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.

Engineered Lipids for Intracellular Reactive Oxygen Species Scavenging in Steatotic Hepatocytes.

Intracellular reactive oxygen species (ROS) in steatotic cells pose a problem due to their potential to cause oxidative stress and cellular damage. Delivering engineered phospholipids to intracellular lipid droplets in steatotic hepatic cells, using the cell's inherent intracellular lipid transport mechanisms are investigated. Initially, it is shown that tail-labeled fluorescent lipids assembled into liposomes are able to be transported to intracellular lipid droplets in steatotic HepG2 cells and HHL-5 cells. Further, an antioxidant, an EUK salen-manganese derivative, which has superoxide dismutase-like and catalase-like activity, is covalently conjugated to the tail of a phospholipid and formulated as liposomes for administration. Steatotic HepG2 cells and HHL-5 cells incubated with these antioxidant liposomes have lower intracellular ROS levels compared to untreated controls and non-covalently formulated antioxidants. This first proof-of-concept study illustrates an alternative strategy to equip native organelles in mammalian cells with engineered enzyme activity.

Cardiac dysfunction in patients with cirrhosis and acute decompensation.

The prevalence of cirrhotic cardiomyopathy (CCM) has been reported as high as 60%-70% in patients with liver cirrhosis and is associated with various negative outcomes. There has been a growing understanding of CCM over recent years. Indeed, the development of imaging techniques has enabled new diagnostic criteria to be proposed by the Cirrhotic Cardiomyopathy Consortium. However, important unanswered questions remain over pathophysiological mechanisms, optimal diagnostic modalities and potential treatment options. While there has been an increasing volume of literature evaluating CCM, there is a lack of clarity on its implications in acute decompensation, acute-on-chronic liver failure and following interventions such as transjugular intrahepatic portosystemic shunt insertion and liver transplantation. This review aims to summarise the literature in these challenging domains and suggest where future research should focus. We conclude that systemic inflammation and structural myocardial changes are likely to be crucial in the pathophysiology of the disease, but the relative contribution of different components remains elusive. Furthermore, future studies need to use standardised diagnostic criteria for CCM as well as incorporate newer imaging techniques assessing both myocardial structure and function. Finally, while specific treatments are currently lacking, therapeutics targeting systemic inflammation, microbial dysbiosis and bacterial translocation are promising targets and warrant further research.

Evaluation of CirrhoCare® - a digital health solution for home management of individuals with cirrhosis.

BACKGROUND & AIMS: Individuals with cirrhosis discharged from hospital following acute decompensation are at high risk of new complications. This study aimed to assess the feasibility and potential clinical benefits of remote management of individuals with acutely decompensated cirrhosis using CirrhoCare®. METHODS: Individuals with cirrhosis with acute decompensation were followed up with CirrhoCare® and compared with contemporaneous matched controls, managed with standard follow-up. Commercially available monitoring devices were linked to the smartphone CirrhoCare® app, for daily recording of heart rate, blood pressure, weight, % body water, cognitive function (CyberLiver Animal Recognition Test [CL-ART] app), self-reported well-being, and intake of food, fluid, and alcohol. The app had 2-way patient-physician communication. Independent external adjudicators assessed the appropriateness of CirrhoCare®-based decisions. RESULTS: Twenty individuals with cirrhosis were recruited to CirrhoCare® (mean age 59 ± 10 years, 14 male, alcohol-related cirrhosis [80%], mean model for end-stage liver disease-sodium [MELD-Na] score 16.1 ± 4.2) and were not statistically different to 20 contemporaneous controls. Follow-up was 10.1 ± 2.4 weeks. Fifteen individuals showed good engagement (≥4 readings/week), 2 moderate (2-3/week), and 3 poor (<2/week). In a usability questionnaire, the median score was ≥9 for all questions. Five CirrhoCare®-managed individuals had 8 readmissions over a median of 5 (IQR 3.5-11) days, and none required hospitalisation for >14 days. Sixteen other CirrhoCare®-guided patient contacts were made, leading to clinical interventions that prevented further progression. Appropriateness was confirmed by adjudicators. Controls had 13 readmissions in 8 individuals, lasting a median of 7 (IQR 3-15) days with 4 admissions of >14 days. They had 6 unplanned paracenteses compared with 1 in the CirrhoCare® group. CONCLUSIONS: This study demonstrates that CirrhoCare® is feasible for community management of individuals with decompensated cirrhosis with good engagement and clinically relevant alerts to new decompensating events. CirrhoCare®-managed individuals have fewer and shorter readmissions justifying larger controlled clinical trials. IMPACT AND IMPLICATIONS: As the burden of cirrhosis grows worldwide, increasing demands are being placed on limited healthcare resources, necessitating the adoption of more sustainable care models that allow for at-home patient management. The CirrhoCare® management system was developed to fill this care gap, deploying a novel combination of hardware, apps, and algorithms, to monitor and intervene in individuals at risk of new decompensation. This study highlights the possibility of reducing hospital readmissions for cirrhosis by optimising specialist community care, reducing the need for interventions such as paracentesis, while providing a more sustainable care pathway that is acceptable to patients. However, given the pilot and non-randomised nature of this study, the outcomes require further validation in a larger randomised controlled trial, to assess both clinical effectiveness and cost-effectiveness. Moreover, the data generated will also facilitate data modelling and further research to refine the CirrhoCare® algorithms to increase their detection sensitivity and utility.

