Restoration of renal function by a novel prostaglandin EP4 receptor-derived peptide in models of acute renal failure.

Acute renal failure (ARF) is a serious medical complication characterized by an abrupt and sustained decline in renal function. Despite significant advances in supportive care, there is currently no effective treatment to restore renal function. PGE(2) is a lipid hormone mediator abundantly produced in the kidney, where it acts locally to regulate renal function; several studies suggest that modulating EP(4) receptor activity could improve renal function following kidney injury. An optimized peptidomimetic ligand of EP(4) receptor, THG213.29, was tested for its efficacy to improve renal function (glomerular filtration rate, renal plasma flow, and urine output) and histological changes in a model of ARF induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 modulated PGE(2)-binding dissociation kinetics, indicative of an allosteric binding mode. Consistently, THG213.29 antagonized EP(4)-mediated relaxation of piglet saphenous vein rings, partially inhibited EP(4)-mediated cAMP production, but did not affect Gα(i) activation or ß-arrestin recruitment. In vivo, THG213.29 significantly improved renal function and histological changes in cisplatin- and renal artery occlusion-induced ARF models. THG213.29 increased mRNA expression of heme-oxygenase 1, Bcl2, and FGF-2 in renal cortex; correspondingly, in EP(4)-transfected HEK293 cells, THG213.29 augmented FGF-2 and abrogated EP(4)-dependent overexpression of inflammatory IL-6 and of apoptotic death domain-associated protein and BCL2-associated agonist of cell death. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP(4) receptor, resulting in improved renal function and integrity following acute renal failure.

Platelet-activating factor in vasoobliteration of oxygen-induced retinopathy.

PURPOSE: To test whether platelet-activating factor (PAF) directly causes retinovascular endothelial cell (EC) death. METHODS: Retinovascular density was calculated in rat pups exposed to 80% O(2) from postnatal days (P)6 to P14 (to produce oxygen-induced retinopathy [OIR]), using the adenosine diphosphatase (ADPase) technique, in animals treated with distinct PAF receptor blockers (PCA-4248, BN52021, or THG315). PAF levels were then measured in the retinas. Viability of ECs from piglets and humans in response to C-PAF (a stable PAF analogue) was determined by the reduction of the tetrazolium salt 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) by viable cells, incorporation of propidium iodide (PI), TUNEL assay, and release of lactate dehydrogenase. Release of thromboxane (TX) was measured in the cell media. RESULTS: PAF levels in retina were markedly increased by exposure of isolated rat retinas to H(2)O(2) (1 micro M) and of rat pups placed in 80% O(2). Exposure to 80% O(2) induced retinal vasoobliteration, which was equally significantly inhibited ( approximately 60%) by all PAF receptor blockers tested. C-PAF increased incorporation of PI by isolated rat retinal microvasculature. Also, C-PAF caused time- and concentration-dependent death of cultured retinal ECs, which was prevented by the PAF receptor antagonist CV-3988. This effect of C-PAF was selective on retinal and neurovascular ECs, but not on other ECs. DNA fragmentation (TUNEL) was hardly detected, and inhibition of apoptosis-related processes by nicotinamide, cyclosporin A, and Z-DEVD-FMK and Z-VAD-FMK (caspase inhibitors) barely protected against death in EC, whereas C-PAF increased release of lactate dehydrogenase, implying that necrosis is the nature of EC death. Finally, C-PAF-induced cell death was preceded by an increase in TXB(2) levels and was prevented by TXA(2) synthase inhibition (with CGS12970). CONCLUSIONS: The data suggest PAF plays a major role in vasoobliteration in OIR by triggering death of neuroretinal microvascular ECs. The cell death seems to be mediated at least in part by TXA(2). These effects of PAF may participate in ischemic retinopathies such as diabetes and retinopathy of prematurity.

THG113: a novel selective FP antagonist that delays preterm labor.

PGF2alpha is an important smooth muscle contractile agent that exerts significant effects on myometrium and is implicated in labor. THG113 was recently identified as a PGF2alpha receptor (FP) antagonist. We characterized the specificity and selectivity of THG113, tested its effects on PGF2alpha-induced smooth muscle contraction, and assessed its efficacy in a model of endotoxin (LPS)-induced preterm labor. [125I]THG113 bound specifically to FP-expressing but not to native (not expressing FP) HEK293 cells. In FP-expressing HEK293 cells, THG113 markedly reduced PGF2alpha-elicited phosphoinositide hydrolysis (IC50 27 nM). Similarly, PGF2alpha-evoked microvascular (retinal) contraction was noncompetitively blocked (by > 90%) by THG113. In contrast, contraction to agonists of homologous prostanoid receptors EP1 and TP (17-phenyl-trinor PGE2 and U46619) was unaffected (< 1%) by high concentrations of THG113 (100 micromol/L); THG113 (100 micromol/L) also did not affect contraction to numerous other agents including platelet activating factor, endothelin, and angiotensin II. Force and duration of PGF2alpha-evoked contractions of myometrial strips of pig (non-pregnant, luteal phase) and mouse (immediately postpartum) were markedly reduced by THG113. In an endotoxin-induced preterm mouse model, lipopolysaccharide (50 microg intraperitioneal) injection at 16 days' gestation resulted in 100% delivery within 15 h; in contrast, 70% of those treated with THG113 (1 mg/day) delivered > 24 h later (at 18 days' gestation; term: 19 days). In addition, in mice injected with lipopolysaccharide and treated 6 h later with THG113 (0.1 mg bolus followed by 1 mg/day) 40% delivered > 48 h later. Fetuses of pregnant mice treated with THG113 were born alive, had higher birth weights (1.6 +/- 0.1 v 1.4 +/- 0.05 g), and appeared healthy. This study describes an effective and selective noncompetitive FP antagonist, THG113, which significantly delays preterm delivery; this provides the basis for future investigations for its use in tocolysis.

Delay of preterm birth in sheep by THG113.31, a prostaglandin F2alpha receptor antagonist.

OBJECTIVE: A novel prostaglandin F2alpha receptor antagonist, THG113.31, was tested for the suppression of uterine contractility and delay of preterm labor in sheep. STUDY DESIGN: We determined the tocolytic effectiveness of THG113.31 on contractions that were stimulated in vitro by prostaglandin F2alpha and E2 in longitudinal and circular myometrial strips. We also tested the ability of THG113.31 in vivo to lower uterine electromyographic activity that was induced by the progesterone receptor blocker, RU486, and to delay preterm birth. RESULTS: THG113.31 suppressed the amplitude of prostaglandin F2alpha, but not prostaglandin E2-induced contractions of both circular and longitudinal myometrium (P<.01). The times to delivery after RU486 were 34.8+/-1.1 hours (saline solution) and 41.9+/-0.5 hours (THG113.31; P<.001) or an average delay of 7.1 hours. There were no changes in fetal blood gases (PaO2 , PaCO2 , pH, or SaO2) because of THG113.31. Fetal cortisol levels rose in each group, and fetal and maternal prostaglandin E2 and F2alpha metabolite concentrations rose similarly in both groups. CONCLUSION: THG113.31 specifically suppresses prostaglandin F2alpha-induced myometrial contractility and delays delivery.