Selective blockade of lysophosphatidic acid LPA 3 receptors reduces murine renal - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:43 |只看该作者 |倒序浏览 |打印

作者：Mark D. Okusa, Hong Ye, Liping Huang, Laura Sigismund, Timothy Macdonald, and Kevin R. Lynch作者单位：Departments of 1 Medicine, 2 Chemistry, and 3 Pharmacology,University of Virginia, Charlottesville, Virginia 22908 5 r6 ]0 {, B5 S% D/ G( _5 a

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      【摘要】- ^% e& P0 [& k& T# a  m, P' l
   Lysophosphatidic acid (LPA) released during ischemia has diverse physiological effects via its G protein-coupled receptors, LPA 1, LPA 2, and LPA 3 (formerly Edg-2, -4, and -7). We testedthe hypothesis that selective blockade of LPA receptors affords protectionfrom renal ischemia-reperfusion (I/R) injury. By real-time PCR,LPA 1-3 receptor mRNAs were expressed in mouse renal cortex, outermedulla, and inner medulla with the following rank order LPA 3 =LPA 2 LPA 1. In C57BL/6 mice whose kidneys weresubjected to ischemia and reperfusion, treatment with a selectiveLPA 3 agonist, oleoyl-methoxy phosphothionate (OMPT), enhancedinjury. In contrast, a dual LPA 1 /LPA 3 -receptorantagonist, VPC-12249, reduced I/R injury, but this protective effect was lostwhen the antagonist was coadministered with OMPT. Interestingly, delayingadministration of VPC-12249 until 30 min after the start of reperfusion didnot alter its efficacy significantly. We conclude that VPC-12249 reduces renalI/R injury predominantly by LPA 3 receptor blockade and could serveas a novel compound in the treatment of ischemia acute renal failure.
      【关键词】 VPC oleoylmethoxy phosphothionate kidney acute renal failure$ e& A: \3 }5 \6 d0 R
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THE SIMPLE PHOSPHOLIPID, lysophosphatidic acid (LPA) is an autacoid that, like prostaglandins and adenosine, can be generated by manycell types and binds to a family of G protein-coupled receptors(LPA 1, LPA 2, and LPA 3 )( 4, 7, 16 ). LPA has heterogeneous functional effects including cellular proliferation, alterations indifferentiation, cell survival, suppression of apoptosis, and plateletaggregation ( 21, 32 ). In addition to theseeffects, LPA is similar to other inflammatory lipid mediators(platelet-activating factor, prostaglandins, thromboxane) and has the capacityto evoke an immune response by attracting and activating immune cells andregulating leukocyte endothelial cell interaction ( 9 ). In the heart, theprecursor to LPA, lysophosphatidyl choline (LPC)( 31 ), is produced byactivation of phospholipase A 2 (PLA 2 ) and yieldsdeleterious 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 appliedto primary explants of proximal tubule cells, leads to cell disruption viadamage to the plasma membrane. These multiple divergent responses are likelymediated by specific LPA receptors that have distinct signaling mechanisms.The high concentration of LPC in plasma and serum suggests that localproduction of LPA could evoke an inflammatory response that may play asignificant role in ischemia-reperfusion (I/R) injury.
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We took advantage of recent advances in the pharmacology and molecularbiology of LPA receptors to determine whether LPA receptors are involved I/Rinjury. Our results highlight a potential role of LPA 3 receptors inmediating injury and a potential novel target for therapeuticintervention.
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METHODS# _. g& V, ^3 M2 @
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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 foodand water until the day of surgery. Mice were anesthetized with a regimen thatconsisted 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-clampedfor 27-32 min. On release of the clamps, the kidneys were observed forreperfusion. Surgical wounds were closed, and mice were returned to cages forup to 24 h. At the end of the experimental period, animals werereanesthetized, blood was obtained by cardiac puncture, and kidneys wereremoved for various analyses., v5 {. `7 Y+ r" R