Randomized, controlled clinical trial of the DIALIVE liver dialysis device versus standard of care in patients with acute-on- chronic liver failure.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterized by severe systemic inflammation, multi-organ failure and high mortality rates. Its treatment is an urgent unmet need. DIALIVE is a novel liver dialysis device that aims to exchange dysfunctional albumin and remove damage- and pathogen-associated molecular patterns. This first-in-man randomized-controlled trial was performed with the primary aim of assessing the safety of DIALIVE in patients with ACLF, with secondary aims of evaluating its clinical effects, device performance and effect on pathophysiologically relevant biomarkers. METHODS: Thirty-two patients with alcohol-related ACLF were included. Patients were treated with DIALIVE for up to 5 days and end points were assessed at Day 10. Safety was assessed in all patients (n = 32). The secondary aims were assessed in a pre-specified subgroup that had at least three treatment sessions with DIALIVE (n = 30). RESULTS: There were no significant differences in 28-day mortality or occurrence of serious adverse events between the groups. Significant reduction in the severity of endotoxemia and improvement in albumin function was observed in the DIALIVE group, which translated into a significant reduction in the CLIF-C (Chronic Liver Failure consortium) organ failure (p = 0.018) and CLIF-C ACLF scores (p = 0.042) at Day 10. Time to resolution of ACLF was significantly faster in DIALIVE group (p = 0.036). Biomarkers of systemic inflammation such as IL-8 (p = 0.006), cell death [cytokeratin-18: M30 (p = 0.005) and M65 (p = 0.029)], endothelial function [asymmetric dimethylarginine (p = 0.002)] and, ligands for Toll-like receptor 4 (p = 0.030) and inflammasome (p = 0.002) improved significantly in the DIALIVE group. CONCLUSIONS: These data indicate that DIALIVE appears to be safe and impacts positively on prognostic scores and pathophysiologically relevant biomarkers in patients with ACLF. Larger, adequately powered studies are warranted to further confirm its safety and efficacy. IMPACT AND IMPLICATIONS: This is the first-in-man clinical trial which tested DIALIVE, a novel liver dialysis device for the treatment of cirrhosis and acute-on-chronic liver failure, a condition associated with severe inflammation, organ failures and a high risk of death. The study met the primary endpoint, confirming the safety of the DIALIVE system. Additionally, DIALIVE reduced inflammation and improved clinical parameters. However, it did not reduce mortality in this small study and further larger clinical trials are required to re-confirm its safety and to evaluate efficacy. CLINICAL TRIAL NUMBER: NCT03065699.

Mechanisms and treatment approaches for ACLF.

