Mechanism of action of cyclosporine in preventing cardiac allograft rejection. II. Graft tissue levels of prostacyclin and thromboxane

Four cyclo-oxygenase products (COP) of arachidonate were measured in tissue homogenates of rat cardiac allografts at intervals after transplantation. In rejecting graft tissue, there was a progressive decrease in the levels of prostaglandin E2 (PGE2) and PGF2 alpha from the time of grafting, which was accompanied by a rise in the levels of prostacyclin (PGI2, measured as its stable hydrolysis product 6-oxo-PGF1 alpha) and thromboxane A2 (TxA2, measured as its stable hydrolysis product TxB2). When the level of each individual COP in rejecting graft tissue was expressed as a percentage of the total COP measured, PGI2 and TxA2 increased from 9% to 36% and from 7% to 25%, respectively, from day three after grafting until the time of graft rejection. In contrast to these changes in COP levels in rejecting grafts, the levels of all four COP in nonrejecting allografts maintained by treatment of the graft recipients with cyclosporine remained normal--i.e., comparable to the levels in the recipients' own hearts.     

Prostaglandin synthesis associated with renal allograft rejection in the dog

Vasospasm and intrarenal thrombosis are characteristics of acute renal allograft rejection. A possible mediator of these phenomena is thromboxane A2. Single kidneys were exchanged between nonimmunosuppressed mongrel dogs. At intervals after transplantation, rejecting and normal kidneys were removed and slices of cortex and medulla were prepared for incubation. The in vitro release of thromboxane B2 (TxB2), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1a (6-keto-PGF1 alpha) into the incubation media was measured by radioimmunoassay. Within 72 hr of transplantation the cortex of rejecting kidneys synthesized 10 to 30 times as much PGE2 and TxB2 as normal controls. A similar increase was not observed for 6-keto-PGF1 alpha synthesis. In the medulla there was a selective reduction in 6-keto-PGF1 alpha production within five days of transplantation. In both cortex and medulla there was a significant increase in the ratio of TxB2 to 6-keto-PGF1 alpha production. Reversal of the normal TxB2:6-keto-PGF1 alpha ratio could induce the widespread intrarenal thrombosis and vasospasm that characterizes acute renal allograft rejection.     

Prostacyclin and thromboxane in acute hemorrhagic pancreatitis in dogs

To study the role of the vasodilatory, antiaggregatory prostacyclin (PGI2) and its endogenous antagonist thromboxane A2 (TxA2) in acute pancreatitis, we measured serum thromboxane B2 (TxB2, which indicates platelet TxA2 production) and plasma 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha, which indicates systemic PGI2 production) from sequential blood samples in trypsin and taurocholate induced acute canine hemorrhagic pancreatitis (AHP). In addition the effect of a prostaglandin synthesis inhibitor, ibuprofen, was studied and systemic (MAP) and pulmonary artery pressure (MPAP) were recorded for 4.5 hr. The animals were divided into a sham-operated group, an AHP group, an ibuprofen prophylaxis group, and an ibuprofen therapy group. In the sham group the parameters remained stable throughout the experiment. In the AHP group MAP decreased steadily and 6-keto-PGF1 alpha rose significantly from 80.0 +/- 7.8 to 956.0 +/- 287.0 pg/ml (P less than 0.001), whereas serum TxB2 and MPAP remained unchanged. Ibuprofen prophylaxis eliminated the initial fall in MAP and the rise of 6-keto-PGF1 alpha. Ibuprofen therapy normalized the initially decreased MAP and depressed the level of 6-keto-PGF1 alpha. We conclude that PGI2 may at least partly mediate the initial hypotension in canine AHP, whereas platelet TxA2 production obviously has a negligible role in the development of hemodynamic changes in AHP.     