Compound administration. A 1 mM stock solution of 1-oleoyl LPA (18:1 LPA), VPC-12249, or oleoyl-methoxy phosphothionate (OMPT) was preparedin a 3% fatty acid-free bovine serum albumin/PBS solution (Sigma, St. Louis,MO). Two protocols were followed for drug administration. For dose-responseexperiments (0.01, 0.1, and 1.0mg·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 todetermine whether delayed treatment altered the efficacy of VPC-12249. In thissecond protocol, the compound administration was initiated 0.5, 1.0, or 1.5 hafter the onset of reperfusion and was continued every 2 h for four additional doses.; y' t; Z$ U* j! p
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Tail cuff blood pressure and heart rate measurement. Systolic blood pressure and heart rate were measured by using a photoelectric sensorfor pulse detection in mouse tail (IITC model 179, IITC/Life ScienceInstruments, Woodland Hills, CA). Mice were allowed to rest quietly for 10 minin a chamber with the temperature controlled at 26°C. Blood pressures weremeasured twice and averaged. Measurements were made at baseline beforecompound administration, 30 and 60 min after intraperitoneal injection.  o/ y! Q4 r' b7 \

Plasma creatinine measurement. Plasma creatinine concentrations were determined using a colorimetric assay according to the manufacturer'sprotocol (Sigma).- x1 b4 z9 b  ?) b
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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 wassnap-frozen in liquid N 2 until time of assay. Kidneys werehomogenized 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 potassiumphosphate 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 supernatantwas added to an equal volume of a solution consisting of o -dianisidine (10 mg/ml), 0.3% H 2 O 2, and 45 mM potassium phosphate, pH 6.0. Absorbance was measured at 460 nm over a periodof 5 min ( 1 ).
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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 andE) and viewed by light microscopy (Zeiss AxioSkop). We quantified the degreeof tubular necrosis using a semiquantitative index of H and E-stained tissuesections. Sections were viewed in a masked fashion (MDO) under x 400magnification, and the percentage of tubules showing epithelial necrosis was assigned the following scoring system: 0 = normal; 1 = , G3 A" f- i1 p& Z* O, T
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Immunohistochemistry. Sections were subjected toimmunohistochemistry using methods previously described( 24 ). We used a rat monoclonal antibody to mouse neutrophils (Clone 7/4, Burlingame, CA). Sections wereincubated with primary antibody (2 µg/ml) followed by a biotinylated goatanti-rat secondary antibody. A peroxidase reaction was performed according tothe manufacturer's protocol (Vectastain ABC Elite kit). Sections were viewedusing 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/contrastwas adjusted.% n" s0 [- L" i9 o# Z! D0 D. u6 ~
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RNA isolation and cDNA synthesis. Kidneys were harvested from micefollowing reperfusion for 24 h. The capsule was removed, and kidneys wereplaced immediately in RNA-later (Ambion, Austin, TX) and stored at -70°Cuntil RNA preparation. Total RNA was extracted from whole kidney or cortex,outer medulla, and inner medulla tissue using the Ultraspec RNA IsolationSystem according to the manufacturer's protocol (Biotecx Laboratories, Houston, TX). cDNA was synthesized from the total RNA isolated from thecortex, outer medulla, and inner medulla tissue using the Thermo-Script RT-PCRSystem according to the manufacturer's protocol (Invitrogen, LifeTechnologies, Carlsbad, CA).
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RT-PCR. The presence of LPA 1, LPA 2, andLPA 3 receptor mRNAs in the cortex, outer medullary, and innermedullary 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). PCRoligonucleotide primers ( Table1 ) 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, andelongation 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 aholding step at 4°C.
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Table 1. Primers for PCR! B/ j; y/ f" X3 A

