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Am J Physiol Renal Physiol 285: F565-F574, 2003. First published May 27, 2003; doi:10.1152/ajprenal.00023.2003
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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


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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.


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  		Table 1. Primers for PCR

 

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.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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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  		Table 2. Quantitative expression of LPA receptor mRNA in mouse kidney

 

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.

 

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.

 

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.

 

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.

 

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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  		Table 3. Renal injury following ischemia-reperfusion

 

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 {Delta}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 ({Delta}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.

 

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.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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{gamma}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. Back


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