Alejandro Esteller

Cholestasis in the rat by means of intravenous administration of cyclosporine vehicle, Cremophor EL

The effect of cyclosporine vehicle, Cremophor EL, on bile flow and biliary bile acids and bilirubin output was studied in anesthetized male Wistar rats. Intravenous administration of Cremophor EL or castor oil as a single bolus reduced bile flow and the biliary output of bile acids and bilirubin. The Cremophor EL-induced cholestasis was an immediate and reversible phenomenon, since at 30-35 min after drug injection all parameters evaluated had returned to control values. A slight increase in serum bilirubin concentrations was observed. Our data indicate that the observed cholestasis is related to a reduction in both bile acid-dependent and bile acid-independent bile flow, probably due to a transitory hepatotoxic effect of Cremophor EL. We conclude that the clinically used vehicle for i.v. administration of cyclosporine, Cremophor EL, has adverse effects on hepatobiliary physiology in the rat and suggest that an alternative vehicle should be used.

Long-term nebivolol administration reduces renal fibrosis and prevents endothelial dysfunction in rats with hypertension induced by renal mass reduction

Objectives D/L-Nebivolol is a lypophilic β1-adrenergic antagonist which is devoid of intrinsic sympathomimetic activity and can increase nitric oxide (NO) bioavailability with its subsequent vasodilating properties. The purpose of the present work was to assess the effect of long-term nebivolol administration on both renal damage and endothelial dysfunction induced by renal mass reduction (RMR) in rats. Atenolol, which does not increase NO bioavailability, was included in the study as a comparative β-adrenoceptor antagonist. Methods Rats were subjected to both right nephrectomy and surgical removal of two-thirds of the left kidney in order to retain approximately one-sixth of the total renal mass. One week after ablation, rats were distributed randomly according to the following experimental groups: control group containing RMR rats without treatment; RMR rats treated daily with nebivolol for 6 months (drinking water, 8 mg/kg per day); and RMR rats treated daily with atenolol for 6 months (drinking water, 80 mg/kg per day). A group of sham-operated animals was also included. Results Administration of either nebivolol or atenolol similarly reduced arterial pressure in comparison with RMR untreated animals; however, animals receiving nebivolol presented lower levels of collagen type I expression as well as lower glomerular and interstitial fibrosis than those receiving atenolol. Urinary excretion of oxidative stress markers were also lower in animals receiving nebivolol than in rats treated with atenolol. Furthermore, nebivolol prevented RMR-induced endothelial dysfunction more efficiently than atenolol. Conclusions Nebivolol protects against renal fibrosis, oxidative stress and endothelial dysfunction better than equivalent doses, in terms of arterial pressure reduction, of atenolol in a hypertensive model of renal damage induced by RMR.

Inhibition of biliary lipid and protein secretion by cyclosporine A in the rat

We investigated the effect of cyclosporine A (CyA) administered as a single i.v. dose of 20 and 40 mg/kg body wt, on biliary secretion of cholesterol, phospholipid, bile acid, and lysosomal marker and canalicular plasma membrane marker enzymes in anaesthetized Wistar rats. CyA reduced the concentration and biliary secretion of cholesterol, phospholipid and bile acid to a considerable extent; the inhibitory effect of CyA on the biliary secretion of phospholipid and bile acid was greater than that on cholesterol. The biliary outputs of acid phosphatase (AcP) and gamma-glutamyltransferase (gamma-GT) were also diminished by the drug, all these effects being dose-dependent. Maximum decreases in bile acid secretion were observed 10 min after administration, whereas those of cholesterol and phospholipid were delayed. Bile acid concentrations and secretion returned to pretest values at 30-50 min after CyA injection whereas those of cholesterol and phospholipid remained significantly reduced at this time point. The greater inhibitory effect of CyA on the biliary outputs of phospholipid and bile acid relative to cholesterol secretion together with the asynchronous fall and recovery of bile acid, cholesterol and phospholipid concentrations and secretion alter the cholesterol/bile acid, phospholipid/bile acid and cholesterol/phospholipid molar ratios as well as the lithogenic index, thus suggesting that CyA would uncouple biliary lipid secretion from bile acid secretion. Since under physiological conditions biliary lipid and gamma-GT secretion is related to and dependent upon bile acid secretion, we propose that the CyA-induced inhibition on lipid and gamma-GT secretion is, at least partly, secondary to the fall in bile acid output caused by the drug. However, since CyA inhibits secretory processes independent of the hepatobiliary flux of bile acid, such as the exocytic discharge of AcP, and because it also uncouples biliary lipid from bile acid secretion, other mechanisms and factors involved in lipid and protein secretion (such as intracellular transport, canalicular membrane fluidity and/or intracanalicular events) might also be altered by this drug.