Prostacyclin and thromboxane A2 moderate postischemic renal failure

Since prostacyclin (PGI2) is known to regulate renal cortical blood flow and since ischemia stimulates thromboxane (Tx) A2 synthesis, the role of these prostanoids in moderating the response to renal ischemia was studied in the rat. At baseline, plasma TxB2 concentration in untreated animals (n = 13) was 357 pg/ml. The left renal pedicle was clamped for 45 minutes after a right nephrectomy (n = 16), which led after 5 minutes of reperfusion to a rise in TxB2 to 2825 pg/ml (p less than 0.001), but there was no change in 6-keto-PGF1 alpha. After 24 hours creatinine levels rose from 0.4 to 3.0 mg/dl (p less than 0.001), and left renal weight rose from 94% to 117% (p less than 0.001) relative to the weight of the right kidney. In nephrectomized but nonischemic sham control rats (n = 7), creatinine level was 0.9 mg/dl and kidney weight 91% after 24 hours. Pretreatment with OKY 046 (n = 13) (2 mg/kg administered intravenously) blocked ischemia-induced TxB2 synthesis, while 6-keto-PGF1 alpha levels rose from 96 to 302 pg/ml (p less than 0.001). There was no increase in creatinine levels or kidney weight relative to the sham group. Pretreatment with ibuprofen (n = 10) (12 mg/kg) or OKY 046 and ibuprofen (n = 9) inhibited TxB2 and 6-keto-PGF1 alpha synthesis, but creatinine levels and renal weight rose (p less than 0.001). Renal histology in OKY 046-pretreated animals was equal to that in nephrectomized controls, while all other ischemic groups showed tubular necrosis. Results indicate that a high PGI2/TxA2 ratio protects against renal ischemia.     

Effect of conjugated estrogens on platelet function and prostacyclin generation in CRF

In a double-blind, randomized, placebo-controlled cross-over study, we investigated in seven patients with chronic renal failure the effect of conjugated estrogens (0.6 mg/kg/day for 5 days) on template bleeding time and on thromboxane A2 (TxA2), beta-thromboglobulin (beta-TG) and prostacyclin (PGI2) concentrations in blood emerging from the template bleeding time incisions. Administration of conjugated estrogens resulted in a significant shortening of the bleeding time in six out of seven patients with a maximum effect 7 and/or 14 days following treatment. Both TxA2 (measured as thromboxane B2, TxB2) and beta-TG release in bleeding time blood were significantly higher following administration of conjugated estrogens as compared to placebo administration. No difference was seen in endothelial PGI2 (measured as 6-keto-prostaglandin F1 alpha) formation when patients were treated with conjugated estrogens as compared to placebo administration over the 28 day observation period. We conclude that in patients with chronic renal failure, infusion of conjugated estrogens results in a significant shortening of the bleeding time together with an increase in platelet reactivity, as indicated by an increase of TxA2 and beta-TG concentration in the microvasculature. No effect was seen on PGI2 production, thereby excluding a major effect on vascular prostaglandin metabolism.     

The role of thromboxane and prostacyclin in ciclosporin-induced nephrotoxicity.

The aim of the study was to determine the influence of ciclosporin (Cs) on prostacyclin and thromboxane generation. Four groups of patients were studied. Group 1: Controls, n = 10. Group 2: Patients without kidney disease treated with Cs, n = 10. Group 3: Patients after transplantation treated with azathioprine, n = 10. Group 4: Renal transplant recipients receiving Cs, n = 10. Parameters investigated: CIn, CPAH, TxB2 in plasma, serum and urine; 6-oxo-PGF1 alpha in plasma and urine, urinary 2,3-dinor-TxB2 excretion. CPAH and CIn were significantly decreased during Cs treatment. Plasma TxB2 levels were enhanced in patients without kidney disease receiving Cs (group 2) amounting to 189 +/- 106 pg/ml as compared to 12 +/- 4 pg/ml in controls (group 1). In patients without kidney disease (group 2), plasma 6-oxo-PGF1 alpha was increased (20 +/- 9 pg/ml) as compared to controls in group 1. Plasma TxB2 and plasma 6-oxo-PGF1 alpha were increased in renal graft recipients without any difference due to different immunosuppressive drugs. Treatment with Cs was associated with impaired renal function and resulted, in patients without kidney disease, in elevated plasma TxB2 and plasma 6-oxo-PGF1 alpha. This effect could not be proven in renal graft recipients. We suggest that the deleterious effect of Cs on kidney function is presumably not paralleled by corresponding changes in prostaglandin and thromboxane formation.     

Renal prostacyclin biosynthesis is reduced in children with hemolytic-uremic syndrome in the context of systemic platelet activation.