Quantitative real-time PCR. Quantitative real-time PCR was performed on the cDNA from the cortex, outer medullary, and inner medullarytissue using the QuantiTect SYBR Green PCR Kit according to the manufacturer'sprotocol (Qiagen, Valencia, CA). PCR oligonucleotide primers( Table 1 ) were designed using the software package Primer Select (Lasergene, DNASTAR). The PCR master mixwas prepared by combining the following reagents to the indicated endconcentrations and the final volume of 50 µl: 1 x QuantiTect SYBRGreen PCR Master Mix, 0.3 µM forward primer, 0.3 µM reverse primer( Table 1 ). The PCR master mixwas combined with 2 µl cDNA (1 µg reverse-transcribed total RNA) in a96-well PCR Plate for iCycler iQ (Bio-Rad Laboratories, Hercules, CA) andsubsequently covered with Optical Quality Sealing Tape for iCycler iQ (Bio-RadLaboratories). The PCR plate was centrifuged and placed in the iCycler iQ Real-Time Detection System (Bio-Rad Laboratories). The following PCR protocolwas used: denaturation program (95°C for 13 min), amplification andquantification program repeated 45 times (94°C for 30 s, 56°C for 30s, 72°C for 30 s with a single fluorescence measurement), melting curveprogram (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 Systemsoftware (Bio-Rad Laboratories). A threshold value of 10 times the meanstandard deviation of fluorescence in all wells over the baseline cycles wascalculated. This threshold is located in the region of exponentialamplification of all samples. The number of cycles required for a sample to cross this threshold was compared with the number of cycles required for GAPDHto cross the threshold. These ratios were plotted and compared. Each samplewas run in duplicate.- Q3 m7 @$ w8 t$ b7 j9 n6 \8 L1 L
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Statistical analysis. A randomized block design was used to analyze the data. In this design, we considered the day of the procedure as ablocking factor. This is done to minimize variables that may affect ourcomparisons. 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 someanalyses, unpaired Student's t -tests were used. P

RESULTS" X9 a/ h0 j1 X/ u: b- b1 [4 ~" j
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LPA and sphingosine 1-phosphate receptor mRNA expression in mousekidneys. The family of Edg receptors was first examined to determine theexpression of LPA and sphingosine 1-phosphate (S1P) receptor mRNA in kidneys. Table 1 lists primers used toamplify LPA and S1P receptor mRNA from mouse kidneys. LPA 1, LPA 2, and LPA 3 mRNA were detected in whole mouse kidneymRNA ( Fig. 1 ). Furthermore,S1P 1, S1P 2, and S1P 3 mRNA were expressed inmouse kidney; however, S1P 4 and S1P 5 mRNA were not found in mouse kidneys. The results of quantitative real-time PCR of LPA receptormRNA in kidney are shown in Table2. The abundance of LPA receptors relative to GAPDH was in thefollowing order LPA 3 = LPA 2 LPA 1 in thecortex, outer medulla, and inner medulla.

Fig. 1. Lysophingolipid receptor mRNA expression in kidney. Kidney mRNA wasanalyzed by the PCR (see METHODS ). Briefly, a PCR master mixconsisted of PCR buffer, primer, and each primer and Platinum Taq DNAPolymerase. 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 finalelongation step (72°C for 7 min) and a holding step at 4°C. Arrowsindicate appropriate size amplification product for lysophosphatidic acid(LPA 1 ), LPA 2, and LPA 3 receptor mRNA. Alsoshown are amplification products for sphingosine 1-phosphate (S1P) receptormRNAs. Primer composition and predicted sizes of amplification products can befound in Table 1.( N' M3 y5 G) @" M. L' ~/ D/ |

Table 2. Quantitative expression of LPA receptor mRNA in mouse kidney: e' G! K" {/ v
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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, wasadministered 2 h before I/R injury followed by four subsequent doses every 2 hfor four additional doses. As shown in Fig.2, 18:1 LPA at the lower doses produced a modest degree ofprotection. 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

Fig. 2. Effect of 18:1 LPA, a nonselective LPA agonist, on renalischemia-reperfusion (I/R) injury. Mouse kidneys were subjected to 32 min ofischemia followed by 24-h reperfusion. After reperfusion, mice were killed andblood was obtained for measurement of plasma creatinine. Mice were treatedwith vehicle or 18:1 LPA (0.01, 0.1, and 1mg·kg - 1 ·dose - 1 )beginning 2 h before ischemia followed by additional doses every 2 h for 4additional doses. Values are means ± SE; n = 4 for each group. * P ; D( K4 ]. T; l! x+ F! R( y/ \

