N. Davies

Serum autotaxin is increased in pruritus of cholestasis, but not of other origin, and responds to therapeutic interventions†‡

Pruritus is a seriously disabling symptom accompanying many cholestatic liver disorders. Recent experimental evidence implicated the lysophospholipase, autotaxin (ATX), and its product, lysophosphatidic acid (LPA), as potential mediators of cholestatic pruritus. In this study, we highlight that increased serum ATX levels are specific for pruritus of cholestasis, but not pruritus of uremia, Hodgkins disease, or atopic dermatitis. Treatment of patients with cholestasis with the bile salt sequestrant, colesevelam, but not placebo, effectively reduced total serum bile salts and fibroblast growth factor 19 levels, but only marginally altered pruritus intensity and ATX activity. Rifampicin (RMP) significantly reduced itch intensity and ATX activity in patients with pruritus not responding to bile salt sequestrants. In vitro, RMP inhibited ATX expression in human HepG2 hepatoma cells and hepatoma cells overexpressing the pregnane X receptor (PXR), but not in hepatoma cells in which PXR was knocked down. Treatment of severe, refractory pruritus by the molecular adsorbents recirculation system or nasobiliary drainage improved itch intensity, which, again, correlated with the reduction of ATX levels. Upon reoccurrence of pruritus, ATX activity returned to pretreatment values. Conclusion: Serum ATX activity is specifically increased in patients with cholestatic, but not other forms of, systemic pruritus and closely correlates with the effectiveness of therapeutic interventions. The beneficial antipruritic action of RMP may be explained, at least partly, by the PXR‐dependent transcriptional inhibition of ATX expression. Thus, ATX likely represents a novel therapeutic target for pruritus of cholestasis. (HEPATOLOGY 2012)

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.

Increased renal expression and urinary excretion of TLR4 in acute kidney injury associated with cirrhosis

Patients with cirrhosis frequently develop renal dysfunction, a proportion of who do not fulfill criteria for hepatorenal syndrome (HRS). We hypothesized that the kidneys in these patients would exhibit histological and biomarker evidence of kidney injury. We looked specifically for TLR expression as they may mediate kidney injury.

'Out-patient' albumin dialysis for cholestatic patients with intractable pruritus

Intractable pruritus is a major problem for some patients with cholestasis. Albumin dialysis has been shown to ameliorate pruritus, but long‐term outcome data are limited.

Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure

BACKGROUND & AIMS In cirrhosis, superimposed inflammation often culminates in acute-on-chronic liver failure (ACLF) but the mechanism underlying this increased sensitivity is not clear. Cx43 is a ubiquitous gap junction protein that allows transmission of signals between cells at a much higher rate than the constitutively expressed gap junctions. The aims of the study were to test the hypothesis that inflammation drives the increased expression of hepatic Cx43 and to determine its role by Cx43 inhibition. METHODS Four weeks after bile-duct ligation (BDL) or sham operation, rats were treated with an anti-TNF antibody, or saline; with or without LPS (1mg/kg); given 3h prior to termination. Biochemistry and cytokines were measured in the plasma and hepatic protein expression (NFkB, TNFα, iNOS, 4HNE, Cx26, 32, and 43) and confocal microscopy (Cx26, 32, and 43) were performed. The effect of a Cx43-specific inhibitory peptide was studied in a mouse BDL model. RESULTS BDL animals administered LPS developed typical features of ACLF but animals administered infliximab were relatively protected. Cx26/32 expression was significantly decreased in BDL animals while Cx43 was significantly increased and increased further following LPS. Infliximab treatment prevented this increase. However, inhibiting Cx43 in BDL mice produced detrimental effects with markedly greater hepatocellular necrosis. CONCLUSIONS The results of this study show for the first time an increased expression of hepatic Cx43 in cirrhosis and ACLF, which was related to the severity of inflammation. This increased Cx43 expression is likely to be an adaptive protective response of the liver to allow better cell-to-cell communication.

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.

Reduction in hyperammonaemia by ornithine phenylacetate prevents lipopolysaccharide‐induced brain edema and coma in cirrhotic rats

In liver failure, inflammation synergistically exacerbates the deleterious cerebral effects of ammonia. The aims were to test whether treatment with the ammonia‐lowering agent ornithine phenylacetate (OP) and/or anti‐TNF‐α (infliximab) prevent the deleterious brain consequences of lipopolysaccharide (LPS) in cirrhotic rats.