Previous studies have reported various abnormalities in prostacyclin (PGI2) synthesis and metabolism in hemolytic-uremic syndrome (HUS). However, the conclusions of most of these studies are based on in vitro or ex vivo experiments that only give an indirect estimate of the actual biosynthesis in vivo. We studied the urinary excretion of PGI2 metabolites, taken as a marker of the actual biosynthesis, in six children with HUS during the acute phase of the disease and again when remission was achieved. Eight age- and sex-matched healthy children were studied as controls. Since HUS is also associated with platelet activation and consumption, we also studied the urinary excretion of thromboxane A2 (TxA2) metabolites. Urinary PGI2 and TxA2 metabolites were assessed by radioimmunoassay after high-performance liquid chromatography (HPLC) purification. Urinary excretion of the PGI2 hydrolysis product, 6-keto-PGF1 alpha, was significantly reduced in children with acute HUS as compared with controls, indicating a defective renal synthesis of PGI2. A significant inverse correlation was found between urinary 6-keto-PGF1 alpha and blood urea nitrogen (BUN), as well as plasma creatinine. At remission, urinary 6-keto-PGF1 alpha levels increased to values higher than those of controls. By contrast, the urinary excretion of the major PGI2 beta-oxidation product, 2,3-dinor-6-keto-PGF1 alpha, was comparable to controls, indicating normal systemic PGI2 biosynthesis. The urinary excretion of both TxA2 hydrolysis product, TxB2, and the major beta-oxidation metabolite, 2,3-dinor-TxB2, were lower than normal in the acute phase of HUS if expressed as absolute values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostacyclin and thromboxane in chronic uremia: effect of hemodialysis

To study the effects of uremia and hemodialysis on the production rates of antiaggregatory prostacyclin (PGI2) and proaggregatory thromboxane A2 (TxA2), we collected serial plasma samples from eight patients with chronic uremia before, during and after hemodialysis and assayed them for 6-keto-PGF1 alpha and TxB2, the stable metabolites of PGI2 and TxA2, respectively. In addition, the capacity of the platelets to produce TxB2 during spontaneous clotting was studied by measuring the TxB2 levels in serum incubated at +37 degrees C for 60 minutes. The PGI2 production of the uremia patients before hemodialysis was less (P less than 0.001) than that of healthy volunteers. It rose significantly following heparinization and remained elevated during hemodialysis. TxB2 generation by platelets during clotting was diminished in uremia. Plasma TxB2 levels were normal before, but increased during hemodialysis. Thus, profound changes in the PGI2/TxA2-system seem to be associated with uremia and hemodialysis.     

Effects of DOCA-salt treatment on the urinary prostaglandins in Sabra rats

To determine whether the increased renal synthesis of thromboxane (Tx)A2 found in genetically hypertensive rats also occurred in rats with a sodium-dependent form of hypertension, the urinary excretion of 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) and of TxB2 was measured by a sensitive and specific radioimmunoassay in hypertension-prone (SBH), -resistant (SBN) and unselected (SB) female rats of the Sabra strains. Rats of the three strains were studied before (9 weeks of age) and after five weeks of deoxycorticosterone (DOCA)-salt treatment. Before treatment, the urinary 6KPGF1 alpha did not differ among the three strains while a higher TxB2 excretion was seen in the SBN rats. After treatment, the urinary excretion of TxB2 increased significantly in SBH and SB but not in SBN rats, while the urinary 6KPGF1 alpha remained unchanged in SBH, increased moderately in SB and markedly in SBN controls. Consequently, DOCA-salt-induced changes in blood pressure and in urinary 6KPGF1 alpha observed in the three strains of rats were inversely related (r = -0.78; P less than 0.001). It is concluded that the high blood pressure developed after DOCA-salt treatment in SBH rats is more likely to depend upon a defect in the renal production of prostacyclin rather than upon an increased synthesis of thromboxane A2.     

Effects of defibrotide on renal function and urinary prostanoid excretion in ciclosporin-treated rats.