Eighteen-to-one LPA is more potent at the LPA 1 andLPA 2 receptors compared with the LPA 3 receptor,suggesting a lower affinity for the latter site( 2 ). The loss of significantprotection 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 LPA 3 receptors, thus the highest dosage may have activated this receptor andnegated the beneficial effects observed at lower doses. To test thishypothesis, we next used OMPT, a highly selective LPA 3 receptoragonist ( 10 ). Mouse kidneys were subjected to 32 min of ischemia followed by 24 h of reperfusion, and OMPTwas administered 2 h before I/R followed by four additional doses every 2 hbeginning at the onset of reperfusion. Unlike 18:1 LPA, OMPT did not exert aprotective effect at any dose tested; indeed, this LPA 3 receptor-selective agonist appeared to aggravate injury at the 0.1-mg/kg dose( Fig. 3 A ). 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-gradeinjury induced by 32 min of ischemia may have prevented our observing a higherdegree 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. Withthis protocol, we found that OMPT (0.1mg·kg - 1 ·dose - 1 ) produced significant injury compared with vehicle( Fig. 3 B ). Plasmacreatinine was 0.39 ± 0.05 ( n = 8) and 0.92 ± 0.17mg/dl ( n = 6) ( P ; _6 _& K' Y. P
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Fig. 3. Effect of oleoyl-methoxy phosphothionate (OMPT), a selectiveLPA 3 agonist on renal I/R injury. A : Mouse kidneys weresubjected to 32 min of ischemia followed by 24-h reperfusion. Afterreperfusion, mice were killed and blood was obtained for measurement of plasmacreatinine. Mice were treated with vehicle or OMPT (0.01, 0.1, and 1mg·kg - 1 ·dose - 1 )beginning 2 h before ischemia followed by 4 additional doses every 2 h. Valuesare means ± SE; n = 4-5 for each group. B : mousekidneys were subjected to 27 min of ischemia followed by 24-h reperfusion.Mice were treated with vehicle ( n = 8) or OMPT (1.0mg·kg - 1 ·dose - 1; n = 8) beginning 2 h before ischemia followed by 4 additional dosesevery 2 h. Values are means ± SE. * P

Dual LPA 1 /LPA 3 antagonist reduced renal I/R injury. We next performed a series ofexperiments to determine the effect of blocking LPA 3 receptors onI/R injury. For these experiments, we used VPC-12249, a dualLPA 1 /LPA 3 antagonist( 14 ). Mouse kidneys weresubjected to 32 min of ischemia/24 h reperfusion and treated with vehicle orVPC-12249 (0.01, 0.1, and 1.0 mg·kg - 1 ·dose - 1 )( Fig. 4 A ). Whereasvehicle treatment led to a marked increase in plasma creatinine (1.49 ± 0.26 mg/dl), VPC-12249 at 0.01, 0.1, and 1.0mg·kg - 1 ·dose - 1 ledto 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 ' \/ i8 l/ Q  s/ C5 t
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Fig. 4. Effect of dose and timing of administration of VPC-12249 on renal I/Rinjury. A : mouse kidneys were subjected to 32-min ischemia and 24-hreperfusion and treated with vehicle or VPC-12249 (0.01, 0.1, and 1.0mg·kg - 1 ·dose - 1 )beginning 2 h before ischemia followed by 4 additional doses every 2 h. Valuesare means ± SE; n = 4 for each group. * P

To determine whether delaying administration until after the start of thereperfusion period can protect kidneys from injury, we administered vehicle orVPC-12249 120 min before ischemia as well as 30, 60, and 90 min after theonset of reperfusion ( Fig.4 B ). This figure shows that the fractional reduction ofplasma is greatest when VPC-12249 is administered 2 h before ischemia.Compared with vehicle treatment, plasma creatinine (fractional change) wasreduced by 0.75 ± 0.09. A similar reduction of plasma creatinine wasobserved when VPC-12249 was delayed by 30 min. In this case, the fractionalchange was 0.56 ± 0.09, which was not significantly different from the75% 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 ofplasma creatinine compared with vehicle treatment was 0.22 ± 0.16( P " u+ Z3 _) T# d$ K8 c1 M/ [' f# j  A

