Clinical, experimental and pathophysiological effects of Yaq-001: a non-absorbable, gut-restricted adsorbent in models and patients with cirrhosis.

OBJECTIVE: Targeting bacterial translocation in cirrhosis is limited to antibiotics with risk of antimicrobial resistance. This study explored the therapeutic potential of a non-absorbable, gut-restricted, engineered carbon bead adsorbent, Yaq-001 in models of cirrhosis and acute-on-chronic liver failure (ACLF) and, its safety and tolerability in a clinical trial in cirrhosis. DESIGN: Performance of Yaq-001 was evaluated in vitro. Two-rat models of cirrhosis and ACLF, (4 weeks, bile duct ligation with or without lipopolysaccharide), receiving Yaq-001 for 2 weeks; and two-mouse models of cirrhosis (6-week and 12-week carbon tetrachloride (CCl4)) receiving Yaq-001 for 6 weeks were studied. Organ and immune function, gut permeability, transcriptomics, microbiome composition and metabolomics were analysed. The effect of faecal water on gut permeability from animal models was evaluated on intestinal organoids. A multicentre, double-blind, randomised, placebo-controlled clinical trial in 28 patients with cirrhosis, administered 4 gr/day Yaq-001 for 3 months was performed. RESULTS: Yaq-001 exhibited rapid adsorption kinetics for endotoxin. In vivo, Yaq-001 reduced liver injury, progression of fibrosis, portal hypertension, renal dysfunction and mortality of ACLF animals significantly. Significant impact on severity of endotoxaemia, hyperammonaemia, liver cell death, systemic inflammation and organ transcriptomics with variable modulation of inflammation, cell death and senescence in the liver, kidneys, brain and colon was observed. Yaq-001 reduced gut permeability in the organoids and impacted positively on the microbiome composition and metabolism. Yaq-001 regulated as a device met its primary endpoint of safety and tolerability in the clinical trial. CONCLUSIONS: This study provides strong preclinical rationale and safety in patients with cirrhosis to allow clinical translation. TRIAL REGISTRATION NUMBER: NCT03202498.

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 a major 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), could reduce inflammation whilst enhancing liver regeneration. METHODS: Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride; lipopolysaccharide (LPS) or galactosamine (GalN) were then administered as extrahepatic or hepatic insults, respectively. G-CSF and/or TAK-242 were administered daily. Treatment durations were 24 hours and 5 days in the LPS model and 48 hours in the GalN model. RESULTS: In a mouse model of LPS-induced ACLF, treatment with G-CSF was associated with significant mortality (66% after 48 hours vs. 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 characterised by p21 overexpression, which is indicative of 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 in markers of hepatocyte regeneration. CONCLUSION: The combination of TAK-242 and G-CSF inhibits inflammation, promotes hepatic regeneration and prevents mortality in models of ACLF; thus, this combination could be a potential treatment option for ACLF. LAY SUMMARY: Acute-on-chronic liver failure is associated with severe liver inflammation and poor short-term survival. Therefore, effective treatments are urgently needed. Herein, we have shown, using mouse models, that the combination of granulocyte-colony stimulating factor (which can promote liver regeneration) and TAK-242 (which inhibits a receptor that plays a key role in inflammation) could be effective for the treatment of acute-on-chronic liver failure.

Hyperammonaemia induces mitochondrial dysfunction and neuronal cell death.

Background & Aims: In 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 hyperammonaemia 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 cocultures of astrocytes and neurons, low concentrations (1 and 5 µM) of NH4Cl were applied. In rats with bile duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonaemia and minimal HE, acute brain slices were studied. One group of BDL rats was treated twice daily with the ammonia scavenger ornithine phenylacetate (OP; 0.3 g/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 with 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 hyperpolarisation of Δψm with consequent overconsumption of adenosine triphosphate 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 hyperammonaemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonaemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development. Lay summary: The impact of hyperammonaemia, 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.

Abnormal brain oxygen homeostasis in an animal model of liver disease.