Defibrotide (DF) has been proposed as a new antithrombotic agent in renal transplantation. Because it was also found to increase prostacyclin synthesis, a reduction in ciclosporin (CS) nephrotoxicity could be supposed. To ascertain this hypothesis, renal function and urinary prostanoids were evaluated in four groups of rats after 10 days of oral treatment (doses in mg/kg/day): CS 50 (group A), CS 50 + DF 400 (group B), DF 400 (group C) and controls (group D). Compared to controls, creatinine clearance (CCR) was significantly lower in groups A and B (In CCr: A = 6.62 +/- 0.28, B = 6.83 +/- 0.24 vs. 8.17 +/- 0.13 microliters/min, p less than 0.01), whereas it did not change in group C (8.03 +/- 0.24 microliters/min). The urinary excretion of prostaglandin E2 (PGE2) was significantly (p less than 0.05) higher in group A (In PGE2: 3.98 +/- 0.98 nmol/mol Cr) and more evidently in groups B and C (6.89 +/- 0.38 and 6.01 +/- 0.32 nmol/mol Cr, respectively) compared to controls (1.43 +/- 0.45 nmol/mol Cr). The urinary excretion of 6-keto-PGF1 alpha and of thromboxane B2 (TxB2) were higher only in groups A and B (ln 6-keto-PGF1 alpha and ln TxB2: A = 6.45 +/- 0.22 and 4.97 +/- 0.20, B = 7.06 +/- 0.31 and 5.43 +/- 0.41 vs. group D = 5.53 +/- 0.22 and 3.79 +/- 0.42 nmol/mol Cr; p less than 0.05). The 6-keto-PGF1 alpha/Tx molar ratio was not significantly affected, although a trend for a reduction in the ratio was found in the treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of a stable prostacyclin analogue on canine renal allograft rejection.

The effect of OP-41483 (Ono Pharmaceutical Co., Osaka, Japan), a stable prostacyclin analogue, on canine renal allograft rejection was investigated. Administration for 4 days after transplantation significantly increased renal cortical blood flow and urine output when compared with untreated dogs with renal allografts. Serum creatinine levels remained relatively low during postoperative days 1-4. Mean animal survival time was prolonged. Vascular lesions and mononuclear cell infiltration were greatly diminished in biopsy specimens removed on day 4. This stable prostacyclin analogue provided a degree of protection against canine renal allograft rejection.     

Prostaglandin and thromboxane synthesis by rat glomerular epithelial cells

Isolated rat glomeruli have been shown to synthesize prostaglandin (PG) and thromboxane (Tx). In this study, we evaluated, by radioimmunoassay and radiochromatographic methods, PG and Tx synthesis by glomerular cells in culture. Transmission and scanning electron microscopy showed polygonal cells, attached by desmosomes, with surface microvilli. These features are typical of glomerular epithelial cells. Incubation of these glomerular epithelial cells with arachidonic acid (C20:4) resulted in an array of endproducts with concentrations of PGE2 greater than TxB2 greater than PGF2 alpha greater than 6-keto-PGF1 alpha . Addition of angiotensin II (AII) to the cultured glomerular cell produced almost exclusive stimulation of PGE2 with PGE2 much much greater than PGF2 alpha greater than TxB2 = 6-keto-PGF1 alpha . AII and AIII (100 micrometer to 1 micrometer ) stimulated PGE2 in glomerular epithelial cells, and the increments of PGE2, as a function of the concentration of AII or AIII, were similar. The sar1-thr8-AII analog inhibited both AII- and AIII-stimulated PGE2 synthesis. The divalent cation ionophore A23187 in concentrations of 0.2 to 2.0 micrometer increased primarily PGE2 and TxB2 synthesis with smaller increases of PGF2 alpha and 6-keto-PGF1 alpha . The relative concentrations of PG and Tx produced by rat glomerular epithelial cells, incubated with C20:4 or A23187, were similar. Our results demonstrate that: (1) the predominant cell grown in culture from the rat glomerulus, after 9 days, is the epithelial cell; (2) this cell is capable of PG and Tx synthesis; (3) stimulation of PG by AII and AIII may be mediated by the same cellular receptor, AII and AIII increase primarily the synthesis of a vasodilatory PG, PGE2; (4) exogenous substrate C20:4 or release of endogenous C20:4 by the divalent cation ionophore A23187 not only stimulates PGE2 but also the vasoconstrictor TxA2; and (5) the PG and Tx endproducts synthesized by epithelial cells may be determined by an intracellular coupling of the specific synthetic enzymes with different pools of C20:4.     

Vasodilating prostaglandins attenuate ischemic renal injury only if thromboxane is inhibited.

