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Background and Aims: The prevalence of chronic liver disease in adults exceeds 30% in some countries and there is significant interest in developing tests and treatments to help control disease progression and reduce healthcare burden. Breath is a rich sampling matrix that offers non-invasive solutions suitable for early-stage detection and disease monitoring. Having previously investigated targeted analysis of a single biomarker, here we investigated a multiparametric approach to breath testing that would provide more robust and reliable results for clinical use. Methods: To identify candidate biomarkers we compared 46 breath samples from cirrhosis patients and 42 from controls. Collection and analysis used Breath Biopsy OMNITM, maximizing signal and contrast to background to provide high confidence biomarker detection based upon gas chromatography mass spectrometry (GC-MS). Blank samples were also analyzed to provide detailed information on background volatile organic compounds (VOCs) levels. Results: A set of 29 breath VOCs differed significantly between cirrhosis and controls. A classification model based on these VOCs had an area under the curve (AUC) of 0.95 +/- 0.04 in cross-validated test sets. The seven best performing VOCs were sufficient to maximize classification performance. A subset of 11 VOCs was correlated with blood metrics of liver function (bilirubin, albumin, prothrombin time) and separated patients by cirrhosis severity using principal component analysis. Conclusions: A set of seven VOCs consisting of previously reported and novel candidates show promise as a panel for liver disease detection and monitoring, showing correlation to disease severity and serum biomarkers at late stage.

INTRODUCTION: Liver cirrhosis and its complication — hepatocellular carcinoma (HCC) — have been associated with increased exhaled limonene. It is currently unclear whether this increase is more strongly associated with the presence of HCC or with the severity of liver dysfunction. METHODS: We compared the exhaled breath of 40 controls, 32 cirrhotic patients, and 12 cirrhotic patients with HCC using the Breath Biopsy platform. Breath samples were analyzed by thermal desorption–gas chromatography–mass spectrometry. Limonene levels were compared between the groups and correlated to bilirubin, albumin, prothrombin time international normalized ratio, and alanine aminotransferase. RESULTS: Breath limonene concentration was significantly elevated in subjects with cirrhosis-induced HCC (M: 82.1 ng/L, interquartile range [IQR]: 16.33–199.32 ng/L) and cirrhosis (M: 32.6 ng/L, IQR: 6.55–123.07 ng/L) compared with controls (M: 6.2 ng/L, IQR: 2.62–9.57 ng/L) (P value = 0.0005 and 0.0001, respectively) with no significant difference between 2 diseased groups (P value = 0.37). Levels of exhaled limonene correlated with serum bilirubin (R2 = 0.25, P value = 0.0016, r = 0.51), albumin (R2 = 0.58, P value = 5.3e-8, r = −0.76), and international normalized ratio (R2 = 0.29, P value = 0.0003, r = 0.51), but not with alanine aminotransferase (R2 = 0.01, P value = 0.36, r = 0.19). DISCUSSION: Exhaled limonene levels are primarily affected by the presence of cirrhosis through reduced liver functional capacity, as indicated by limonene correlation with blood metrics of impaired hepatic clearance and protein synthesis capacity, without further alterations observed in subjects with HCC. This suggests that exhaled limonene is a potential non-invasive marker of liver metabolic capacity (see Visual abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A388).