Acute-on-chronic liver failure (ACLF) is a life-threatening syndrome characterized by decompensation of cirrhosis, severe systemic inflammation and organ failures. ACLF is frequently triggered by intra- and/or extrahepatic insults, such as bacterial infections, alcohol-related hepatitis or flares of hepatic viruses. The imbalance between systemic inflammation and immune tolerance causes organ failures through the following mechanisms: (i) direct damage of immune cells/mediators; (ii) worsening of circulatory dysfunction resulting in organ hypoperfusion and (iii) metabolic alterations with prioritization of energetic substrates for inflammation and peripheral organ 'energetic crisis'. Currently, the management of ACLF includes the support of organ failures, the identification and treatment of precipitating factors and expedited assessment for liver transplantation (LT). Early LT should be considered in patients with ACLF grade 3, who are unlikely to recover with the available treatments and have a mortality rate > 70% at 28 days. However, the selection of transplant candidates and their prioritization on the LT waiting list need standardization. Future challenges in the ACLF field include a better understanding of pathophysiological mechanisms leading to inflammation and organ failures, the development of specific treatments for the disease and personalized treatment approaches. Herein, we reviewed the current knowledge and future perspectives on mechanisms and treatment of ACLF.

A novel smartphone scleral-image based tool for assessing jaundice in decompensated cirrhosis patients.

BACKGROUND AND AIM: Serum bilirubin is an established marker of liver disease. Reliable tools for non-invasive assessment of jaundice in cirrhosis patients, at risk of clinical decompensation, are highly desirable. While smartphone-based imaging has been described in neonatal jaundice, it has not been investigated in advanced cirrhosis patients. METHODS: We included 46 hospitalized patients with acute cirrhosis decompensation and jaundice. Scleral images using an Android smartphone were taken to derive "Scleral Color Values (SCV)," which were matched with same day serum bilirubin measurements. In 29 patients, repeat SCV and bilirubin measurements were performed over time. We analyzed the relationship of SCV and its dynamics with serum bilirubin, clinical scores, and patient outcomes. RESULTS: Of 46 patients, 26 (57%) had alcoholic hepatitis as the decompensation precipitant. Seven patients died during admission; a further 12 following hospital discharge. SCV had an excellent linear correlation with serum bilirubin (rho = 0.90, P < 0.001); changes in SCV and serum bilirubin across different time points, were also closely associated (rho = 0.77, P < 0.001). SCV correlated significantly with CLIF Consortium Acute Decompensation score (rho = 0.38, P < 0.001) and grade of Acute-on-Chronic Liver Failure (rho = 0.42, P = 0.039). SCV was higher in patients who died, however, not significantly (86.1 [IQR 83.0-89.7] vs 82.3 [IQR 78.5-83.3], P = 0.22). The associations of SCV with clinical parameters mirrored those of serum bilirubin. CONCLUSION: Smartphone-based assessment of jaundice shows excellent concordance with serum bilirubin and is associated with clinical parameters in acute cirrhosis decompensation. This approach offers promise for remote assessment of cirrhosis patients at-risk of decompensation, post hospital discharge.

The role of brain inflammation and abnormal brain oxygen homeostasis in the development of hepatic encephalopathy.

Hepatic encephalopathy (HE) is a frequent complication of chronic liver disease (CLD) and has a complex pathogenesis. Several preclinical and clinical studies have reported the presence of both peripheral and brain inflammation in CLD and their potential impact in the development of HE. Altered brain vascular density and tone, as well as compromised cerebral and systemic blood flow contributing to the development of brain hypoxia, have also been reported in animal models of HE, while a decrease in cerebral metabolic rate of oxygen and cerebral blood flow has consistently been observed in patients with HE. Whilst significant strides in our understanding have been made over the years, evaluating all these mechanistic elements in vivo and showing causal association with development of HE, have been limited through the practical constraints of experimentation. Nonetheless, improvements in non-invasive assessments of different neurophysiological parameters, coupled with techniques to assess changes in inflammatory and metabolic pathways, will help provide more granular insights on these mechanisms. In this special issue we discuss some of the emerging evidence supporting the hypothesis that brain inflammation and abnormal oxygen homeostasis occur interdependently during CLD and comprise important contributors to the development of HE. This review aims at furnishing evidence for further research in brain inflammation and oxygen homeostasis as additional therapeutic targets and potentially diagnostic markers for HE.

The Use of Rifaximin in Patients With Cirrhosis.