Background & Aims: Increased plasma ammonia concentration and consequent disruption of brain energy metabolism could underpin the pathogenesis of hepatic encephalopathy (HE). Brain energy homeostasis relies on effective maintenance of brain oxygenation, and dysregulation impairs neuronal function leading to cognitive impairment. We hypothesised that HE is associated with reduced brain oxygenation and we explored the potential role of ammonia as an underlying pathophysiological factor. Methods: In a rat model of chronic liver disease with minimal HE (mHE; bile duct ligation [BDL]), brain tissue oxygen measurement, and proton magnetic resonance spectroscopy were used to investigate how hyperammonaemia impacts oxygenation and metabolic substrate availability in the central nervous system. Ornithine phenylacetate (OP, OCR-002; Ocera Therapeutics, CA, USA) was used as an experimental treatment to reduce plasma ammonia concentration. Results: In BDL animals, glucose, lactate, and tissue oxygen concentration in the cerebral cortex were significantly lower than those in sham-operated controls. OP treatment corrected the hyperammonaemia and restored brain tissue oxygen. Although BDL animals were hypotensive, cortical tissue oxygen concentration was significantly improved by treatments that increased arterial blood pressure. Cerebrovascular reactivity to exogenously applied CO2 was found to be normal in BDL animals. Conclusions: These data suggest that hyperammonaemia significantly decreases cortical oxygenation, potentially compromising brain energy metabolism. These findings have potential clinical implications for the treatment of patients with mHE. Lay summary: Brain dysfunction is a serious complication of cirrhosis and affects approximately 30% of these patients; however, its treatment continues to be an unmet clinical need. This study shows that oxygen concentration in the brain of an animal model of cirrhosis is markedly reduced. Low arterial blood pressure and increased ammonia (a neurotoxin that accumulates in patients with liver failure) are shown to be the main underlying causes. Experimental correction of these abnormalities restored oxygen concentration in the brain, suggesting potential therapeutic avenues to explore.

Inhibition of TLR7 and TLR9 Reduces Human Cholangiocarcinoma Cell Proliferation and Tumor Development.

BACKGROUND: Toll-like receptors (TLRs) are key players in innate immunity and modulation of TLR signaling has been demonstrated to profoundly affect proliferation and growth in different types of cancer. However, the role of TLRs in human intrahepatic cholangiocarcinoma (ICC) pathogenesis remains largely unexplored. AIMS: We set out to determine if TLRs play any role in ICCs which could potentially make them useful treatment targets. METHODS: Tissue microarrays containing samples from 9 human ICCs and normal livers were examined immunohistochemically for TLR4, TLR7, and TLR9 expression. Proliferation of human ICC cell line HuCCT1 was measured by MTS assay following treatment with CpG-ODN (TLR9 agonist), imiquimod (TLR7 agonist), chloroquine (TLR7 and TLR9 inhibitor) and IRS-954 (TLR7 and TLR9 antagonist). The in vivo effects of CQ and IRS-954 on tumor development were also examined in a NOD-SCID mouse xenograft model of human ICC. RESULTS: TLR4 was expressed in all normal human bile duct epithelium but absent in the majority (60%) of ICCs. TLR7 and TLR9 were expressed in 80% of human ICCs. However, TLR7 was absent in all cases of normal human bile duct epithelium and only one was TLR9 positive. HuCCT1 cell proliferation in vitro significantly increased following IMQ or CpG-ODN treatment (P < 0.03 and P < 0.002, respectively) but decreased with CQ (P < 0.02). In the mouse xenograft model there was significant reduction in size of tumors from CQ and IRS-954 treated mice compared to untreated controls. CONCLUSION: TLR7 and TLR9 should be further explored for their potential as actionable targets in the treatment of ICC.

The role of RIPK1 mediated cell death in acute on chronic liver failure.

