Hyperammonemia induces mitochondrial dysfunction and neuronal cell death

BACKGROUND & AIMS: In liver cirrhosis, astrocytic swelling is believed to be the principal mechanism of ammonia neurotoxicity leading to hepatic encephalopathy (HE). The role of neuronal dysfunction in HE is not clear. We aimed to explore the impact of hyperammonemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging. METHODS: To primary co-cultures of astrocytes and neurons, low concentrations (1 and 5μM) of NH4Cl were applied. In rats with bile-duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonemia and minimal HE, acute brain slices were studied. One group of BDL rats were treated twice daily with the ammonia scavenger ornithine phenylacetate (OP, 0.3g/kg). Fluorescence measurements of changes in mitochondrial membrane potential (ΔΨm), cytosolic and mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LP) rates, and cell viability were performed using confocal microscopy. RESULTS: Neuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction and reduced cell viability (27.8±2.3% and 41.5±3.7%, respectively) compared to untreated cultures (15.7±1.0%, both p<0.0001). BDL led to increased cerebral LP (p=0.0003) and cytosolic ROS generation (p<0.0001), which was restored by OP (both p<0.0001). Mitochondrial function was severely compromised in BDL resulting in hyperpolarization of ΔΨm with consequent overconsumption of ATP and augmentation of mitochondrial ROS production. Administration of OP restored ΔΨm. In BDL animals, neuronal loss was observed in hippocampal areas, which was partially prevented by OP. CONCLUSIONS: Our results elucidate that low-grade hyperammonemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development. LAY SUMMARY: The impact of hyperammonemia, a common finding in patients with liver cirrhosis, on brain mitochondrial function was investigated in this study. The results show that ammonia in concentrations commonly seen in patients induces severe mitochondrial dysfunction, overproduction of damaging oxygen molecules and profound injury and death of neurons in rat brain cells. These findings point towards a novel mechanism of ammonia-induced brain injury in liver failure and potential novel therapeutic targets

Combination of G-CSF and a TLR4 inhibitor reduce inflammation and promote regeneration in a mouse model of ACLF

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is an unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with Granulocyte-Colony Stimulating Factor (G-CSF) targets inflammation whilst enhancing liver regeneration. METHODS: Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride followed by either lipopolysaccharide (LPS) or galactosamine (GalN) as extrahepatic or hepatic insults, respectively. G-CSF and/or TLR4-antagonist, TAK-242, were administered daily. The treatment duration was 24h and 5d in the LPS model and 48h in the GalN model, respectively. RESULTS: In a LPS-induced ACLF mouse model treatment with G-CSF was associated with a significant mortality of 66% after 48 hours compared with 0% without G-CSF. Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterized by p21 over-expression suggesting hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase of markers of hepatocyte regeneration. CONCLUSION: TLR4 inhibition with TAK-242 rescued G-CSF-driven cell death, inflammation, enhanced tissue repair, and significantly induced regeneration thus suggesting that the combination of G-CSF and TAK-242 is a novel approach for the treatment of ACLF. LAY SUMMARY: The combinatorial therapy of Granulocyte-Colony Stimulating Factor and TAK-242, a Toll-like Receptor-4 inhibitor, achieves the dual aim of reducing hepatic inflammation and inducing liver regeneration for the treatment of acute-on-chronic liver failure

Plasma ammonia levels predict hospitalisation with liver-related complications and mortality in clinically stable outpatients with cirrhosis

BACKGROUND AND AIMS: Hyperammonaemia is central in the pathogenesis of hepatic encephalopathy, but also has pleiotropic deleterious effects on several organ systems, impacting on immune function, sarcopenia, energy metabolism and portal hypertension. This study was performed to test the hypothesis that severity of hyperammonaemia is a risk factor for liver-related complications in clinically stable outpatients with cirrhosis. METHODS: We collected data from 754 clinically stable outpatients with cirrhosis from 3 independent liver units. Baseline ammonia levels were corrected to the upper limit of normal (AMM-ULN) for the reference laboratory. The primary endpoint was hospitalisation with liver-related complications (a composite endpoint of bacterial infection, variceal bleeding, overt hepatic encephalopathy, or new onset or worsening of ascites). Multivariable competing risk frailty analysis and fast unified random forest were performed to predict complications and mortality. External validation was carried out using prospective data from 130 cirrhotic patients in an independent tertiary liver centre. RESULTS: Overall, 260 (35%) patients were hospitalised with liver-related complications. On multivariable analysis, AMM-ULN was an independent predictor of both liver-related complications (HR=2.13; 95%CI=1.89-2.40; p<0.001) and mortality (HR=1.45; 95%CI=1.20-1.76; p<0.001). AUROC of AMM-ULN was 77.9% for 1-year complications, higher than traditional severity scores. Statistical differences in survival were found between high and low levels of AMM-ULN both for complications and mortality (p<0.001) using 1.4 as the optimal cut-off from the training set. AMM-ULN remained a key variable for the prediction of complications within the random forests model in the derivation cohort and upon external validation. CONCLUSION: Ammonia is an independent predictor of hospitalisation with liver-related complications and mortality in clinically stable outpatients with cirrhosis and performs better than traditional prognostic scores in predicting complications. LAY SUMMARY: We conducted a prospective cohort study evaluating the association of blood ammonia levels with the risk of adverse outcomes in 754 patients with stable cirrhosis across 3 independent liver units. We found that ammonia is a key determinant that helps to predict which patients will be hospitalised, develop liver-related complications and die; this was confirmed in an independent cohort of patients