Rifaximin is an oral nonsystemic antibiotic with minimal gastrointestinal absorption and broad-spectrum antibacterial activity covering both gram-positive and gram-negative organisms. Rifaximin is currently used worldwide in patients with cirrhosis for preventing recurrent HE because its efficacy and safety have been proven by large randomized clinical trials. In the last decade, experimental and clinical evidence suggest that rifaximin could have other beneficial effects on the course of cirrhosis by modulating the gut microbiome and affecting the gut-liver axis, which in turn can interfere with major events of the pathophysiological cascade underlying decompensated cirrhosis, such as systemic inflammatory syndrome, portal hypertension, and bacterial infections. However, the use of rifaximin for prevention or treatment of other complications, including spontaneous bacterial peritonitis or other bacterial infections, is not accepted because evidence by clinical trials is still very weak. The present review deals in the first part with the potential impact of rifaximin on pathogenic mechanisms in liver diseases, whereas in the second part, its clinical effects are critically discussed. It clearly emerges that, because of its potential activity on multiple pathogenic events, the efficacy of rifaximin in the prevention or management of complications other than HE deserves to be investigated extensively. The results of double-blinded, adequately powered randomized clinical trials assessing the effect of rifaximin, alone or in combination with other drugs, on hard clinical endpoints, such as decompensation of cirrhosis, acute-on-chronic liver failure, and mortality, are therefore eagerly awaited.

Non-alcoholic Fatty Liver Disease: Also a Disease of the Brain? A Systematic Review of the Preclinical Evidence.

Non-alcoholic fatty liver disease (NAFLD) currently affects 25% of the global adult population. Cognitive impairment is a recently recognised comorbidity impeding memory, attention, and concentration, affecting the patients' activities of daily living and reducing their quality of life. This systematic review provides an overview of the evidence for, and potential pathophysiological mechanisms behind brain dysfunction at a neurobiological level, in preclinical NAFLD. We performed a systematic literature search for animal models of NAFLD studying intracerebral conditions using PubMed, Embase and Scopus. We included studies that reported data on neurobiology in rodent and pig models with evidence of steatosis or steatohepatitis assessed by liver histology. 534 unique studies were identified, and 30 studies met the selection criteria, and were included. Findings of neurobiological changes were divided into five key areas: (1) neuroinflammation, (2) neurodegeneration, (3) neurotransmitter alterations, (4) oxidative stress, and (5) changes in proteins and synaptic density. Despite significant heterogeneity in the study designs, all but one study of preclinical NAFLD reported changes in one or more of the above key areas when compared to control animals. In conclusion, this systematic review supports an association between all stages of NAFLD (from simple steatosis to non-alcoholic steatohepatitis (NASH)) and neurobiological changes in preclinical models.

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.

Management of the major complications of cirrhosis: Beyond guidelines.

Along with a growing understanding of the pathophysiology of cirrhosis and its complications, new therapies and management strategies have emerged in recent years. Many of these advances have helped inform the current EASL clinical practice guidelines1 on the management of some of the key complications of cirrhosis, such as ascites, variceal bleeding and infection. However, there are still some aspects of management where the evidence base is less clear, and/or where opinions amongst practitioners remain divided. Some of these more controversial areas are explored in this section, wherein we present evidence culminating in a suggested management approach based on expert opinion and extending beyond the current guidelines.

Determinants of mortality in patients with cirrhosis and uncontrolled variceal bleeding.