Background Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension. Methods and findings To establish preclinical proof of concept, we developed two independent rat models of cirrhosis that were characterized by progressive reduction in renal blood flow and glomerular filtration rate and showed evidence of renal endothelial dysfunction. We then set out to further explore and validate our hypothesis in a phase 2 randomized open-label parallel-group study in male and female patients with alcohol-related cirrhosis and portal hypertension. Forty patients were randomized 1:1 to treatment with serelaxin intravenous (i.v.) infusion (for 60 min at 80 μg/kg/d and then 60 min at 30 μg/kg/d) or terlipressin (single 2-mg i.v. bolus), and the regional hemodynamic effects were quantified by phase contrast magnetic resonance angiography at baseline and after 120 min. The primary endpoint was the change from baseline in total renal artery blood flow. Therapeutic targeting of renal vasoconstriction with serelaxin in the rat models increased kidney perfusion, oxygenation, and function through reduction in renal vascular resistance, reversal of endothelial dysfunction, and increased activation of the AKT/eNOS/NO signaling pathway in the kidney. In the randomized clinical study, infusion of serelaxin for 120 min increased total renal arterial blood flow by 65% (95% CI 40%, 95%; p < 0.001) from baseline. Administration of serelaxin was safe and well tolerated, with no detrimental effect on systemic blood pressure or hepatic perfusion. The clinical study’s main limitations were the relatively small sample size and stable, well-compensated population. Conclusions Our mechanistic findings in rat models and exploratory study in human cirrhosis suggest the therapeutic potential of selective renal vasodilation using serelaxin as a new treatment for renal dysfunction in cirrhosis, although further validation in patients with more advanced cirrhosis and renal dysfunction is required. Trial registration ClinicalTrials.gov NCT01640964, In a Research Article, Jonathan Fallowfield and colleagues study serelaxin as a possible treatment for renal vasoconstriction in liver cirrhosis., Author summary Why was this study done? Hepatorenal syndrome type 1 is a rapidly progressive, but potentially reversible, form of acute kidney injury occurring in patients with liver cirrhosis, characterized by severe renal vasoconstriction and a very poor prognosis. There is a significant unmet need for a widely approved, safe, and effective pharmacological treatment. Serelaxin, a recombinant form of the human peptide hormone relaxin-2, has been shown to increase renal perfusion in healthy human volunteers. We tested whether serelaxin could ameliorate renal vasoconstriction and renal dysfunction in cirrhosis. What did the researchers do and find? Administration of serelaxin improved renal blood flow, oxygenation, and function in two independent animal models of cirrhosis through reversal of endothelial dysfunction and increased activation of nitric oxide signaling in the kidney. In an exploratory phase 2 clinical trial in patients with cirrhosis and portal hypertension, serelaxin infusion induced a significant increase in renal blood flow and was safe and well tolerated, with no adverse effects on systemic blood pressure or hepatic perfusion. What do these findings mean? Our data indicate that selective targeting of renal vasoconstriction using serelaxin may modulate renal dysfunction in cirrhosis without affecting systemic blood pressure. Further studies in patients with more advanced cirrhosis and renal dysfunction are required to confirm whether there are beneficial effects on renal function and patient outcomes.

Liver fibrosis and its end stage, cirrhosis, represent the final common pathway of virtually all chronic liver diseases. As our understanding of the pathogenesis of liver fibrosis has progressed, it has become evident that the liver provides a useful generic model of inflammation and repair, demonstrating interplay between the epithelial, inflammatory, myofibroblast and extracellular matrix components of the mammalian wound healing response. In this review, the paradigm that liver fibrosis is a potentially reversible process-demonstrating both fibrosis (scarring) and resolution with remodeling and restitution of normal or near-normal tissue architecture-will be explored. The remarkable progress in unraveling the complexities of liver fibrosis has been due to developments in technologies including the isolation of discrete liver cell populations which have facilitated studies of their behavior in tissue culture and in vivo. More recently, animal models that mimic chronic liver diseases have been established. These models are tractable and can be applied in gene knockout and transgenic mice. This article will highlight recent studies that reveal key mechanisms mediating the regression of liver fibrosis which have derived from the use of such complementary animal and human model systems and describe how our greater understanding of this dynamic process is likely to inform the development of directed and effective anti-fibrotic approaches.

Portal hypertension is the main cause of complications in patients with cirrhosis. However, evaluating the development and progression of portal hypertension represents a challenge for clinicians. There has been considerable focus on the potential role of noninvasive markers of portal hypertension that could be used to stratify patients with respect to the stage of portal hypertension and to monitor disease progression or treatment response in a longitudinal manner without having to undertake repeated invasive assessment. The pathogenesis of portal hypertension is increasingly understood and emerging knowledge of the vascular processes that underpin portal hypertension has paved the way for exploring novel biomarkers of vascular injury, angiogenesis, and endothelial dysfunction. In this paper we focus on the pathogenesis of portal hypertension and potential non-invasive biomarkers with particular emphasis on serum analytes.

