Oral presentation at ISHEN 2014!

ISHEN symposium

We wish an awesome congress to all participants of ISHEN 2014!

This biennial conference organized by the ISHEN (International Society for Hepatic Encephalopathy and Nitrogen metabolism) gathers all researchers involved in the field of hepatic encephalopathy. It will be held from 10 to 14 September 2014, London, UK.

We particularly wish to Marc-André to give a great talk, for whom this will be his first oral presentation at an international conference. Dr Bemeur will present and Dr. Rose, who organized a round table. In addition, we will present three posters of our recent work.

Related Publications

Oral

The novel glutaminase inhibitor CB-839 prevents oral glutamine-induced hyperammonemia in portacaval shunted rats.

Cristina R. Bosoi, Marc-André Clément, Mélanie Tremblay, Christopher F. Rose.

Background: Ammonia plays a major role in the pathogenesis of hepatic encephalopathy (HE) and therefore ammonia-lowering treatments remain a primary therapeutic strategy. Glutamine deamidation by the mitochondrial enzyme glutaminase (GLS) is believed to a major source of ammonia production in cirrhotic patients and increased intestinal GLS activity has been shown to be linked to minimal HE and to an increased risk of developing overt HE. CB-839 is a potent, selective and orally bioavailable GLS inhibitor (Gross et al., Mol Cancer Ther 13:890) that is currently in Phase 1 clinical trials for the treatment of cancer (clinicaltrials.gov). Aim: To evaluate the effect of CB-839, a GLS inhibitor, in preventing the onset of hyperammonemia following an oral glutamine challenge (OGC) in rats with portacaval anastomosis (PCA). Methods: Four week PCA rats received a single dose of CB-839 (gavage, 200 mg/kg diluted in 5ml/kg of vehicle). Control PCA rats received equivalent volumes of vehicle. Four hours after CB-839 or vehicle administration, PCA rats received an oral glutamine challenge (gavage, 100 mg/kg). Repeated aortic blood samples were obtained at baseline, 0.5h, 1h, 1.5h, 2.5h, 2.5h, 3h and 4h following OGC. Glutamine and ammonia were measured using commercial available kits. Results: Baseline ammonia levels were similar in both PCA groups. Following OGC, blood ammonia increased in vehicle-treated PCA rats with a peak at 2h (2.3-fold increase vs baseline, p<0.05). In CB-839 treated-PCA rats, ammonia levels did not change compared to the baseline value and were significantly decreased compared to non-treated PCA rats (p<0.05). At 4h, ammonia levels returned to baseline values in both groups. Baseline glutamine levels were not significantly different between treated and non-treated PCA rats. Following OGC, no significant difference between glutamine levels was observed in non-treated PCA rats compared to baseline values. However, in PCA rats treated with CB-839 glutamine levels significantly increased compared to non-treated PCA controls attaining a peak at 2.5 h (1.59 ± 0.40 mM vs 0.60 ± 0.15 mM, p<0.05). At 4h, glutamine levels remained significantly increased. Conclusions: CB-839 treatment inhibited glutamine induced hyperammonemia in PCA rats. These preliminary results strongly suggest CB-839 is an effective agent to attenuate GLS-induced ammonia production. Further studies are warranted to evaluate CB-839 as a novel agent for the treatment of HE.

Oral

Bile-duct ligated rats are susceptible to hypotension-induced neuronal cell loss: Implications for persisting neurological complications following liver transplantation.

Marc-André Clément, Cristina Bosoi, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.

Background: Hepatic encephalopathy (HE) is a major neuropsychiatric complication caused by liver disease characterized by cognitive and motor dysfunction. The only curative treatment to date remains liver transplantation (LT). Historically, HE has always been considered to be a reversible metabolic disorder and has therefore been expected to completely resolve following LT. However, even following the implantation of a new liver, persisting neurological complications remain a common problem affecting as many as 47% (8 47%) of liver transplant recipients. LT is a major surgical procedure accompanied by intraoperative stress and confounding factors, including blood loss (hypovolumia) and hypotension. We hypothesize, in the setting of MHE, that the compromised brain becomes predisposed to what would normally be an innocuous hypotensive insult, resulting in cell injury and death. Methods: Using 6-week bile-duct ligated rats and respective controls, blood is withdrawn from the femoral artery (inducing hypovolemia) until an arterial pressure of 30 mmHg (hypotension) and maintained for 150 minutes. Upon sacrifice, brains are perfused and extracted for western blotting and immunohistochemistry. Results: Both BDL rats and SHAM-operated controls without hypotension do not display any neuronal loss. However, BDL rats following hypotension demonstrated a significant decrease in neuronal cell count in the frontal cortex using NeuN+DAPI and Cresyl Violet compared to hypotensive SHAM-operated controls. In addition, neuronal loss was associated with an increased in cellular stress protein, hsp32, hsp70 and caspase-3, suggesting apoptotic cell death. Discussion: These findings suggest that patients with HE are more susceptible to hypotension-induced neuronal cell loss and this may explain why transplanted patients are experiencing persisting neurological complications. Aside from cirrhotic patients having a stroke, these results also suggest a patient with HE (even MHE) with a “frail brain”, fare worse during transplantation leading to poor neurological outcome. This implies MHE should not be ignored and therefore treated pre-LT.