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

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.

Albumin Regeneration for Extracorporeal Liver Support Using Prometheus: A Step in the Right Direction

The incidence of liver disease is increasing worldwide and about 1 million patients die from liver ailure each year. Mortality rates in patients with liver ailure are about 50% and liver transplantation is the nly effective treatment known to prolong the life of hese patients.1 The expanding gap between the number of patients on the waiting list for transplantation and the number of organs available highlights the need for a temporary liver support system that could be used on an emergency basis either as a bridge to liver regeneration or to liver transplantation. To achieve these effects, the device should be able to lower blood levels of toxic substances that accumulate in liver failure so that further organ injury can be avoided and an environment for liver regeneration created. For such a device to be clinically useful, it must be efficacious, cost effective, and relatively straightforward to use.2 Such a device is an unmet need that the Prometheus system, which is essentially a detoxification system based on the principles of albumin regeneration, described in a paper in this issue of GASTROENTEROLOGY sought to address.3 Liver support devices are traditionally categorized into 2 main types. Purely artificial devices are essentially detoxification systems, whereas bioartificial devices contain living hepatocytes. Early studies with both types of devices have demonstrated biological effects both on the removal of toxic substances that accumulate in liver failure as well as on organ function. Hemodialysis and hemofiltration are capable of removing toxic substances from the circulation, but their efficacy is low because most substances that accumulate in liver failure such as toxic bile acids, bilirubin, endotoxin, and cytokines are protein bound or have a high molecular weight. Although activated charcoal-based detoxification systems have been used in the past, the present artificial detoxification devices rely on the premise of detoxifying albumin either directly, as in the Prometheus, or indirectly via toxin exchange in the molecular adsorbent recirculating system (MARS).1,2,5–7 The concept is based around the discovery that albumin is not merely a fluid and a protein that provides oncotic properties to the plasma, but also is a very important circulating detoxification molecule that carries toxic wastes to the liver. In liver failure, the data suggest that

PTH-095 Oral carbon therapy is associated with a selective modulation of the microbiome in cirrhotic rats which is associated with a significant reduction in inflammatory activation

Introduction The host-microbiome interaction is pathological in cirrhosis promoting a dysregulated inflammatory response resulting in organ injury and diminished survival. Yaq-001 is an oral non-absorpable carbon shown to result in improvement in hepatic haemodynamics and markers of liver injury but the mechanism of how this is achieved in unknown. The aims of this study were to determine whether the beneficial effects of Yaq-001 was related to the composition and associated functional state of the microbiome in bile-duct ligated cirrhotic animals. Method BDL (n = 12) or sham (n = 12) rats were randomised to treatment with Yaq-001 (Yaqrit Ltd. UK) from weeks 2–4. Analysis of urine samples was performed using 1NMRs. Bacterial DNA from stool was extracted, amplified and sequenced. Arterial plasma was co-incubated with HEK-Blue hTLR4 and IL-1β/IL-18 reporter cell lines. Constitutive ROS and LPS-induced ROS production from circulating monocyte populations was determined using flow cytometry. Results BDL was associated with a significant reduction in Peptidostreptococcaceae and Clostridium XI (p < 0.05) in stool compared to sham controls. Carbon therapy was associated with significant increases in firmicutes, in particular clostridia populations in stool with significant reductions in bacteroides populations (p < 0.05). Functionally, BDL rats were found to have significantly higher urinary bile acids, Trimethylamine N-oxide, benzoate, glycine, acetate and lactate than sham controls (p < 0.05). Significantly lower levels of citrate, dimethylamine (DMA) and creatinine were observed in BDL compared to sham animals (p < 0.05). Carbon treatment results in a significant increase in urinary creatinineand bile acids and reduction in urinary glycine (p < 0.05). A significant increase in IL18/IL1B expression was observed in untreated BDL rats compared to sham which was significantly attenuated with carbon treatment (p < 0.05). Monocyte LPS-induced ROS production was significantly higher in BDL rats but attenuated with carbon treatment (p < 0.05). Conclusion The results of this study show that Yaq-001, which is a non-specific adsorbent has substantial effects on the composition and function of the microbiome in cirrhotic rats and positively modulates the function of monocytes regarding ROS production and inflammasome activation. These data provide compelling evidence that the gut bacterial products are important in mediating immune dysfunction in cirrhosis and is a target of therapy. Disclosure of interest None Declared.