Acute-on-chronic liver failure (ACLF) is characterized predominantly by non-apoptotic forms of hepatocyte cell death. Necroptosis is a form of programmed lytic cell death in which receptor interacting protein kinase (RIPK) 1, RIPK3 and phosphorylated mixed lineage kinase domain-like (pMLKL) are key components. This study was performed to determine the role of RIPK1 mediated cell death in ACLF. RIPK3 plasma levels and hepatic expression of RIPK1, RIPK3, and pMLKL were measured in healthy volunteers, stable patients with cirrhosis, and in hospitalized cirrhotic patients with acutely decompensated cirrhosis, with and without ACLF (AD). The role of necroptosis in ACLF was studied in two animal models of ACLF using inhibitors of RIPK1, necrostatin-1 (NEC-1) and SML2100 (RIPA56). Plasma RIPK3 levels predicted the risk of 28- and 90-day mortality (AUROC, 0.653 (95%CI 0.530-0.776), 0.696 (95%CI 0.593-0.799)] and also the progression of patients from no ACLF to ACLF [0.744 (95%CI 0.593-0.895)] and the results were validated in a 2nd patient cohort. This pattern was replicated in a rodent model of ACLF that was induced by administration of lipopolysaccharide (LPS) to bile-duct ligated rats and carbon tetrachloride-induced fibrosis mice administered galactosamine (CCL4/GalN). Suppression of caspase-8 activity in ACLF rodent model was observed suggesting a switch from caspase-dependent cell death to necroptosis. NEC-1 treatment prior to administration of LPS significantly reduced the severity of ACLF manifested by reduced liver, kidney, and brain injury mirrored by reduced hepatic and renal cell death. Similar hepato-protective effects were observed with RIPA56 in a murine model of ACLF induced by CCL4/GalN. These data demonstrate for the first time the importance of RIPK1 mediated cell death in human and rodent ACLF. Inhibition of RIPK1 is a potential novel therapeutic approach to prevent progression of susceptible patients from no ACLF to ACLF.

The Lipopolysaccharide-Sensing Caspase(s)-4/11 Are Activated in Cirrhosis and Are Causally Associated With Progression to Multi-Organ Injury.

BACKGROUND AND AIMS: The development of multi-organ injury in cirrhosis is associated with increased intestinal permeability, translocation of gut-derived bacterial products [e.g., lipopolysaccharide (LPS)] into the circulation, and increased non-apoptotic hepatocyte cell death. Pyroptosis is a non-apoptotic, lytic form of cell death mediated by the LPS-sensing caspase(s)-4/11 (caspase-4 in humans, caspase-11 in mice), which leads to activation of the effector protein Gasdermin D (GSDMD) and subsequent formation of pores in the plasma membrane. Endoplasmic reticulum (ER) stress, a feature of cirrhosis, has been identified as a factor promoting the activation of caspase-11, thus increasing sensitivity of the cell to LPS-mediated pyroptosis. The aim of this study was to determine the role of bacterial LPS in the activation of hepatic caspase(s)-4/11 and progression of hepatic and extra-hepatic organ injury in cirrhosis. MATERIALS AND METHODS: Human liver samples from patients with stable cirrhosis (SC) or acutely decompensated cirrhosis (AD) were analyzed for caspase-4 activation by immunohistochemistry. Wild-type and Casp11 -/- mice underwent CCl4 treatment by gavage to induce advanced liver fibrosis, and subsequently low-dose injection of LPS to mimic bacterial translocation and induce multi-organ injury. Liver, kidney, and brain function were assessed by plasma ALT/creatinine and brain water respectively. The activity of inflammatory caspases was assessed by fluorometric assay and the occurrence of pyroptosis and overall cell death in liver tissue by GSDMD cleavage and TUNEL assay, respectively. Primary human hepatocytes were cultured according to standard techniques. RESULTS: Human liver samples demonstrated increased caspase-4 activation in AD cirrhosis. Caspase-4 activation was associated with MELD score and circulating levels of LDH. Wild-type mice treated with CCl4 developed significant multi-organ injury (increased ALT, creatinine, and brain water) upon LPS injection, and showed increased hepatic GSDMD cleavage compared to mice treated with CCl4 alone. Primary human hepatocytes could be sensitized to pyroptosis by pre-treatment with the ER-stress inducer tunicamycin and LPS. Casp11 -/- mice treated with CCl4 + LPS were significantly protected from multi-organ injury compared to wild-type CCl4 + LPS. CONCLUSION: These data demonstrate for the first time a causal relationship between LPS-mediated activation of caspase(s)-4/11 and development of hepatic and extra-hepatic injury in cirrhosis.