Ischemia-induced renal injury is prevented by inhibition of thromboxane (Tx) synthesis. This protection was believed to be secondary to a high prostaglandin (PG)/TxA2 ratio. This study tests whether increasing the PG/Tx ratio by administration of vasodilating PGs protects the reperfused ischemic kidney. Anesthetized rats underwent right nephrectomy and 45 minutes of left renal pedicle clamping. Beginning 10 minutes before clamp release, animals were treated intravenously with the following: saline placebo (n = 10); the cyclooxygenase inhibitor ibuprofen (Ibu), 12.5 mg/Kg in a bolus (n = 8); a stable analogue of prostacyclin (PGI2), 500 ng/kg/minute for 2 hours (n = 9); PGE1, 400 ng/kg/minute for 2 hours (n = 8); the combination Ibu and PGI2 (n = 8) or PGE1 (n = 8). In saline treated ischemic controls, 5 minutes after reperfusion plasma, thromboxane (TxB2) and 6-keto-PGF1 levels were 2537 and 317 pg/ml, respectively--higher than the TxB2 and 6-keto-PGF1 levels of 750 and 80 pg/ml, respectively, in nephrectomized but nonischemic sham controls (n = 7) (p less than 0.05). In ischemic control animals at 24 hours, creatinine levels were 4.6 mg/dl, relative to 0.9 ml/dl in sham animals (p less than 0.05); the weight of the left (L) ischemic kidney relative to the right (R) normal kidney was 118%, compared with 99% in sham animals (p less than 0.05); and renal histology of ischemic control animals at 24 hours showed acute tubular necrosis (ATN) relative to normal findings in sham animals. Pretreatment with Ibu led to: TxB2 and 6-keto-PGF1 levels of 116 and 40 pg/ml, lower than those of sham animals (p less than 0.05); creatinine levels of 4.6 mg/dl, L/R renal weight of 119%; and ATN similar to that of ischemic controls. Treatment with a PGI2 analogue or PGE1 was not protective and led to increases in TxB2, 6-keto-PGF1, creatinine, L/R renal weight, and ATN similar to that of ischemic controls. The combination of Ibu and either PGI2 or PGE1 led to: reduced levels of TxB2 and 6-keto-PGF1 (p less than 0.05); attenuated increases in creatinine to 2.2 and 2.3 mg/dl, respectively (p less than 0.05); and limited ATN (p less than 0.05). These data indicate that the vasodilating PG protect the ischemic reperfused kidney only when Tx is inhibited.     

Improved survival after donor pretreatment with anti-lymphoblast globulin (ALG) in perfused kidneys: Experimental findings

Although donor pretreatment has been utilized both experimentally and clinically, its role in conjunction with subsequent hypothermic pulsatile perfusion of renal allografts prior to transplantation has not been clearly defined. This study tests two modes of donor pretreatment and their compatibility with perfusion. Donor dogs were either (1) untreated, (2) pretreated with cyclophosphamide (CY) and methylprednisolone (MP), or (3) pretreated with equine anti-canine antilymphoblast globulin (ALG). Survival and rejection of perfused and nonperfused kidney allografts were compared in an unrelated mongrel dog population using CY and MP or ALG donor pretreatment. Results indicate that the beneficial effects of donor pretreatment with CY and MP are abrogated when kidneys are perfused for 24 hr prior to transplantation (CY + MP, nonperfused, survival = 17.23 ± 4.75, CY + MP, perfused 24 hr, survival = 12.83 ± 3.94 days). Dogs receiving kidneys from donors pretreated with ALG in conjunction with 24-hr pulsatile perfusion, however, demonstrated prolonged renal allograft survival (24.20 ± 10.46 days) (nonpretreated controls, survival = 12.60 ± 3.79 days) (P < 0.001), and the onset of rejection was also significantly delayed (ALG + 24-hr perfusion, graft rejection = 17.87 ± 7.72 days) (non-pretreated controls, graft rejection = 7.87 ± 2.59 days) (P < 0.001). Donor pretreatment of renal allografts with ALG, therefore, appears to be an attractive modality for reduction of graft immunogenicity when perfusion is also utilized prior to transplant.     

