IntroductionMetabolic syndrome induces endothelial dysfunction, a surrogate marker of cardiovascular disease. In parallel, metabolic syndrome is frequently associated with non-alcoholic fatty liver disease (NAFLD), which may progress to cirrhosis. The aim of the present study was to evaluate intrahepatic endothelial dysfunction related to cyclooxygenase end products and oxidative stress as possible mechanisms involved in the pathophysiology of NAFLD.Materials and MethodsSprague-Dawley rats were fed standard diet (control-diet, CD) or high-fat-diet (HFD) for 6 weeks. Metabolic syndrome was assessed by recording arterial pressure, lipids, glycemia and rat body weight. Splanchnic hemodynamics were measured, and endothelial dysfunction was evaluated using concentration-effect curves to acetylcholine. Response was assessed with either vehicle, L-NG-Nitroarginine (L-NNA), indomethacin, tempol, or a thromboxane receptor antagonist, SQ 29548. We quantified inflammation, fibrosis, oxidative stress, nitric oxide (NO) bioavailability and thromboxane B2 levels.ResultsHFD rats exhibited metabolic syndrome together with the presence of NAFLD. Compared to control-diet livers, HFD livers showed increased hepatic vascular resistance unrelated to inflammation or fibrosis, but with decreased NO activity and increased oxidative stress. Endothelial dysfunction was observed in HFD livers compared with CD rats and improved after cyclooxygenase inhibition or tempol pre-incubation. However, pre-incubation with SQ 29548 did not modify acetylcholine response.ConclusionsOur study provides evidence that endothelial dysfunction at an early stage of NAFLD is associated with reduced NO bioavailability together with increased cyclooxygenase end products and oxidative stress, which suggests that both pathways are involved in the pathophysiology and may be worth exploring as therapeutic targets to prevent progression of the disease.

Gap junctions may contribute to modulating portal pressure and intrahepatic vascular relaxation. Liver gap junctions may represent a new therapeutic target in cirrhotic portal hypertension.

Chronic intermittent hypoxia (CIH) occurs with obstructive sleep apnea syndrome (OSAS) and provokes systemic endothelial dysfunction, which is associated with oxidative stress and low nitric oxide (NO) bioavailability. Cirrhotic livers exhibit intrahepatic endothelial dysfunction, which is characterized by an impaired endothelium-dependent response to vasodilators and hyperresponse to vasoconstrictors. We hypothesized that CIH may also contribute to intrahepatic endothelial dysfunction in cirrhosis. Normal and cirrhotic rats were exposed for 14 days to repetitive cycles of CIH mimicking OSAS in humans, or caged with room air (handled controls [HC]). Hepatic endothelial function was assessed in isolated and perfused rat livers by dose-response curves to acetylcholine (ACh) and methoxamine (Mtx). In a group of cirrhotic rats, in vivo systemic and hepatic hemodynamic parameters were evaluated at baseline and after volume expansion. In addition, liver samples were obtained to assess endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), NO bioavailability, and nitrotyrosinated proteins as a marker of oxidative stress. Cirrhotic rats exposed to CIH exhibited an attenuated vasodilatory response to ACh and hyperresponse to Mtx compared with HC rats. During volume expansion, similar portal pressure increases were observed in CIH and HC rats, although the mean arterial pressure increase was lower after CIH. These functional responses were associated with the presence of increased hepatic oxidative stress without changes in p-eNOS after CIH exposure. In normal rats, no hemodynamic changes were found. Conclusion: CIH exacerbates intrahepatic endothelial dysfunction in cirrhotic rats, which is associated with increased oxidative stress that may reduce NO bioavailability. Clinical studies are needed to assess whether OSAS contributes to endothelial impairment in human patients with cirrhosis. (HEPATOLOGY 2013;57:1564-1574 I ntrahepatic endothelial dysfunction is regarded as a key early event in liver cirrhosis. This impairment is characterized by an abnormal nitric oxide (NO) endothelium-dependent relaxation and an exaggerated response to vasoconstrictors in the hepatic vascular bed. Both factors contribute to increase hepatic vascular resistance, leading to portal hypertension and its complications. 1,2 Obstructive sleep apnea syndrome (OSAS) is characterized by chronic intermittent hypoxia (CIH) and also provokes systemic endothelial dysfunction, as suggested by reduced endothelium-dependent vasodilation. Indeed, several clinical studies have demonstrated reduced flowmediated dilation 3 and blunted vasodilation in response to acetylcholine (ACh), 4 which acts on the endothelium and causes vasodilation through an NO-dependent pathway. In addition, experimental studies using animal models of CIH have shown attenuation in the vascular response to ACh in different vessels 5 and increased vasoconstriction. 6 These responses have been attributed to increased scavenging of NO by reactive oxygen species (ROS), bec...

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Background/Aims: Glomerular filtration rate (GFR) is the best index for evaluating renal function. We aimed to develop a simplified iohexol plasma clearance procedure for GFR measurement in rats without urine collection, animal catheterization or anesthesia, with limited sampling and requiring blood instead of plasma, to further reduce the sample volume and improve animal welfare. Methods: After iohexol injection (129.4 mg), samples were drawn according to 2-compartment kinetics and analyzed by high performance liquid chromatography. Healthy male Lewis rats were used to find a correction factor (CF) to obtain the ‘reference clearance' from the simplified 1-comparment model. This approach was validated using male or female (Lewis, Sprague-Dawley) rats and animals with renal mass reduction (RMR). In additional rats, different simplified approaches were evaluated. Results: Iohexol concentrations in blood and plasma strongly correlated (r = 0.9784, p < 0.0001). A CF of 0.90 enabled the calculation of the reference GFR. Validation results in male Lewis rats were 0.99 ± 0.27 for the reference GFR and 1.03 ± 0.29 ml/min/100 g for the simplified approach. Results in female Sprague-Dawley rats confirmed the suitability of the proposed method. In RMR rats, GFR was 0.14 ± 0.05 and 0.14 ± 0.04 ml/min/100 g for the reference and simplified model, respectively. Conclusion: The procedure we set up to measure GFR in conscious rats was proven to be reliable, required a small volume of blood at only 4 selected time points, without the need to collect urine or catheterize the animals, was applicable to rats from different strains and sexes, both healthy and with renal function impairment. Moreover, the procedure enables the monitoring of GFR changes over time in the same animal, thereby reducing the number of animals to be used.