Influence of acute ethanol administration on hepatic glutathione metabolism in the rat.

The effect of acute ethanol administration on the hepatic metabolism of glutathione was studied in male Wistar rats. Animals fasted for 18 hr received ethanol (5 g/kg body wt.) through a gastric tube as a 20% (w/v) solution in 0.154 NaCl. Four hours after administration of ethanol liver glutathione content was decreased by 21% when compared to saline-treated controls. A significant reduction (28%) was also found in gamma-glutamylcysteine synthetase activity and plasma glutathione levels were increased non significantly by 17% with respect to control rats. Glutathione S-transferase activity in the liver of ethanol-treated animals was decreased by 28% but no change was found in total glutathione peroxidase activity. The results indicate that the lowered glutathione synthesis could be an important factor contributing to the reduction of hepatic glutathione concentration following the acute ingestion of ethanol.

Effect of chronic ethanol feeding on glutathione and glutathione-related enzyme activities in rat liver

The effects of chronic ethanol consumption on liver glutathione concentrations and glutathione-related enzyme activities were studied in rats over a period of 1-9 weeks. The animals received a liquid diet containing 36% of calories as ethanol or isocaloric carbohydrate. Glutathione concentrations were significantly enhanced following ethanol intake with increases of 99% after 3 weeks and a progressive decrease thereafter. Glutathione S-transferase activity reached a maximum increase of 36% after 2 weeks of ethanol feeding. Glutathione peroxidase activity remained unchanged for the first 6 weeks of treatment, with a tendency to decrease in the last weeks of ethanol consumption. Our findings indicate that chronic ethanol administration profoundly modifies the hepatic metabolism of glutathione and may thus have important effects on the detoxification of xenobiotics by the liver.

Role of prostanoids and nitric oxide inhibition in rats with experimental hepatic fibrosis.

Nitric oxide (NO) and prostaglandins have been proposed as vasodilator substances involved in peripheral vasodilatation characteristic of the liver cirrhosis. A link between NO and prostanoids has been suggested. The present study investigated the effect of simultaneous blockade of both, NO synthase (NOS) and cyclooxigenase (COX) in sham-operated (SO), or rats with bile-duct ligation (BDL) in the development of liver fibrosis. Animals were distributed in two groups SO (n=15) or BDL (n=15). Treatments (5 days) started three weeks after surgical procedure. Both, SO and BDL animals were treated with indomethacin (INDO) (5 mg/kg/day) alone, with NG-nitro-L-arginine-methyl-ester (NAME) (4 mg/kg/day) alone or with INDO and NAME combination at the same doses. At the end of follow-up body weight, packed cell volume, mean arterial blood pressure (MAP) and heart rate were measured. Liver tissue was processed for histological studies. In this study, BDL animals showed a decreased MAP. Treatment with L-NAME in BDL rats increased MAP. The chronic COX inhibition alone did not play an important role in the haemodynamic changes. The BDL produced a loss of hepatic structure, with ductular metaplasia that occupied the greater part of the hepatic parenchyma. Also, an important degree of fibrosis was observed. Both NO and PG synthesis inhibitors, alone or in combination, induced enhancing collagen fiber deposition in the hepatic parenchyma. These findings support the notion that the interaction between the NOS and COX pathways should be relevant in hepatic cirrhosis in which both NOS and COX are induced.ResumenEn la vasodilatación periférica característica de la cirrosis hepática han sido involucrados diferentes vasodilatadores como son el óxido nítrico (NO) y las prostaglandinas, entre los cuales se ha sugerido que existe una estrecha relación. En este estudio se investiga el efecto del bloqueo simultáneo de la NO sintasa (NOS) y de la ciclooxigenasa (COX) en ratas con cirugía simulada (SO) o con ligadura del conducto biliar (LCB). Los animales se distribuyen en dos grupos SO (n=15) y LCB (n=15). Los tratamientos (5 días) comienzan tres semanas después de la cirugía. Tanto los animales SO como los LCB se tratan con indometacina (5 mg/kg/día), con NG-nitro-L-arginina-metiléster (NAME) (4 mg/kg/día), o con la combinación de ambos a la misma dosis. Al final del tratamiento se mide el peso corporal, el hematocrito, la presión arterial media (PAM) y la frecuencia cardiaca. Se realizan estudios histológicos en el tejido hepático. Los animales LCB muestran un descenso de la PAM. No se observan cambios hemodinámicos con el tratamiento con indometacina mientras que el NAME eleva la PAM. La LCB produce una pérdida de la estructura hepática, con metaplasia ductular que ocupa la mayor parte del parénquima hepático y un importante grado de fibrosis. Los animales tratados con NAME y/o indometacina muestran un empeoramiento del grado de fibrosis. Nuestros resultados indican que la interacción entre las vías de la NOS y la COX puede ser relevante en la cirrosis hepática, en la cual ambos sistemas están activados.