BACKGROUND & AIMS: Failure to control oesophago-gastric variceal bleeding (OGVB) and acute-on-chronic liver failure (ACLF) are both important prognostic factors in cirrhosis. The aims of this study were to determine whether ACLF and its severity define the risk of death in OGVB and whether insertion of rescue transjugular intrahepatic shunt (TIPS) improves survival in patients with failure to control OGVB and ACLF. METHODS: Data on 174 consecutive eligible patients, with failure to control OGVB between 2005 and 2015, were collected from a prospectively maintained intensive care unit registry. Rescue TIPS was defined as technically successful TIPS within 72 hours of presentation with failure to control OGVB. Cox-proportional hazards regression analyses were applied to explore the impact of ACLF and TIPS on survival in patients with failure to control OGVB. RESULTS: Patients with ACLF (n = 119) were significantly older, had organ failures and higher white cell count than patients with acute decompensation (AD, n = 55). Mortality at 42-days and 1-year was significantly higher in patients with ACLF (47.9% and 61.3%) than in those with AD (9.1% and 12.7%, p <0.001), whereas there was no difference in the number of endoscopies and transfusion requirements between these groups. TIPS was inserted in 78 patients (AD 21 [38.2%]; ACLF 57 [47.8%]; p = 0.41). In ACLF, rescue TIPS insertion was an independent favourable prognostic factor for 42-day mortality. In contrast, rescue TIPS did not impact on the outcome of patients with AD. CONCLUSIONS: This study shows that in patients with failure to control OGVB, the presence and severity of ACLF determines the risk of 42-day and 1-year mortality. Rescue TIPS is associated with improved survival in patients with ACLF. LAY SUMMARY: Variceal bleeding that is not controlled by initial endoscopy is associated with high risk of death. The results of this study showed that in the occurrence of failure of the liver and other organs defines the risk of death. In these patients, insertion of a shunt inside the liver to drain the portal vein improves survival.

Endpoints and design of clinical trials in patients with decompensated cirrhosis: Position paper of the LiverHope Consortium.

Management of decompensated cirrhosis is currently geared towards the treatment of complications once they occur. To date there is no established disease-modifying therapy aimed at halting progression of the disease and preventing the development of complications in patients with decompensated cirrhosis. The design of clinical trials to investigate new therapies for patients with decompensated cirrhosis is complex. The population of patients with decompensated cirrhosis is heterogeneous (i.e., different etiologies, comorbidities and disease severity), leading to the inclusion of diverse populations in clinical trials. In addition, primary endpoints selected for trials that include patients with decompensated cirrhosis are not homogeneous and at times may not be appropriate. This leads to difficulties in comparing results obtained from different trials. Against this background, the LiverHope Consortium organized a meeting of experts, the goal of which was to develop recommendations for the design of clinical trials and to define appropriate endpoints, both for trials aimed at modifying the natural history and preventing progression of decompensated cirrhosis, as well as for trials aimed at managing the individual complications of cirrhosis.

PREDICT identifies precipitating events associated with the clinical course of acutely decompensated cirrhosis.

BACKGROUND & AIMS: Acute decompensation (AD) of cirrhosis may present without acute-on-chronic liver failure (ACLF) (AD-No ACLF), or with ACLF (AD-ACLF), defined by organ failure(s). Herein, we aimed to analyze and characterize the precipitants leading to both of these AD phenotypes. METHODS: The multicenter, prospective, observational PREDICT study (NCT03056612) included 1,273 non-electively hospitalized patients with AD (No ACLF = 1,071; ACLF = 202). Medical history, clinical data and laboratory data were collected at enrolment and during 90-day follow-up, with particular attention given to the following characteristics of precipitants: induction of organ dysfunction or failure, systemic inflammation, chronology, intensity, and relationship to outcome. RESULTS: Among various clinical events, 4 distinct events were precipitants consistently related to AD: proven bacterial infections, severe alcoholic hepatitis, gastrointestinal bleeding with shock and toxic encephalopathy. Among patients with precipitants in the AD-No ACLF cohort and the AD-ACLF cohort (38% and 71%, respectively), almost all (96% and 97%, respectively) showed proven bacterial infection and severe alcoholic hepatitis, either alone or in combination with other events. Survival was similar in patients with proven bacterial infections or severe alcoholic hepatitis in both AD phenotypes. The number of precipitants was associated with significantly increased 90-day mortality and was paralleled by increasing levels of surrogates for systemic inflammation. Importantly, adequate first-line antibiotic treatment of proven bacterial infections was associated with a lower ACLF development rate and lower 90-day mortality. CONCLUSIONS: This study identified precipitants that are significantly associated with a distinct clinical course and prognosis in patients with AD. Specific preventive and therapeutic strategies targeting these events may improve outcomes in patients with decompensated cirrhosis. LAY SUMMARY: Acute decompensation (AD) of cirrhosis is characterized by a rapid deterioration in patient health. Herein, we aimed to analyze the precipitating events that cause AD in patients with cirrhosis. Proven bacterial infections and severe alcoholic hepatitis, either alone or in combination, accounted for almost all (96-97%) cases of AD and acute-on-chronic liver failure. Whilst the type of precipitant was not associated with mortality, the number of precipitant(s) was. This study identified precipitants that are significantly associated with a distinct clinical course and prognosis of patients with AD. Specific preventive and therapeutic strategies targeting these events may improve patient outcomes.