Introduction Hepatorenal syndrome (HRS) is a feared complication of cirrhosis characterised by intense renal vasoconstriction. The pathophysiology remains unclear, pharmacotherapy is limited and mortality is high. We investigated vascular responsiveness and the pathogenesis of renal vasoconstriction in models of advanced rat cirrhosis. Additionally,we determined the mechanism of action of the vasoactive peptide relaxin (RLN), previously shown to increase renal blood flow (RBF) in experimental cirrhosis (Snowdon V et al ., BSG 2013). Methods We induced cirrhosis,reduced RBF and renal dysfunction in male SD rats by carbon tetrachloride (16 wk) or bile duct ligation (4 wk). Arteries from renal (renal, segmental, interlobar) and splanchnic circulation were isolated for functional assessment using wire myography. qPCR array for vasoactive signalling genes,western blot for eNOS signalling proteins and NOS activity assay were undertaken in cirrhotic and control kidneys. Markers of oxidative stress and inflammatory cytokines were measured in serum by ELISA. We studied the effects of s.c. infusion of recombinant human RLN(seralaxin;72 h, 4 µg/h) on these parameters. Doppler USS measured changes in cardiac output (CO) and renal arterial resistive index (RRI) in response to i.v. RLN (4 µg). Kidney endothelial morphology was assessed by electron microscopy, H+E and PAS stained kidney by light microscopy. Results In renal arteries from control and cirrhotic rats endothelial vasodilatation was eNOS-dependent. In cirrhotic rats endothelium-dependent relaxation (acetylcholine; 10–9–3 × 10–5 M) was dramatically reduced (p Conclusion Renal vascular endothelial dysfunction characterises experimental cirrhosis, through a reduction in renal eNOS activity. This impairment may contribute to the renal vasoconstriction seen in cirrhosis and is a promising target for therapeutic modulation. RLN treatment restored renal endothelial vasodilatation. The potential for recombinant forms of RLN as a haemodynamic modulator in human cirrhosis and HRS merits investigation in translational studies. Disclosure of Interest None Declared.

Introduction Hepatorenal syndrome (HRS) is a feared complication of cirrhosis with a high mortality rate and limited treatment options. The hallmark features of HRS are profound renal vasoconstriction, resulting in a functional renal failure but with normal kidney histology. The peptide hormone relaxin (RLN) mediates maternal haemodynamic adaptations to pregnancy, including increased renal blood flow (RBF) and glomerular filtration rate (GFR). We hypothesised that RLN could beneficially modulate RBF in cirrhosis and HRS. Methods Cirrhosis, with reduced RBF, was induced in rats by 16 weeks of intraperitoneal (i.p.) carbon tetrachloride (CCl4) and decompensated biliary cirrhosis by 3 weeks bile duct ligation (BDL). We measured the effect of acute intravenous (i.v.) and extended (72 hr) subcutaneous (s.c.) RLN on systemic haemodynamics, RBF, GFR and organ histology. Subgroups of rats were co-treated with the nitric oxide (NO) synthase inhibitor L-NAME. Blood oxygen dependent-magnetic resonance imaging (BOLD-MRI) was used to quantify changes in renal oxygenation. Tissue expression and distribution of RLN receptor (RXFP1) was determined by qPCR and immunofluorescence. Expression of vasoconstrictor genes was quantified by qPCR array. Results RXFP1 was detected on glomerular podocytes, renal pericytes, and endothelial cells of the renal, segmental and interlobar arteries of cirrhotic rats. In CCl4 cirrhosis, acute i.v. RLN (4µg) induced a 50% increase in RBF after 60 minutes ( p n = 6). BOLD-MRI showed increased tissue oxygenation at the same timepoint in renal cortex and medulla. Extended s.c. RLN increased RBF by 54% in CCl4 ( p n = 8) and 57% in BDL ( p n = 5) and increased GFR by 138% in CCl4 ( p n = 8) and 103% in BDL ( p n = 5). Mean arterial pressure was unaffected by RLN. L-NAME (250mg/L) orally (p.o.) abrogated the effect of RLN on RBF and GFR. The relative expression of vasoconstrictor genes in the kidney was markedly reduced by RLN treatment. Conclusion RLN increases RBF in experimental cirrhosis. Crucially, RLN also improves renal function and oxygenation but does not induce systemic hypotension even in decompensated disease. The effects of RLN are mediated via augmentation of NO and downregulation of vasoconstrictor genes known to be important in the pathogenesis of HRS. RLN has potential as a treatment for HRS and further translational studies are warranted. Disclosure of Interest None Declared.

Background Hepatorenal syndrome is a feared complication of cirrhosis, with a high mortality rate and limited treatment options. The hallmarks of the disorder are functional renal failure, normal kidney histological findings, and profound renal vasoconstriction. The hormone relaxin mediates haemodynamic adaptations to pregnancy including increased renal blood flow and glomerular filtration rate (GFR). We hypothesised that relaxin could be used to modulate renal blood flow in cirrhosis.