Bile pigment formation and excretion in the rabbit

Bile and plasma levels of biliverdin and bilirubin, together with the hepatic biliverdin reductase and bilirubin UDP-glucuronosyl transferase activities, were studied in the rabbit. No biliverdin could be detected in the blood plasma. The bilirubin concentration in blood was 7.81 +/- 0.79 mumol/l. Biliverdin was the predominant pigment in bile (63%). Hepatic biliverdin reductase activity was 0.086 +/- 0.016 nmol/mg protein/hr. The synthesis of bilirubin was apparently limited by the enzyme activity. Most of the bilirubin in bile was conjugated (90%) with monoconjugates predominating (75%). Hepatic UDP-glucuronosyl transferase activity was 2.65 +/- 0.18 and 1.14 +/- 0.16 mumol/mg protein/hr with and without activation, respectively.

Changes in biliary secretion and lactate metabolism induced by diethyl maleate in rabbits.

Diethyl maleate is a compound which binds with glutathione by means of a glutathione S-transferase and is excreted into bile leading to a rapid depletion of hepatic glutathione. In the rabbit, the activity of the enzyme is fairly low and we were thus prompted to study the possible effects of diethyl maleate on biliary secretion and metabolic status in this species. The administration of diethyl maleate induced a transient choleresis followed by cholestasis. The choleresis coursed with increases in the biliary output of sodium and unaccounted anions, whereas those of chloride, bicarbonate and bile acids were unaffected. Our data seem to confirm that choleresis is due to the osmotic activity of diethyl maleate compounds excreted into bile, as has been reported in rats and dogs. The cholestasis observed coursed with falls in the outputs of sodium, chloride and bicarbonate though that of bile acids remained constant. Following diethyl maleate administration, a metabolic acidosis appeared with progressive increases of blood lactate concentration. In bile the concentration of this anion closely followed that of plasma. The cholestasis is attributed to a lowered biliary secretion of bicarbonate probably secondary to the metabolic alteration. The hepatic values of cytoplasmatic and mitochondrial NADH/NAD ratios and of adenine nucleotide concentrations suggest that the increase in blood lactate results rather from a fall in its hepatic utilization that from an increase in its production.

Effects of melatonin on fuel utilization in exercised rats: role of nitric oxide and growth hormone.

We have previously reported that melatonin modifies carbohydrate and lipid utilization in exercised rats, maintaining glycemia and reducing plasma and liver lactate and plasma β‐hydroxybutyrate. This study was undertaken to determine whether effects on fuel metabolism were related to changes in nitric oxide (NO) production or growth hormone (GH) secretion. Male Wistar rats received melatonin i.p. at a dose of 0.5 mg/kg body weight 30 min before being exercised to exhaustion on a treadmill at a speed of 24 m/min and a 12% slope. Melatonin ameliorated the decrease in plasma glucose and the increase in plasma urea, free fatty acid, β‐hydroxybutyrate, and nitrite induced by exercise. Melatonin‐treated exercised rats had significantly elevated liver glycogen content and hepatic tissue showed a lowered expression of both inducible and constitutive NO synthase (iNOS and cNOS). Administration of the NO inhibitor NG‐nitro‐l‐arginine (l‐NAME) to exercised rats caused a significant reduction in plasma nitrite, but liver glycogen and biochemical parameters in blood did not significantly differ from untreated exercised animals, indicating the absence of a direct association between melatonin effects on fuel metabolism and NO levels. Although results of treatment with pyridostigmine, a cholinergic agonist drug that stimulates GH release, partially differed from that of melatonin, modulation of GH secretion could play a role in the metabolic actions of the hormone because effects of melatonin on exercised rats were almost completely blocked by simultaneous administration of l‐NAME.

Role of glucose reabsorption from bile on hyperglycaemia-induced cholestasis in the rabbit

The effect of glucose administration on bile secretion of glucose and bile flow and composition was studied in the rabbit. After intravenous glucose infusion at 83 mumol/kg/min a mean bile concentration of 12.7 +/- 1.8 mg/dl was reached. Intraportal administration of phlorizin enhanced bile glucose concentration to 169.6 +/- 18.1 mg/dl, suggesting the presence of a system for transferring glucose from bile to liver in the biliary tree of the rabbit. A significant correlation between bile flow and plasma glucose levels could be demonstrated. A cholestatic effect appeared in glucose-infused rabbits with a decrease in bile flow by about 40% during the second hour of infusion. Both bile acid and inorganic electrolyte output were significantly lowered. Cholestasis was maintained after phlorizin administration. Possible explanations for this effect are discussed.