Recombinant Alkaline Phosphatase Prevents Acute on Chronic Liver Failure.

The lipopolysaccharide (LPS)- toll-like receptor-4 (TLR4) pathway plays an important role in liver failure. Recombinant alkaline phosphatase (recAP) deactivates LPS. The aim of this study was to determine whether recAP prevents the progression of acute and acute-on-chronic liver failure (ACLF). Eight groups of rats were studied 4-weeks after sham surgery or bile duct ligation and were injected with saline or LPS to mimic ACLF. Acute liver failure was induced with Galactosamine-LPS and in both models animals were treated with recAP prior to LPS administration. In the ACLF model, the severity of liver dysfunction and brain edema was attenuated by recAP, associated with reduction in cytokines, chemokines, liver cell death, and brain water. The activity of LPS was reduced by recAP. The treatment was not effective in acute liver failure. Hepatic TLR4 expression was reduced by recAP in ACLF but not acute liver failure. Increased sensitivity to endotoxins in cirrhosis is associated with upregulation of hepatic TLR4, which explains susceptibility to development of ACLF whereas acute liver failure is likely due to direct hepatoxicity. RecAP prevents multiple organ injury by reducing receptor expression and is a potential novel treatment option for prevention of ACLF but not acute liver failure.

Toll-like receptor 4 is a therapeutic target for prevention and treatment of liver failure.

BACKGROUND & AIMS: Toll-like receptor 4 (TLR4) plays an essential role in mediating organ injury in acute liver failure (ALF) and acute-on-chronic liver failure (ACLF). Herein, we assess whether inhibiting TLR4 signaling can ameliorate liver failure and serve as a potential treatment. METHODS: Circulating TLR4 ligands and hepatic TLR4 expression were measured in plasma samples and liver biopsies from patients with cirrhosis. TAK-242 (TLR4 inhibitor) was tested in vivo (10 mg/kg intraperitoneally) in rodent models of ACLF (bile duct ligation + lipopolysaccharide [LPS]; carbon tetrachloride + LPS) and ALF (galactosamine + LPS) and in vitro on immortalized human monocytes (THP-1) and hepatocytes (HHL5). The in vivo therapeutic effect was assessed by coma-free survival, organ injury and cytokine release and in vitro by measuring IL-6, IL-1ß or cell injury (TUNEL), respectively. RESULTS: In patients with cirrhosis, hepatic TLR4 expression was upregulated and circulating TLR4 ligands were increased (p <0.001). ACLF in rodents was associated with a switch from apoptotic cell death in ALF to non-apoptotic forms of cell death. TAK-242 reduced LPS-induced cytokine secretion and cell death (p = 0.002) in hepatocytes and monocytes in vitro. In rodent models of ACLF, TAK-242 administration improved coma-free survival, reduced the degree of hepatocyte cell death in the liver (p <0.001) and kidneys (p = 0.048) and reduced circulating cytokine levels (IL-1ß, p <0.001). In a rodent model of ALF, TAK-242 prevented organ injury (p <0.001) and systemic inflammation (IL-1ß, p <0.001). CONCLUSION: This study shows that TLR4 signaling is a key factor in the development of both ACLF and ALF; its inhibition reduces the severity of organ injury and improves outcome. TAK-242 may be of therapeutic relevance in patients with liver failure. LAY SUMMARY: Toll-like receptor 4 (or TLR4) mediates endotoxin-induced tissue injury in liver failure and cirrhosis. This receptor sensitizes cells to endotoxins, which are produced by gram-negative bacteria. Thus, inhibiting TLR4 signaling with an inhibitor (TAK-242) ameliorates organ injury and systemic inflammation in rodent models of acute and acute-on-chronic liver failure.

Ammonia Scavenging Prevents Progression of Fibrosis in Experimental Nonalcoholic Fatty Liver Disease.