Background Hepatorenal syndrome is a feared complication of cirrhosis, with a high mortality rate and limited treatment options. The hallmarks of the disorder are functional renal failure, normal kidney histological findings, and profound renal vasoconstriction. The hormone relaxin mediates haemodynamic adaptations to pregnancy including increased renal blood flow and glomerular filtration rate (GFR). We hypothesised that relaxin could be used to modulate renal blood flow in cirrhosis. Methods Cirrhosis was induced in male rats by 16 weeks of carbon tetrachloride (intraperitoneally) and decompensated biliary cirrhosis by bile duct ligation. The effect of acute intravenous or extended (72 h) subcutaneous relaxin versus placebo was determined by analysis of systemic haemodynamics, renal blood flow, GFR, and organ histology. The effect of co-treatment with L-NG-nitroarginine methyl ester (L-NAME) was measured in subgroups of relaxin or placebo treated rats. Blood oxygen dependent-MRI (BOLD-MRI) was used to non-invasively detect changes to renal oxygenation. Hepatic and renal expression of RXFP1 (relaxin receptor) was determined by IHC and quantitative PCR (qPCR). A qPCR array was used to quantify changes in renal vasodilator/vasoconstrictor gene expression in response to relaxin. Findings RXFP1 was detected in glomerular podocytes, renal pericytes, and renal, segmental, and interlobar arteries of cirrhotic rats. In carbon tetrachloride (CCl 4 ) induced cirrhosis, acute intravenous relaxin (4 μg) induced a 50% increase in renal blood flow after 60 min (p 4 (p 4 (p vs placebo, n=8) and 70% in bile duct ligated animals (p vs placebo, n=3). Mean arterial pressure was unaffected by relaxin. L-NAME (250 mg/L) orally abrogated the effect of relaxin on renal blood flow and GFR. Relative expression of vasoconstrictor genes in kidney was markedly reduced by relaxin treatment. Interpretation Relaxin increases renal blood flow in experimental models of cirrhosis. Crucially, relaxin also improves renal function and oxygenation and does not induce systemic hypotension even in advanced disease. These effects are modulated via augmentation of nitric oxide and downregulation of vasoconstrictors, pivotal in the pathogenesis of hepatorenal syndrome. Relaxin has potential as a novel therapy for hepatorenal syndrome, and further translational studies are warranted. Funding Wellcome Trust through the Scottish Translational Medicine and Therapeutics Initiative.

Introduction We have previously reported that the multifunctional hormone relaxin (RLX) downregulated the activation state and contractility of hepatic myofibroblasts and reduced portal hypertension (PHT) in cirrhotic rats (Fallowfield J et al BASL 2010). RLX has been shown to induce a range of haemodynamic effects in different organs and species, largely through effects on nitric oxide (NO). In cirrhosis, there is hepatic NO deficiency and hyporesponsiveness. We postulated that the effects of RLX on PHT were, at least in part, mediated by activation of the NO pathway. Methods Cirrhosis and PHT was induced in age-matched male Sprague-Dawley rats by 8 weeks biweekly i.p. CCl4, before randomisation to the following groups: (1) recombinant human H2-relaxin (H2-RLX) s.c. for 72 h; (2) placebo s.c. for 72 h; (3) H2-RLX s.c. + L-NAME p.o. for 72 h; (4) placebo s.c. + L-NAME p.o. for 72 h; n =5–10/group. NO levels in serum were determined by quantitative immunoassay for total nitrite and hepatic NO bioavailability by cGMP immunoassay. Relative levels of Ser 473 phosphorylated Akt (p-Akt) and Ser 1179 phosphorylated eNOS (p-eNOS) protein in whole liver extracts were quantified by Western blotting. Rho-kinase activity was assessed by phosphorylation of the endogenous Rho-kinase substrate moesin (Thr 558 ). Portal pressure (PP) and mean arterial pressure (MAP) were measured under general anaesthesia by direct cannulation. Results Rats treated with CCl4 for 8 weeks developed micronodular cirrhosis, splenomegaly and PHT. There was no difference in mean serum nitrite levels between H2-RLX and placebo treated rats. However, H2-RLX increased hepatic cGMP production (p Conclusion A reduction in NO bioavailability is considered to be a major factor increasing intrahepatic vascular tone in cirrhosis. Our data indicate that H2-RLX was capable of stimulating intrinsic (but not systemic) NO generation in fibrotic liver by activating the Akt/eNOS/cGMP pathway. Furthermore, inhibition of this axis with L-NAME ablated the portal hypotensive effect of H2-RLX, suggesting that it could represent a novel liver-specific NO donor in cirrhotic PHT. Competing interests None declared.