ABSTRACT
Title
Farnesoid-X-receptor (FXR) agonist INT-747 restores hepatic DDAH activity after ischemia/reperfusion injury.
Authors
A. Bianchi1, A. Ferrigno1, V. Rizzo2, P. Richelmi1, M. Vairetti1.
1Dept. of Internal Medicine and Therapeutics and 2Dept. of Biochemistry, IRCCS San Matteo, University of Pavia; Pavia, Italy.
1Dept. of Internal Medicine and Therapeutics and 2Dept. of Biochemistry, IRCCS San Matteo, University of Pavia; Pavia, Italy.
Abstract
Background-Aims: The liver plays a crucial role in the metabolism of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, by degradation via dimethylarginine dimethylaminohydrolase (DDAH) (Nijveldt et al. Clin Nutr 2003). DDAH, widely distributed in rats and humans, is a highly oxidation-sensitive enzyme that may be inhibited by oxidative stress (Tain and Baylis. Kidney Int. 2007). Recently, Farnesoid X receptor (FXR) has been found to control blood levels of ADMA in mouse liver via the coordinated regulation of DDAH and the cationic amino acid transporters, CAT-1, (Hu et al., J Biol Chem. 2006). The goals of our study were: first, to investigate the effects of hepatic ischemia-reperfusion (I/R) injury on DDAH activity, second, whether the administration of INT -747, a FXR agonist, restores the DDAH activity.
Methods: The effects of I/R were studied in vivo in a normothermic hepatic partial I/R model. Ischemia to the left and median lobe was induced by clamping the portal vein and hepatic artery with microvascular clips for 60 min, then the clips were removed and the liver was reperfused for 60 min. To prevent postsurgical dehydration and hypotension 1 ml of saline was injected in the inferior vena cava. Sham-operated control animals had similar manipulation of the liver hilus without vascular occlusion and were kept under anesthesia for an equal length of time. To investigate the effect of FXR activation on DDAH activity, rats were orally administered 10 mg/kg/day of the INT-747 (Intercept Pharmaceuticals Inc.), a FXR agonist, in vehicle (methylcellulose 1%) for 5 days or with vehicle alone. Serum levels of AST, ALT, Alkaline Phosphatase (AP), ADMA and Arginine were determined. Hepatic biopsies were used for the determination of DDAH activity and the tissue oxidative status by evaluating reduced and oxidized glutathione (GSH/ GSSG) and lipid peroxidation.
Results: A marked increase in serum AST, ALT and AP levels after I/R confirmed the hepatic damage. An increase in ADMA levels and a decrease in Arginine concentrations were observed in serum samples at the end of reperfusion, supporting previous finding (Trocha et al., Pharmacol Rep 2010). A significant reduction in DDAH activity was found after I/R, but no changes in GSH/GSSG ratio and lipid peroxidation were observed as compared with sham-operated rats. The INT-747 administration completely prevented the decrease in DDAH activity after I/R damage .
Conclusions: ADMA and DDAH are widely distributed in tissues and appear to play a central role in regulating NO synthesis in physiological and/or pathological states. In this study we found that hepatic I/R injury elevates ADMA levels by impairing DDAH activity. The reduction in DDAH, for the first time documented after I/R injury, do not seems oxidative-stress-mediated as suggested by GSH/GSSG ratio and lipid peroxidation levels comparable with those observed in sham-operated rats. Interestingly, the INT-747 treatment, an agonist of FXR, restores the DDAH activity after I/R injury. FXR is a member of the nuclear receptor superfamily of ligand-activated transcription factors that functions as an endogenous sensor for bile acids and it could represent a new chance in the control of the complex mechanisms involved in the hepatic I/R injury.
Nijveldt et al. (2003).Clin Nutr 22:99-104.
Tain and Baylis (2007).Kidney Int. 72:886-9.
Hu et al. (2006). J Biol Chem. 281:39831-8.
Trocha et al. (2010). Pharmacol Rep 62:343-51.
Methods: The effects of I/R were studied in vivo in a normothermic hepatic partial I/R model. Ischemia to the left and median lobe was induced by clamping the portal vein and hepatic artery with microvascular clips for 60 min, then the clips were removed and the liver was reperfused for 60 min. To prevent postsurgical dehydration and hypotension 1 ml of saline was injected in the inferior vena cava. Sham-operated control animals had similar manipulation of the liver hilus without vascular occlusion and were kept under anesthesia for an equal length of time. To investigate the effect of FXR activation on DDAH activity, rats were orally administered 10 mg/kg/day of the INT-747 (Intercept Pharmaceuticals Inc.), a FXR agonist, in vehicle (methylcellulose 1%) for 5 days or with vehicle alone. Serum levels of AST, ALT, Alkaline Phosphatase (AP), ADMA and Arginine were determined. Hepatic biopsies were used for the determination of DDAH activity and the tissue oxidative status by evaluating reduced and oxidized glutathione (GSH/ GSSG) and lipid peroxidation.
Results: A marked increase in serum AST, ALT and AP levels after I/R confirmed the hepatic damage. An increase in ADMA levels and a decrease in Arginine concentrations were observed in serum samples at the end of reperfusion, supporting previous finding (Trocha et al., Pharmacol Rep 2010). A significant reduction in DDAH activity was found after I/R, but no changes in GSH/GSSG ratio and lipid peroxidation were observed as compared with sham-operated rats. The INT-747 administration completely prevented the decrease in DDAH activity after I/R damage .
Conclusions: ADMA and DDAH are widely distributed in tissues and appear to play a central role in regulating NO synthesis in physiological and/or pathological states. In this study we found that hepatic I/R injury elevates ADMA levels by impairing DDAH activity. The reduction in DDAH, for the first time documented after I/R injury, do not seems oxidative-stress-mediated as suggested by GSH/GSSG ratio and lipid peroxidation levels comparable with those observed in sham-operated rats. Interestingly, the INT-747 treatment, an agonist of FXR, restores the DDAH activity after I/R injury. FXR is a member of the nuclear receptor superfamily of ligand-activated transcription factors that functions as an endogenous sensor for bile acids and it could represent a new chance in the control of the complex mechanisms involved in the hepatic I/R injury.
Nijveldt et al. (2003).Clin Nutr 22:99-104.
Tain and Baylis (2007).Kidney Int. 72:886-9.
Hu et al. (2006). J Biol Chem. 281:39831-8.
Trocha et al. (2010). Pharmacol Rep 62:343-51.