Bumetanide normalizes brain edema in rats with bile duct ligation: evidence for increased bbb permeability via NKCC1.

Jimmy Huynh, Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.

Background: Brain edema is a serious complication associated with hepatic encephalopathy (HE) due to chronic liver disease. An increase in BBB ion permeability (increase uptake of ions and accompanied by water fluxes towards the brain) can occur across an intact BBB through alterations in transport mechanisms. Endothelial cells that comprise the BBB secrete up to 40% of brain interstitial fluid as they transport Na+, K+ and 2 Cl- across the BBB. NH4+ has very similar ionic properties to K+ (similar ionic radius and diffusion coefficient) and can be transported through K+ channels and cotransporters, implying that hyperammonemia could result in BBB hyperpermeability. An increase in BBB permeability via transport Na+-K+-2Cl- (NKCC1) has shown to promote brain edema and astrocyte swelling under pathophysiological conditions such as ischemia. Aim: To study the BBB integrity (vasogenic vs cytotoxic) and the role of NKCC1 in the pathogenesis of brain edema in cirrhotic rats. Methods: Two distinct animal models of chronic liver failure and HE are used in the present study; 1) biliary cirrhosis model (6 weeks bile duct ligation (BDL)). 2) portacaval shunt model (4 weeks portacaval anastomosis (PCA)). Both models develop hyperammonemia however brain edema is only observed in BDL. BBB breakdown was assessed by measuring brain extravasation of Evans blue and sodium fluorescein (injected i.v). Expression of BBB tight junction proteins (occludin, claudin-5, ZO-1 and ZO-2) were assessed by Western blot. Bumetanide was administered (i.p) for 10 days in BDL and BDL SHAM. Brain water content was measured in the frontal cortex using the specific gravimetric method. Levels of brain NKCC1 mRNA were evaluated by RT-PCR in cerebral microvessels. Results: Extravasation of Evans blue and sodium fluorescein was not detected and there was no significant change in all tight junction protein levels measured in both BDL and PCA models. Brain water content was reduced in bumetanide-treated BDL rats compared to control (77.66±0.15% vs 78.12±0.21%). In brain microvessels, NKCC mRNA increased in BDL rats compared to BDL SHAM (0.78±0.09 vs. 1.92±0.42) whereas no change was found in PCA compared to PCA SHAM (1.72±0.52 vs. 1.53±0.23). Conclusions: BDL rats did not demonstrate a change in BBB integrity or in expression of BBB tight junction proteins. This suggests brain edema in BDL is not of vasogenic origin. Furthermore, since brain edema was only observed in BDL rats (vs PCA), this implies additional factors aside ammonia, are involved in the pathogenesis of brain edema. Moreover, an increase of NKCC1 mRNA and an attenuation of brain edema following bumetanide treatment were demonstrated in BDL rats suggesting NKCC1 plays a role in the development of brain edema in chronic liver disease. Furthermore, these results demonstrate the potential therapeutic use of bumetanide for the treatment of HE.

Beneficial effects of muscle mass optimization using leucine supplementation and exercise training in the prevention of hepatic encephalopathy in experimental cirrhosis.

Chantal *Bémeur, Sara Ghezzal, Marc-André Clément, Cristina R. Bosoi, Roxanne Beauchamp, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.

Background: Malnutrition is an important prognostic factor potentially influencing clinical outcome of patients suffering from chronic liver disease (cirrhosis; CLD). Malnutrition, considered a consequence of metabolic disturbances (hypermetabolism), exacerbates severe muscle loss and hepatic encephalopathy (complex neuropsychiatric disorder) in cirrhotic patients. New management strategies focussing on improving nutritional status and attenuating CLD-related complications are an unmet clinical need. We hypothesize supplementation with branched-chain amino acid leucine (LEU) and exercise training (EX) could possibly attenuate muscle mass loss and prevent hepatic encephalopathy (characterized by brain edema as well as cognitive and psychomotor impairments) in CLD. Methods: CLD was induced in rats following 6-week bile-duct ligation (BDL). Five experimental groups were tested; 1) BDL; 2) BDL + LEU; 3) BDL + EX; 4) BDL + LEU + EX; 5) Sham-operated rats. One week following BDL, rats were submitted to 15 min EX (10 cm/s) every other day and BDL rats receiving LEU, were gavaged daily (1.35 mg/kg) for 5 weeks. Body weight, muscle (gastrocnemius) mass, metabolic state (calculation of energy expenditure independent of food intake and fecal mass), cerebral edema (specific gravity method) and cognitive/psychomotor function (open-field test; anxiety-like behavior assessment and novel object recognition test; memory testing) were measured in all groups. Results: BDL rats gained less body weight and muscle mass compared to sham-operated rats. LEU-treated BDL rats display an improvement in brain edema, muscle mass and circumference and metabolic activity, which was further ameliorated with EX. In addition, BDL rats receiving LEU and EX exhibited less anxiety-like behavior as well as better novel object recognition memory. Conclusion: Our results demonstrate that supplemental LEU along with EX reduces body weight and muscle mass loss, improves metabolic activity, attenuates brain edema and improve cognitive and psychomotor function. These findings suggest that strategies aiming at improving nutritional status will attenuate muscle mass loss, reduce the risk of developing hepatic encephalopathy and therefore improve quality of life and decrease mortality in CLD. LEU supplementation and EX could rapidly be translated into clinical practice.