Protective effect of VPC-12249 on mice subjected to renal I/R injury ismediated by LPA 3 receptors. To determinewhether the protective effect of VPC-12249 was due to blockade ofLPA 3 and/or LPA 1 receptors, we coadministered OMPT andVPC-12249 ( Fig. 5 ). Weperformed renal I/R in mice treated with vehicle, VPC-12249 (0.1mg·kg - 1 ·dose - 1 ), orthe combination of OMPT (1.0mg·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
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Fig. 5. Effect of VPC-12249 on renal injury is mediated by LPA 3 receptors. Mouse kidneys were subjected to 32-min ischemia and 24-hreperfusion and treated with vehicle, VPC-12249 (0.1mg·kg - 1 ·dose - 1 ), orVPC-12249 (0.1mg·kg - 1 ·dose - 1 )  OMPT (1.0mg·kg - 1 ·dose - 1 ).Values are means ± SE. NS, not significant.( Q% F& M' n' R0 j% L& o5 S

VPC-12249 reduces tubular injury and ischemic necrosis following I/Rinjury in mice. Light microscopic analysis of the outer medulla followingI/R injury revealed a loss of brush-border villi, tubular necrosis, andobstruction of proximal tubule cells in outer medulla of vehicle-treated mice( Fig. 6 C ). The severity of the injury was reduced markedly in kidneys from mice treated withVPC-12249 ( Fig. 6 D ).Similar reduction of tissue injury was evident from the cortex and outermedullary ( Fig. 6, A and B, vehicle and VPC-12249, respectively) and innermedullary ( Fig. 6, E and F, vehicle and VPC-12249, respectively) sections. Table 3 demonstrates injuryquantitatively in the cortex, outer medulla, and inner medulla of kidneys.VPC-12249 reduced injury significantly in the outer medulla." X/ `: c6 F  P/ E6 M3 J2 c) U5 m

Fig. 6. Effect of VPC-12249 on renal morphology following I/R. Mouse kidneys weresubjected to 32-min ischemia and 24-h reperfusion and were treated withvehicle ( A, C, D ) or VPC-12249 (1mg·kg - 1 ·dose - 1; B, D, F ) beginning 2 h before ischemia followed by 4 additional dosesevery 2 h. Kidneys were fixed and paraffin embedded, and 4-µm sections werecut and stained with hematoxylin and eosin. A and B : cortex; C and D : outer medulla; and E and F : innermedulla. Magnification x 200.& r- D" }# x9 O8 @) ]4 }" ?) Y) A0 }

Table 3. Renal injury following ischemia-reperfusion* |  h" c0 c2 a& d! k4 Y

VPC-12249 reduces leukocyte infiltration in kidneys subjected to I/Rinjury in rats. Leukocytes are thought to contribute to renal injuryfollowing I/R, therefore we examined the degree of leukocyte infiltration byusing MPO activity. We found that I/R injury produced an increase in MPOactivity in mice following 24 h of reperfusion. VPC-12249 reduced kidney MPOactivity; MPO activity was 1.50 ± 0.10 ( n = 15) and 0.84± 0.14 OD 460 ·g - 1 ·min - 1 at 24 h ( n = 15) in vehicle- and VPC-12249-treated mice, respectively( P

Fig. 7. Effect of VPC-12249 on myeloperoxidase (MPO) activity of kidneys subjectedto I/R. Mouse kidneys were subjected to 32-min ischemia followed by 24-hreperfusion. VPC-12249 (1mg·kg - 1 ·dose - 1 ) wasadministered intraperitoneally beginning 2 h before I/R followed by 4additional doses every 2 h. MPO activity( OD 460 ·g - 1 ·min - 1 )for vehicle (filled bar; n = 15) or VPC-12249 treated (hatched bar; n = 15). Values are means ± SE. * P ' e: n4 E5 o$ I# k
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Fig. 8. Effect of VPC-12249 on neutrophil accumulation in the outer medulla ofkidneys subjected to I/R. Mouse kidneys were subjected to 32-min ischemiafollowed by 24-h reperfusion. Vehicle ( A and7 L: h+ y7 Y) l0 s4 t
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C ) or VPC-12249 (1mg·kg - 1 ·dose - 1; B and D ) was administered intraperitoneally beginning 2 hbefore 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 outermedulla. Magnification x 400.6 O4 i1 Z3 H3 k. N/ j
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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 effectof VPC-12249 and OMPT; therefore, we measured blood pressure and heart rate atbaseline and 30 and 60 min after intraperitoneal administration of vehicle,VPC-12249 (0.1mg·kg - 1 ·dose - 1 ),OMPT (1.0mg·kg - 1 ·dose - 1 ), orVPC-12249 OMPT. Blood pressure ( Fig.9 A ) and heart rate ( Fig. 9 B ) were notaffected by any of the treatment regimens at any of the time points.