Potassium deficiency decreases the capacity for urea synthesis and markedly increases ammonia in rats.

Our study provides novel findings of experimental hypokalemia reducing urea cycle functionality and thereby severely increasing plasma ammonia. This is pathophysiologically interesting because plasma ammonia increases during hypokalemia by a hitherto unknown mechanism, which may be particular important in relation to the unexplained link between hypokalemia and hepatic encephalopathy. Potassium deficiency decreases gene expression, protein synthesis, and growth. The urea cycle maintains body nitrogen homeostasis including removal of toxic ammonia. Hyperammonemia is an obligatory trait of liver failure, increasing the risk for hepatic encephalopathy, and hypokalemia is reported to increase ammonia. We aimed to clarify the effects of experimental hypokalemia on the in vivo capacity of the urea cycle, on the genes of the enzymes involved, and on ammonia concentrations. Female Wistar rats were fed a potassium-free diet for 13 days. Half of the rats were then potassium repleted. Both groups were compared with pair- and free-fed controls. The following were measured: in vivo capacity of urea-nitrogen synthesis (CUNS); gene expression (mRNA) of urea cycle enzymes; plasma potassium, sodium, and ammonia; intracellular potassium, sodium, and magnesium in liver, kidney, and muscle tissues; and liver sodium/potassium pumps. Liver histology was assessed. The diet induced hypokalemia of 1.9 ± 0.4 mmol/L. Compared with pair-fed controls, the in vivo CUNS was reduced by 34% (P < 0.01), gene expression of argininosuccinate synthetase 1 (ASS1) was decreased by 33% (P < 0.05), and plasma ammonia concentrations were eightfold elevated (P < 0.001). Kidney and muscle tissue potassium contents were markedly decreased but unchanged in liver tissue. Protein expressions of liver sodium/potassium pumps were unchanged. Repletion of potassium reverted all the changes. Hypokalemia decreased the capacity for urea synthesis via gene effects. The intervention led to marked hyperammonemia, quantitatively explainable by the compromised urea cycle. Our findings motivate clinical studies of patients with liver disease.

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.

Liver perfusion MRI in a rodent model of cirrhosis: Agreement with bulk-flow phase-contrast MRI and noninvasive evaluation of inflammation in chronic liver disease using flow-sensitive alternating inversion recovery arterial spin labelling and tissue T1.

Noninvasive measurements of liver perfusion and fibrosis in cirrhotic small animals can help develop treatments for haemodynamic complications of liver disease. Here, we measure liver perfusion in cirrhotic rodents using flow-sensitive alternating inversion recovery arterial spin labelling (FAIR ASL), evaluating agreement with previously validated caval subtraction phase-contrast magnetic resonance imaging (PCMRI) total liver blood flow (TLBF). Baseline differences in cirrhotic rodents and the haemodynamic effects of acute inflammation were investigated using FAIR ASL and tissue T1. Sprague-Dawley rats (nine bile duct ligated [BDL] and ten sham surgery controls) underwent baseline hepatic FAIR ASL with T1 measurement and caval subtraction PCMRI (with two-dimensional infra-/supra-hepatic inferior vena caval studies), induction of inflammation with intravenous lipopolysaccharide (LPS) and repeat liver FAIR ASL with T1 measurement after ~90 minutes. The mean difference between FAIR ASL hepatic perfusion and caval subtraction PCMRI TLBF was -51 ± 30 ml/min/100 g (Bland-Altman 95% limits-of-agreement ±258 ml/min/100 g). The FAIR ASL coefficient of variation was smaller than for caval subtraction PCMRI (29.3% vs 50.1%; P = .03). At baseline, FAIR ASL liver perfusion was lower in BDL rats (199 ± 32 ml/min/100 g vs sham 316 ± 24 ml/min/100 g; P = .01) but liver T1 was higher (BDL 1533 ± 50 vs sham 1256 ± 18 ms; P = .0004). Post-LPS FAIR ASL liver perfusion response differences were observed between sham/BDL rats (P = .02), approaching significance in sham (+78 ± 33 ml/min/100 g; P = .06) but not BDL rats (-49 ± 40 ml/min/100 g; P = .47). Post-LPS differences in liver tissue T1 were nonsignificant (P = .35). FAIR ASL hepatic perfusion and caval subtraction PCMRI TLBF agreement was modest, with significant baseline FAIR ASL liver perfusion and tissue T1 differences in rodents with advanced cirrhosis compared with controls. Following inflammatory stress, differences in hepatic perfusion response were detected between cirrhotic/control animals, but liver T1 was unaffected. Findings underline the potential of FAIR ASL in the assessment of vasoactive treatments for patients with chronic liver disease and inflammation.