BACKGROUND AND AIMS: In nonalcoholic fatty liver disease (NAFLD), fibrosis is the most important factor contributing to NAFLD-associated morbidity and mortality. Prevention of progression and reduction in fibrosis are the main aims of treatment. Even in early stages of NAFLD, hepatic and systemic hyperammonemia is evident. This is due to reduced urea synthesis; and as ammonia is known to activate hepatic stellate cells, we hypothesized that ammonia may be involved in the progression of fibrosis in NAFLD. APPROACH AND RESULTS: In a high-fat, high-cholesterol diet-induced rodent model of NAFLD, we observed a progressive stepwise reduction in the expression and activity of urea cycle enzymes resulting in hyperammonemia, evidence of hepatic stellate cell activation, and progressive fibrosis. In primary, cultured hepatocytes and precision-cut liver slices we demonstrated increased gene expression of profibrogenic markers after lipid and/or ammonia exposure. Lowering of ammonia with the ammonia scavenger ornithine phenylacetate prevented hepatocyte cell death and significantly reduced the development of fibrosis both in vitro in the liver slices and in vivo in a rodent model. The prevention of fibrosis in the rodent model was associated with restoration of urea cycle enzyme activity and function, reduced hepatic ammonia, and markers of inflammation. CONCLUSIONS: The results of this study suggest that hepatic steatosis results in hyperammonemia, which is associated with progression of hepatic fibrosis. Reduction of ammonia levels prevented progression of fibrosis, providing a potential treatment for NAFLD.

Impaired brain glymphatic flow in experimental hepatic encephalopathy.

BACKGROUND & AIMS: Neuronal function is exquisitely sensitive to alterations in the extracellular environment. In patients with hepatic encephalopathy (HE), accumulation of metabolic waste products and noxious substances in the interstitial fluid of the brain is thought to result from liver disease and may contribute to neuronal dysfunction and cognitive impairment. This study was designed to test the hypothesis that the accumulation of these substances, such as bile acids, may result from reduced clearance from the brain. METHODS: In a rat model of chronic liver disease with minimal HE (the bile duct ligation [BDL] model), we used emerging dynamic contrast-enhanced MRI and mass-spectroscopy techniques to assess the efficacy of the glymphatic system, which facilitates clearance of solutes from the brain. Immunofluorescence of aquaporin-4 (AQP4) and behavioural experiments were also performed. RESULTS: We identified discrete brain regions (olfactory bulb, prefrontal cortex and hippocampus) of altered glymphatic clearance in BDL rats, which aligned with cognitive/behavioural deficits. Reduced AQP4 expression was observed in the olfactory bulb and prefrontal cortex in HE, which could contribute to the pathophysiological mechanisms underlying the impairment in glymphatic function in BDL rats. CONCLUSIONS: This study provides the first experimental evidence of impaired glymphatic flow in HE, potentially mediated by decreased AQP4 expression in the affected regions. LAY SUMMARY: The 'glymphatic system' is a newly discovered brain-wide pathway that facilitates clearance of various substances that accumulate in the brain due to its activity. This study evaluated whether the function of this system is altered in a model of brain dysfunction that occurs in cirrhosis. For the first time, we identified that the clearance of substances from the brain in cirrhosis is reduced because this clearance system is defective. This study proposes a new mechanism of brain dysfunction in patients with cirrhosis and provides new targets for therapy.

Immunomodulatory and antioxidant function of albumin stabilises the endothelium and improves survival in a rodent model of chronic liver failure.

BACKGROUND & AIMS: Liver failure is characterized by endothelial dysfunction, which results in hemodynamic disturbances leading to renal failure. Albumin infusion improves hemodynamics and prevents renal dysfunction in advance liver failure. These effects are only partly explained by the oncotic properties of albumin. This study was designed to test the hypothesis that albumin exerts its beneficial effects by stabilising endothelial function. METHODS: In vivo: systemic hemodynamics, renal function, markers of endothelial dysfunction (ADMA) and inflammation were studied in analbuminaemic and Sprague-Dawley rats, 6-weeks after sham/bile duct ligation surgery. In vitro: human umbilical vein endothelial cells were stimulated with LPS with or without albumin. We studied protein expression and gene expression of adhesion molecules, intracellular reactive oxygen species, and cell stress markers. RESULTS: Compared to controls, analbuminaemic rats had significantly greater hemodynamic deterioration after bile duct ligation, resulting in worse renal function and shorter survival. This was associated with significantly greater plasma renin activity, worse endothelial function, and disturbed inflammatory response. In vitro studies showed that albumin was actively taken up by endothelial cells. Incubation of albumin pre-treated endothelial cells with LPS was associated with significantly less activation compared with untreated cells, decreased intracellular reactive oxygen species, and markers of cell stress. CONCLUSIONS: These results show, for the first time, that absence of albumin is characterised by worse systemic hemodynamics, renal function and higher mortality in a rodent model of chronic liver failure and illustrates the important non-oncotic properties of albumin in protecting against endothelial dysfunction.