Fig. 9. Effect of VPC-12249 and OMPT on blood pressure and heart rate. Mice wereadministered intraperitoneal vehicle, VPC-12249 (0.1mg·kg - 1 ·dose - 1 ),OMPT (1.0mg·kg - 1 ·dose - 1 ), orVPC-12249 OMPT every 2 h, and blood pressure ( A ) and heart rate( B ) were measured at baseline before injection, 30 and 60 min aftereach injection. There was no significant difference between groups at each ofthe time points. SE were omitted for clarity; n = 4 for eachgroup.
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DISCUSSION6 M. B/ a( J0 F3 T0 ~

We found that LPA had a paradoxical effect in our model of kidney I/Rinjury. That is, at lower doses, 18:1 LPA was protective, but this protectionwas lost at a higher dosage. However, when a LPA 3 receptor-selective agonist was substituted for 18:1 LPA, there was noprotective effect at any dose; indeed, administration of this agonistexacerbated moderate injury. Interestingly, a LPA 3 -receptorantagonist reduced injury in a dose-dependent fashion and this protection waslessened by collision with the LPA 3 receptor agonist. Because twoLPA receptor types predominate in the kidney and one of these(LPA 2 ) appears to have a distinctly higher affinity than the other(LPA 3 ), we explain our results by ascribing a protective effect toagonist occupation of the LPA 2 receptor while agonist occupation ofthe other (LPA 3 ) receptor facilitates I/R injury. There is abiphasic effect of a nonselective agonist (i.e., 18:1 LPA) because protection occurs when the "protective" LPA receptor (LPA 2 ) isoccupied, but this is reversed at increased doses as the LPA 3 receptor is occupied. The beneficial action of the antagonist is due toblockade of the "injury-promoting" receptor (LPA 3 ).This explanation predicts that a selective LPA 2 -receptorantagonist, were it available, would not be protective and might worsen injury.
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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[ 35 S]GTP S binding, etc.) showed consistently that theEC 50 values for LPA at LPA 3 are one to two log ordershigher than those measured at the LPA 1 and LPA 2 receptors. 1 Weinterpret these data as meaning that the LPA 3 receptor is alow-affinity LPA binding site compared with the LPA 1 andLPA 2 receptors. However, some of the difference might be due topoorer coupling of the LPA 3 receptor to G proteins.7 O) P7 H+ h" p2 a$ o/ v+ \/ h$ m
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VPC-12249 behaves as a competitive antagonist, thus its binding affinitycan be determined by Schild analyses; i.e., a radioligand binding assay is notrequired. The K i values so determined are LPA 1 = 125 nM, LPA 2 5,000 nM, and LPA 3 = 425 nM( 22 ). Note that although theaffinity of LPA 3 for VPC-12249 is lower than that ofLPA 1, VPC-12249 might be a "better" blocker at LPA 3, if we are correct in our assumption that this receptor has alower affinity for LPA. A more subtle point is that the rank-order potency of16:0 LPA and 18:1 LPA is different at various LPA receptors. For example,LPA 1 16:0 = 18:1; LPA 2 16.0 = 18:1; and LPA 3 16:0
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OMPT is an agonist at LPA 3; in all assays, EC 50 forOMPT = EC 50 for 18:1 LPA, whereas at the other two LPA receptors, OMPT
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Our results are significant on several levels. First, our data extend anunderstanding of LPA actions in the kidney. In general, LPA has been viewed asa survival factor ( 5, 8, 15, 18, 21, 27 ); indeed, LPA treatment ofprimary cultures of dispersed mouse proximal tubule cells opposed apoptosis ofthese cells ( 18 ). This conceptis in line with our observation that lower doses of LPA are protective in theI/R injury model. Our present results suggest that LPA levels rise in thekidney 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 increaseduring injury ( 20 ). WhetherLPC or LPA is increased in other forms of renal injury is unknown.Furthermore, ischemic injury is known to lead to metabolism of membranephospholipids in the heart( 28 ), brain( 26 ), and liver( 3 ). The recent discovery of alysophospholipid-preferring phospholipase D (lysoPLD), autotaxin( 31 ) [also known asectonucleotide phosphodiesterase pyrophophatase (ENPP) type 2], suggests thatLPC is a direct precursor of LPA. Interestingly, an isotype of this enzyme, gp130 RB13-6 (ENPP-3)( 29 ), which also exhibitslysoPLD activity (Lynch KR, unpublished observations), is highly expressed in the mouse kidney (Okusa MD, unpublished observations). Investigations of theregulation and of this enzyme and the effect of its blockade on I/R injury areactive areas of investigation in our laboratories.
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The mechanism for protection is not known as multiple factors are known toparticipate in I/R injury. Inflammatory infiltration appears to play animportant role in the pathogenesis of I/R injury( 23, 30 ). LPA participates in theinflammatory process by acting as a chemoattractant, by activating monocytes( 6, 17 ), and by increasing tissueinflammatory cell infiltration ( 11 ). Agents that reduceinflammatory cell infiltration have reduced renal I/R injury. Both biochemicalmeasurements of MPO and histological staining of neutrophils determined that VPC-12249 reduced leukocyte infiltration. This may lead to a decrease ininflammation, leukocyte-induced stasis of the medullary circulation, andreduce renal I/R injury. Whether a direct effect of VPC-12249 on proximaltubules or inflammatory cells mediates this protective effect is notknown.8 _" O7 J0 t$ k4 D' H

