affinity for LPA. A more subtle point is that the rank-order potency of
16:0 LPA and 18:1 LPA is different at various LPA receptors. For example,
LPA1 16:0 = 18:1; LPA2 16.0 = 18:1; and LPA3
16:0 < 18:1. Therefore, if the ischemic kidney produces 16:0 LPA
preferentially over 18:1 LPA, VPC-12249 will be an even better blocker at the
LPA3 receptor site. Note that the two most prominent LPA species in
serum are 16:0 and 18:1 LPA. Moreover, it should be noted that the expression
of LPA1 mRNA is barely detectable
(Fig. 1 and
Table 2), thus it is possible
that LPA1 receptors are expressed in a very low density. These data
support our contention that VPC-12249 is a better blocker at LPA3
than LPA1 and LPA2.
OMPT is an agonist at LPA3; in all assays, EC50 for
OMPT = EC50 for 18:1 LPA, whereas at the other two LPA receptors,
OMPT <<< LPA.
Our results are significant on several levels. First, our data extend an
understanding of LPA actions in the kidney. In general, LPA has been viewed as
a survival factor (5,
8,
15,
18,
21,
27); indeed, LPA treatment of
primary cultures of dispersed mouse proximal tubule cells opposed apoptosis of
these cells (18). This concept
is in line with our observation that lower doses of LPA are protective in the
I/R injury model. Our present results suggest that LPA levels rise in the
kidney during I/R injury. Although we do not have direct measurements of renal
LPA levels, a related lipid and precursor of LPA, LPC, is reported to increase
during injury (20). Whether
LPC or LPA is increased in other forms of renal injury is unknown.
Furthermore, ischemic injury is known to lead to metabolism of membrane
phospholipids in the heart
(28), brain
(26), and liver
(3). The recent discovery of a
lysophospholipid-preferring phospholipase D (lysoPLD), autotaxin
(31) [also known as
ectonucleotide phosphodiesterase pyrophophatase (ENPP) type 2], suggests that
LPC is a direct precursor of LPA. Interestingly, an isotype of this enzyme,
gp130RB13-6 (ENPP-3)
(29), which also exhibits
lysoPLD activity (Lynch KR, unpublished observations), is highly expressed in
the mouse kidney (Okusa MD, unpublished observations). Investigations of the
regulation and of this enzyme and the effect of its blockade on I/R injury are
active areas of investigation in our laboratories.
The mechanism for protection is not known as multiple factors are known to
participate in I/R injury. Inflammatory infiltration appears to play an
important role in the pathogenesis of I/R injury
(23,
30). LPA participates in the
inflammatory process by acting as a chemoattractant, by activating monocytes
(6,
17), and by increasing tissue
inflammatory cell infiltration
(11). Agents that reduce
inflammatory cell infiltration have reduced renal I/R injury. Both biochemical
measurements of MPO and histological staining of neutrophils determined that
VPC-12249 reduced leukocyte infiltration. This may lead to a decrease in
inflammation, leukocyte-induced stasis of the medullary circulation, and
reduce renal I/R injury. Whether a direct effect of VPC-12249 on proximal
tubules or inflammatory cells mediates this protective effect is not
known.
Second, our results suggest functions for two LPA receptors. LPA evokes a
bewildering variety of responses when applied to cells in culture, but
heretofore receptor antagonists and selective agonists have not been available
to assign these various responses to individual receptors. Although the tool
compounds that we used in this study are imperfect, their activities do
suggest assignment of opposing activities to the LPA2 and
LPA3 receptors. The LPA2 receptor, which is occupied at
lower LPA concentrations, exerts a protective (perhaps antiapoptotic) effect,
whereas the LPA3 receptor, which is occupied only when the
LPA2 site is saturated, is injurious, perhaps by sending a
proapoptotic signal. Thus the evolution of a LPA receptor type with a
distinctly lower affinity for LPA (i.e., the LPA3 receptor) might
be explained in that it is a sensor to countermand the signals sent through
the higher-affinity LPA receptors (i.e., LPA1 and LPA2)
when LPA levels rise above a certain threshold. It will be interesting to
learn whether this bimodal action is operative in other pathologies such as
cancer, where the opposite effect of LPA (proapoptosis) is desirable.
Third and finally, the activity of our LPA-receptor antagonist, VPC-12249,
in preventing renal I/R injury in the mouse model suggests the LPA3
receptor as a therapeutic target for an important set of pathologies. We are
particularly encouraged by the ability of VPC-12249 to remain efficacious in
preventing ischemic injury even when administered 30 min after the start of
reperfusion. If verified, LPA receptor blockers might include drugs that would
be useful not only prophylactically (i.e., abdominal surgery) but also
reactively (i.e., trauma). Despite the development in animals of new compounds
for the treatment and prevention of acute renal failure, results in human
studies have been disappointing. There are many reasons for this observation
including severity of illness of humans in acute renal failure studies, lack
of sensitive markers of renal function, and the design of clinical trials.
Certainly, major emphasis is now placed on the design of clinical trials and
new early markers of acute renal failure. This effort should lead to better
trial design, and we believe that innovative compounds such as VPC-12249 could
lead to improved renal protection from ischemia injury in humans.
 |
DISCLOSURES
|
---|
This work was supported by grants from the National Institutes of Health
[R-01-DK-056223 (to M. D. Okusa), R-01-GM-052722 (to K. R. Lynch), and
R-01-CA-088994 (to K. R. Lynch)].
 |
ACKNOWLEDGMENTS
|
---|
The authors gratefully acknowledge the careful reading of the manuscript by
Dr. D. L. Rosin, Department of Pharmacology, University of Virginia. OMPT was
a gift from Dr. G. Mills, Department of Experimental Therapeutics, MD Anderson
Cancer Center.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: M. D. Okusa, Division
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.
1 Despite much effort by several groups, there is not yet a credible
radioligand binding assay for LPA receptors. Therefore, we do not know
affinity constants (Kd values) for agonist ligands, rather
we have rank order potencies (EC50 values) of a variety of agonist
ligands at each of three LPA receptors. 
 |
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