Effect of toll-like receptor 7 and 9 targeted therapy to prevent the development of hepatocellular carcinoma.

BACKGROUND & AIMS: Chronic liver disease is a predisposing factor for development of hepatocellular carcinoma (HCC). Toll-like receptors play a crucial role in immunity against microbial pathogens and recent evidence suggests that they may also be important in pathogenesis of chronic liver disease. The purpose of this study was to determine whether TLR7 and TLR9 are potential targets for prevention and progression of HCC. METHODS: Tissue microarrays containing liver samples from patients with cirrhosis, viral hepatitis and HCC were examined for expression of TLR7 and TLR9 and the data obtained was validated in liver specimens from the hospital archives. Proliferation of human HCC cell lines was studied following stimulation of TLR7 and TLR9 using agonists (imiquimod and CpG-ODN respectively) and inhibition with a specific antagonist (IRS-954) or chloroquine. The effect of these interventions was confirmed in a xenograft model and diethylnitrosamine (DEN)/nitrosomorpholine (NMOR)-induced model of HCC. RESULTS: TLR7 and TLR9 expression was up-regulated in human HCC tissue. Proliferation of HuH7 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 and TLR9 inhibition using IRS-954 or chloroquine significantly reduced HuH7 cell proliferation in vitro and inhibited tumour growth in the mouse xenograft model. HCC development in the DEN/NMOR rat model was also significantly inhibited by chloroquine (P < 0.001). CONCLUSION: The data suggest that inhibiting TLR7 and TLR9 with IRS-954 or chloroquine could potentially be used as a novel therapeutic approach for preventing HCC development and/or progression in susceptible patients.

Prevention of acute kidney injury in a rodent model of cirrhosis following selective gut decontamination is associated with reduced renal TLR4 expression.

BACKGROUND & AIMS: Superimposed infection and/or inflammation precipitate renal failure in cirrhosis. This study aimed at testing the hypothesis that increased gut bacterial translocation in cirrhosis primes the kidney to the effect of superimposed inflammation by upregulating expression of Toll-like receptor 4 (TLR4), NFκB, and cytokines. A well-characterized bile-duct ligated (BDL) model of cirrhosis, which develops renal failure following superimposed inflammatory insult with lipopolysaccharide (LPS), was used and selective gut decontamination was performed using norfloxacin. METHODS: Sprague-Dawley rats were studied: Sham, Sham+LPS; BDL, BDL+LPS; an additional BDL and BDL+LPS groups were selectively decontaminated with norfloxacin. Plasma biochemistry, plasma renin activity (PRA) and cytokines and, protein expression of TLR4, NFκB, and cytokines were measured in the kidney homogenate. The kidneys were stained for TLR4, TLR2, and caspase-3. Endotoxemia was measured using neutrophil burst and Limulus amoebocyte lysate (LAL) assays. RESULTS: The groups treated with norfloxacin showed significant attenuation of the increase in plasma creatinine, plasma and renal TNF-α and renal tubular injury on histology. The increased renal protein expression of TLR4, NFκB, and caspase-3 in the untreated animals was significantly attenuated in the norfloxacin treated animals. PRA was reduced in the treated animals and severity of endotoxemia was also reduced. CONCLUSIONS: The results show for the first time that kidneys in cirrhosis show an increased expression of TLR4, NFκB, and the pro-inflammatory cytokine TNF-α, which makes them susceptible to a further inflammatory insult. This increased susceptibility to LPS can be prevented with selective decontamination, providing novel insights into the pathophysiology of renal failure in cirrhosis.