Splanchnic prostanoid production: effect of hemorrhagic shock

The effect of hemorrhagic shock on rat splanchnic prostanoid synthesis was examined in vitro. Male rats were bled to a mean pressure of 30 mm Hg for 30 min. At that time a midline laparotomy was performed and the superior mesenteric artery (SMA) was cannulated and removed in continuity with its end organ (SV + SI). The SV + SI was perfused at 3 ml/min with oxygenated Krebs-Henseleit buffer at 37 degrees C. Venous effluent (3 ml) was collected at 15, 30, 60, 90, 120, 150, and 180 min and frozen for analysis of basal prostanoid release. Levels of 6-keto-PGF1 alpha (6-keto), PGE2, thromboxane B2 (TxB2), and PGF2 alpha were analyzed by radioimmunoassay and compared in the shock (SK) and sham-operated control (SM) animals. SK 6-keto and TxB2 increased significantly at 15 min of perfusion and then decreased to a lower baseline from 60 through 120 min of perfusion. The major prostanoid released at all time periods was 6-keto (2 to 20 times higher than other prostanoids). One-way ANOVA (pooled times) showed that 6-keto significantly increased in the SK group compared to the SM group (P less than 0.02). These data suggest that the splanchnic bed attempts to compensate for hemorrhagic shock by the marked release of the endogenous vasodilator prostacyclin. Release of the other prostanoids (including TxB2) from the splanchnic bed was modest in our model.     

Assessment of prostacyclin and thromboxane A2 release during reperfusion after global ischemia induced by crystalloid cardioplegia--comparison between warm and cold ischemia

The metabolites of prostacyclin (PGI2) and thromboxane A2 (TxA2), 6-keto-PGF1 alpha and thromboxane B2 (TxB2), were investigated during reperfusion (RP) following warm (37 degrees C, 60 min, n = 9) or cold (15 degrees C, 120 min, n = 11) ischemia induced by cold (4 degrees C) or normothermic (30 degrees C) K+ cardioplegia (CP) in isolated canine hearts subjected to global ischemia and RP. 6-Keto-PGF1 alpha flux was significantly higher (p less than 0.025) in the warm group at 1, 5, and 10 min of RP (4,202 +/- 1,412, 2,475 +/- 1,875, and 1,255 +/- 633 pg/g.min, mean +/- SD) compared to those in the cold group (1,504 +/- 1,245, 434 +/- 641, and 370 +/- 329 pg/g.min). TxB2 flux was small in amount compared to 6-keto-PGF1 alpha in both groups. Regarding the coronary hemodynamics, the cold group alone showed statistically significant relationships of coronary sinus blood flow to TxB2 level and TxB2/6-keto-PGF1 alpha ratio in coronary sinus blood. Also, coronary vascular resistance showed linear relations to these two parameters of the metabolites. In a supplementary experiment only with cold ischemia for 180 min, 6-keto-PGF1 alpha was released at each coronary flush-out by CP and the incremental amount showed a gradual increase during ischemia. These results indicated that significant production and release of PGI2 occurred during ischemia and RP following CP arrest and these related to the degree of myocardial damage while the response of TxA2 seemed less significant. The role of PGI2 release during RP following cardioplegic arrest was discussed.     

Roles for thromboxane A2 and leukotrienes in endotoxin-induced acute renal failure

Bolus i.v. administration of 100 micrograms/kg of E. Coli lipopolysaccharide endotoxin (LPS) to adult male Munich-Wistar rats (N = 18) resulted in a progressive fall in RBF and GFR from 6.9 +/- 0.2 SE and 1.1 +/- 0.05 ml/min to minimal values at 50 minutes of 3.8 +/- 0.4 and 0.32 +/- 0.08 (P less than 0.05), respectively, without a fall in mean arterial pressure. At 50 minutes, renal cortical generation rates of PGE2 (1075 +/- 108 pg/mg tissue), 6 keto PGF1 alpha (221 +/- 41 pg/mg), and TxB2 (106 +/- 12 pg/mg) were significantly higher than those of vehicle-treated control rats (N = 10, PGE2 = 466 +/- 107, 6 keto PGF1 alpha = 94 +/- 3, and TxB2 = 35 +/- 3 pg/mg), and morphologic examination revealed normal histology with notable absence of leukocytes and platelets. Pretreatment of a third group of nine rats with TxA2 synthetase inhibitor UK-37.248 (dazoxiben, 10 mg/kg) selectively abolished the LPS-induced rise in TxB2 (29 +/- 3 pg/mg), but not PGE2 (837 +/- 62 pg/mg) or 6 keto PGF1 alpha (179 +/- 5 pg/mg), prevented the fall in RBF at 50 minutes (6.3 +/- 0.4 ml/min), and allowed for significant preservation of GFR (0.67 +/- 0.08 ml/min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased thromboxane biosynthesis in childhood hemolytic uremic syndrome