Role of ammonia in NAFLD: An unusual suspectKey points

Summary: Mechanistically, the symptomatology and disease progression of non-alcoholic fatty liver disease (NAFLD) remain poorly understood, which makes therapeutic progress difficult. In this review, we focus on the potential importance of decreased urea cycle activity as a pathogenic mechanism. Urea synthesis is an exclusive hepatic function and is the body’s only on-demand and definitive pathway to remove toxic ammonia. The compromised urea cycle activity in NAFLD is likely caused by epigenetic damage to urea cycle enzyme genes and increased hepatocyte senescence. When the urea cycle is dysfunctional, ammonia accumulates in liver tissue and blood, as has been demonstrated in both animal models and patients with NAFLD. The problem may be augmented by parallel changes in the glutamine/glutamate system. In the liver, the accumulation of ammonia leads to inflammation, stellate cell activation and fibrogenesis, which is partially reversible. This may be an important mechanism for the transition of bland steatosis to steatohepatitis and further to cirrhosis and hepatocellular carcinoma. Systemic hyperammonaemia has widespread negative effects on other organs. Best known are the cerebral consequences that manifest as cognitive disturbances, which are prevalent in patients with NAFLD. Furthermore, high ammonia levels induce a negative muscle protein balance leading to sarcopenia, compromised immune function and increased risk of liver cancer. There is currently no rational way to reverse reduced urea cycle activity but there are promising animal and human reports of ammonia-lowering strategies correcting several of the mentioned untoward aspects of NAFLD. In conclusion, the ability of ammonia-lowering strategies to control the symptoms and prevent the progression of NAFLD should be explored in clinical trials

Copeptin as an Indicator of Hemodynamic Derangement and Prognosis in Liver Cirrhosis

BACKGROUND: Advanced liver cirrhosis is associated with systemic hemodynamic derangement leading to the development of severe complications associated with increased mortality. Copeptin is a stable cleavage product of the precursor of arginine vasopressin, a key-regulator in hemodynamic homeostasis. Copeptin is currently considered a reliable prognostic marker in a wide variety of diseases other than cirrhosis. The present study aimed to assess copeptin, both experimentally and clinically, as a potential biomarker of hemodynamic derangement and to evaluate its prognostic significance in cirrhosis. MATERIALS AND METHODS: Two studies were executed: 1) in 18 thioacetamide-induced cirrhotic rats and 5 control rats, plasma copeptin and hemodynamic measurements were performed, 2) in 61 cirrhotic patients, serum copeptin concentration was measured in samples collected at time of registration at the waiting list for liver transplantation. In 46 patients, also a second copeptin measurement was performed during follow-up while registered at the waiting list for liver transplantation. To determine the association of serum copeptin and clinical data with outcome, Cox proportional hazard regression analysis and Kaplan Meier analysis were performed. RESULTS: Plasma copeptin concentration was significantly higher in cirrhotic rats than in controls (1.6 ± 0.5 vs. 0.9 ± 0.1 pmol/L, p< 0.01) and was negatively correlated to the mean arterial blood pressure (r = -0.574, p = 0.013). In cirrhotic patients, serum copeptin concentration was high [11.0 (5.2-24.0) pmol/L] and increased significantly during the time of registration at the waiting list for liver transplantation. MELD and MELD-sodium score were significantly correlated to serum copeptin [MELD: (r = 0.33, p = 0.01), MELD-sodium: (r = 0.29, p = 0.02)], also at time of the second copeptin measurement [MELD and MELD-sodium: r = 0.39, p< 0.01]. In cirrhotic humans, serum copeptin concentration was significantly associated with outcome, independently of the MELD and MELD-sodium score. Patients with a low serum copeptin concentration at time of registration at the liver transplant waiting list had significantly better transplant-free survival rates at 3, 6 and 12 months of follow-up as compared to those with a high serum copeptin concentration (Log-rank: p< 0.01, p< 0.01 and p = 0.02 respectively). CONCLUSIONS: Circulating copeptin levels are elevated in rats and humans with cirrhosis. Copeptin is independently associated with outcome in cirrhotic patients awaiting liver transplantation.status: publishe