Ammonia reduction with ornithine phenylacetate restores brain eNOS activity via the DDAH-ADMA pathway in bile duct-ligated cirrhotic rats.

Ammonia is central in the pathogenesis of hepatic encephalopathy, which is associated with dysfunction of the nitric oxide (NO) signaling pathway. Ornithine phenylacetate (OP) reduces hyperammonemia and brain water in cirrhotic animals. This study aimed to determine whether endothelial NO synthase activity is altered in the brain of cirrhotic animals, whether this is associated with changes in the endogenous inhibitor, asymmetric-dimethylarginine (ADMA) and its regulating enzyme, dimethylarginine-dimethylaminohydrolase (DDAH-1), and whether these abnormalities are restored by ammonia reduction using OP. Sprague-Dawley rats were studied 4-wk after bile duct ligation (BDL) (n = 16) or sham operation (n = 8) and treated with placebo or OP (0.6 g/kg). Arterial ammonia, brain water, TNF-α, plasma, and brain ADMA were measured using standard techniques. NOS activity was measured radiometrically, and protein expression for NOS enzymes, ADMA, DDAH-1, 4-hydroxynonenol ((4)HNE), and NADPH oxidase (NOX)-1 were measured by Western blotting. BDL significantly increased arterial ammonia (P < 0.0001), brain water (P < 0.05), and brain TNF-α (P < 0.01). These were reduced significantly by OP treatment. The estimated eNOS component of constitutive NOS activity was significantly lower (P < 0.05) in BDL rat, and this was significantly attenuated in OP-treated animals. Brain ADMA levels were significantly higher and brain DDAH-1 significantly lower in BDL compared with sham (P < 0.01) and restored toward normal following treatment with OP. Brain (4)HNE and NOX-1 protein expression were significantly increased in BDL rat brain, which were significantly decreased following OP administration. We show a marked abnormality of NO regulation in cirrhotic rat brains, which can be restored by reduction in ammonia concentration using OP.

L-ornithine and phenylacetate synergistically produce sustained reduction in ammonia and brain water in cirrhotic rats.

UNLABELLED: Treatment of hyperammonemia and hepatic encephalopathy in cirrhosis is an unmet clinical need. The aims of this study were to determine whether L-ornithine and phenylacetate/phenylbutyrate (administered as the pro-drug phenylbutyrate) (OP) combined are synergistic and produce sustained reduction in ammonia by L-ornithine acting as a substrate for glutamine synthesis, thereby detoxifying ammonia, and the phenylacetate excreting the ornithine-derived glutamine as phenylacetylglutamine in the urine. Sprague-Dawley rats were studied 4 weeks after bile duct ligation (BDL) or sham operation. Study 1: Three hours before termination, an internal carotid sampling catheter was inserted, and intraperitoneal saline (placebo), OP, phenylbutyrate, or L-ornithine were administered after randomization. BDL was associated with significantly higher arterial ammonia and brain water and lower brain myoinositol (P < 0.01, respectively), compared with sham-operated controls, which was significantly improved in the OP-treated animals; arterial ammonia (P < 0.001), brain water (P < 0.05), brain myoinositol (P < 0.001), and urinary phenylacetylglutamine (P < 0.01). Individually, L-ornithine or phenylbutyrate were similar to the BDL group. In study 2, BDL rats were randomized to saline or OP administered intraperitoneally for 6 hours or 3, 5, or 10 days and were sacrificed between 4.5 and 5 weeks. The results showed that the administration of OP was associated with sustained reduction in arterial ammonia (P < 0.01) and brain water (P < 0.01) and markedly increased arterial glutamine (P < 0.01) and urinary excretion of phenylacetylglutamine (P < 0.01) in each of the OP treated groups. CONCLUSION: The results of this study provide proof of the concept that L-ornithine and phenylbutyrate/phenylacetate act synergistically to produce sustained improvement in arterial ammonia, its brain metabolism, and brain water in cirrhotic rats.