Vascular endothelial cell damage plays a central role in the pathogenesis of the hemolytic uremic syndrome (HUS), resulting in intravascular platelet activation and thrombotic microangiopathy. A deficiency of the antiaggregatory prostacyclin (PGI2) has been postulated by experiments under ex vivo conditions. However, this observation has not been confirmed in vivo. The pathophysiological contribution of thromboxane (Tx)A2, a potent vasoconstrictor and platelet-aggregating prostanoid which is predominantly produced by platelets, has not been elucidated so far. In order to quantitate endogenous formation of TxA2 in children with HUS, plasma concentrations of the enzymatic metabolite 11-dehydro-TxB2 of TxA2 and urinary excretion rates of three major TxA2 metabolites, TxB2, 11-dehydro-TxB2 and 2,3-dinor-TxB2 were analyzed using gas chromatography/mass spectrometry. PGI2 biosynthesis was assessed by measuring urinary excretion of an index metabolite of its systemic production, 2,3-dinor-6-keto-prostaglandin (PG) F1 alpha, and an index of its renal production, 6-keto-PGF1 alpha. TxA2 biosynthesis was markedly elevated in the acute phase of HUS. This activation could be detected for a longer period of time than the presence of thrombocytopenia. Concomitantly in the acute phase, renal PGI2 formation was significantly elevated and systemic PGI2 formation was elevated in 50% of the patients. These data indicate that TxA2 formation is increased in the acute phase in patients with HUS. This enhanced biosynthesis is consistent with increased platelet activation, whereas the increased PGI2 biosynthesis reflects predominantly renal endothelial cell damage.     

Effect of antioxidant therapy on cyclooxygenase-derived eicosanoid release during intestinal ischemia-reperfusion.

Conflicting data have been reported on the relationship between reactive oxygen intermediates and the formation of oxygenase-derived eicosanoids. Plasma levels of prostacyclin (PGI2, measured as the stable metabolite 6-keto-PGF1 alpha) and thromboxane A2 (TxA2, measured as TxB2) in the effluent blood of a canine ileal segment were determined following 1 or 2 h of ischemia. The synthesis of both eicosanoids was significantly stimulated during reperfusion, but extension of the ischemic interval from 60 to 120 min was not followed by a further increase. The role of oxidants potentially involved in the process was investigated by using materials that inactivate the xanthine-oxidase-generated intermediates. Previous studies on the same in vivo animal model had demonstrated the effectiveness of antioxidant therapy in reducing the postischemic histamine release. There was no significant alteration in the amount of eicosanoids synthesized following oral allopurinol, catalase, dimethylsulfoxide, mannitol or desferrioxamine treatment. Intravenously administered allopurinol, however, significantly elevated the postischemic 6-keto-PGF1 alpha/TxB2 ratio. The results suggest that these antioxidants at doses inhibitory to histamine liberation are not effective in influencing the postischemic eicosanoid release. Intravenously administered allopurinol could exert a potentially beneficial effect through a mechanism other than the blockade of xanthine oxidase.     

Endothelin and eicosanoid synthesis in cultured mesangial cells

We investigated the effect of endothelin on the generation of eicosanoids, which are known to regulate basal and stimulated mesangial cell tone. The results showed that endothelin is a potent stimulus of prostaglandin E2 (PGE2), prostacyclin (PGI2), and thromboxane A2 (TxA2) synthesis by bovine mesangial cells. Percentage increases in eicosanoid synthesis induced by endothelin (10(-10) to 10(-6) M), were 50 to 275% for PGE2, 28 to 168% for PGI2 and 42 to 111% for TxA2, respectively. Endothelin-induced eicosanoid synthesis in mesangial cells was concentration, but not time dependent. Aspirin (500 microM) completely prevented endothelin-induced eicosanoid synthesis. The calcium entry blocker nitrendipine (10(-8) M) failed to inhibit endothelin-induced eicosanoid synthesis. These data suggest that endothelin-induced changes in renal circulation and glomerular function in normal and disease conditions may be modulated by the concomitant stimulation of mesangial eicosanoid synthesis.