Haemodynamic changes in cirrhosis following terlipressin and induction of sepsis-a preclinical study using caval subtraction phase-contrast and cardiac MRI.

OBJECTIVES: Effects of liver disease on portal venous (PV), hepatic arterial (HA), total liver blood flow (TLBF), and cardiac function are poorly understood. Terlipressin modulates PV flow but effects on HA, TLBF, and sepsis/acute-on-chronic liver failure (ACLF)-induced haemodynamic changes are poorly characterised. In this study, we investigated the effects of terlipressin and sepsis/ACLF on hepatic haemodynamics and cardiac function in a rodent cirrhosis model using caval subtraction phase-contrast (PC) MRI and cardiac cine MRI. METHODS: Sprague-Dawley rats (n = 18 bile duct-ligated (BDL), n = 16 sham surgery controls) underwent caval subtraction PCMRI to estimate TLBF and HA flow and short-axis cardiac cine MRI for systolic function at baseline, following terlipressin and lipopolysaccharide (LPS) infusion, to model ACLF. RESULTS: All baseline hepatic haemodynamic/cardiac systolic function parameters (except heart rate and LV mass) were significantly different in BDL rats. Following terlipressin, baseline PV flow (sham 181.4 ± 12.1 ml/min/100 g; BDL 68.5 ± 10.1 ml/min/100 g) reduced (sham - 90.3 ± 11.1 ml/min/100 g, p < 0.0001; BDL - 31.0 ± 8.0 ml/min/100 g, p = 0.02), sham baseline HA flow (33.0 ± 11.3 ml/min/100 g) increased (+ 92.8 ± 21.3 ml/min/100 g, p = 0.0003), but BDL baseline HA flow (83.8 ml/min/100 g) decreased (- 34.4 ± 7.5 ml/min/100 g, p = 0.11). Sham baseline TLBF (214.3 ± 16.7 ml/min/100 g) was maintained (+ 2.5 ± 14.0 ml/min/100 g, p > 0.99) but BDL baseline TLBF (152.3 ± 18.7 ml/min/100 g) declined (- 65.5 ± 8.5 ml/min/100 g, p = 0.0004). Following LPS, there were significant differences between cohort and change in HA fraction (p = 0.03) and TLBF (p = 0.01) with BDL baseline HA fraction (46.2 ± 4.6%) reducing (- 20.9 ± 7.5%, p = 0.03) but sham baseline HA fraction (38.2 ± 2.0%) remaining unchanged (+ 2.9 ± 6.1%, p > 0.99). Animal cohort and change in systolic function interactions were significant only for heart rate (p = 0.01) and end-diastolic volume (p = 0.03). CONCLUSIONS: Caval subtraction PCMRI and cardiac MRI in a rodent model of cirrhosis demonstrate significant baseline hepatic haemodynamic/cardiac differences, failure of the HA buffer response post-terlipressin and an altered HA fraction response in sepsis, informing potential translation to ACLF patients. KEY POINTS: Caval subtraction phase-contrast and cardiac MRI demonstrate: • Significant differences between cirrhotic/non-cirrhotic rodent hepatic blood flow and cardiac systolic function at baseline. • Failure of the hepatic arterial buffer response in cirrhotic rodents in response to terlipressin. • Reductions in hepatic arterial flow fraction in the setting of acute-on-chronic 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.