Second, our results suggest functions for two LPA receptors. LPA evokes abewildering variety of responses when applied to cells in culture, butheretofore receptor antagonists and selective agonists have not been availableto assign these various responses to individual receptors. Although the tool compounds that we used in this study are imperfect, their activities dosuggest assignment of opposing activities to the LPA 2 and LPA 3 receptors. The LPA 2 receptor, which is occupied atlower LPA concentrations, exerts a protective (perhaps antiapoptotic) effect,whereas the LPA 3 receptor, which is occupied only when theLPA 2 site is saturated, is injurious, perhaps by sending aproapoptotic signal. Thus the evolution of a LPA receptor type with adistinctly lower affinity for LPA (i.e., the LPA 3 receptor) mightbe explained in that it is a sensor to countermand the signals sent throughthe higher-affinity LPA receptors (i.e., LPA 1 and LPA 2 )when LPA levels rise above a certain threshold. It will be interesting tolearn whether this bimodal action is operative in other pathologies such ascancer, where the opposite effect of LPA (proapoptosis) is desirable.6 G$ D& ~* L7 [
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Third and finally, the activity of our LPA-receptor antagonist, VPC-12249,in preventing renal I/R injury in the mouse model suggests the LPA 3 receptor as a therapeutic target for an important set of pathologies. We areparticularly encouraged by the ability of VPC-12249 to remain efficacious inpreventing ischemic injury even when administered 30 min after the start ofreperfusion. If verified, LPA receptor blockers might include drugs that wouldbe useful not only prophylactically (i.e., abdominal surgery) but alsoreactively (i.e., trauma). Despite the development in animals of new compoundsfor the treatment and prevention of acute renal failure, results in humanstudies have been disappointing. There are many reasons for this observation including severity of illness of humans in acute renal failure studies, lackof sensitive markers of renal function, and the design of clinical trials.Certainly, major emphasis is now placed on the design of clinical trials andnew early markers of acute renal failure. This effort should lead to bettertrial design, and we believe that innovative compounds such as VPC-12249 couldlead to improved renal protection from ischemia injury in humans." K* s, K! V2 |: k; c4 h

DISCLOSURES) m8 s9 E# l# N# x& o$ z* x

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), andR-01-CA-088994 (to K. R. Lynch)].
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ACKNOWLEDGMENTS5 C( }. Z2 K  m; m. [1 X

The authors gratefully acknowledge the careful reading of the manuscript byDr. D. L. Rosin, Department of Pharmacology, University of Virginia. OMPT wasa gift from Dr. G. Mills, Department of Experimental Therapeutics, MD AndersonCancer Center.
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