A. Habtesion

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.

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.

294 TLR 9 INHIBITION: A NOVEL TARGET OF THERAPY FOR PRIMARY LIVER CANCER

Introduction Toll like receptor 9 (TLR9) is a member of the nucleotide-sensing endosomal TLR family which is critical to the innate immune defense against invading pathogens. TLR9 is activated by unmethylated CpG which is highly specific for bacterial DNA. Upon activation, TLR9 traffics from the endoplasmic reticulum (ER) to endosomes TLR9 signalling is inhibited by the aminoquinolone drug chloroquine. Aims (1) assess changes in TLR9 subcellular distribution. (2) Detect any changes in the endolysosomal system. (3) Determine the effects on cell proliferation in hepatocellular carcinoma (HCC) and cholangio carcinoma cell (CC) lines upon stimulation and inhibition of TLR9 signalling in each case. Methods Huh7D and HUCCT cells were treated with unmethylated CpG (ODN 2006) to stimulate, or chloroquine and Dynavax; IRS compound to inhibit TLR9 signalling. Cells were also treated with the TLR9 antagonist iODN. Cell growth was assessed and confocal immunofluorescence microscopy was used to determine TLR9 subcellular localisation using EEA1 and LAMP1, markers of the endolysosomal system. Results Confocal microscopy indicated a marked nuclear translocation of TLR9 in HUCCT and Huh7D when stimulated with CpG, while unstimulated controls showed cytoplasmic TLR9 localisation. TLR9 inhibition by iODN and chloroquine resulted in decreased cytoplasmic TLR9 meanwhile Dynavax treatment caused translocation of TLR9 to the perinuclear membranes. Dramatic changes were also observed in the distribution of LAMP1 and EEA1, which were found to be localise to juxtanuclear punctae on TLR9 stimulation. While following inhibition they translocated to perinuclear membranes. Huh7D cell counts the CpG treated cells, iODN, chloroquine and Dynavax compound were 4.5×10 5 2.1×10 5 , 1.5×10 5 and 1.7×10 5 per ml respectively, compared with the untreated cells 3×10 5 per ml which indicate a significant increase in proliferation with increased TLR9 stimulation and a significant decrease with TLR9 inhibition (p CpG treated cells, iODN, chloroquine and Dynavax were respectively 3.3×10 5 , 1.8×10 5 , 1.4×10 5 and 1.5×10 5 per ml compared with the untreated cells at 1.7×10 5 per ml. Conclusion Our study indicates that TLR9 activation increases cell proliferation whereas inhibition reduces it. Our data suggest that TLR9 may be associated with tumour proliferation and may provide a potential target for therapy in liver tumours. Competing interests None declared.

293 SELECTIVE GUT DECOMTAMINATION REDUCES HEPATIC EXPRESSION OF TOLL-LIKE RECEPTOR (TLR) 4 AND DEVELOPMENT OF CIRRHOSIS BUT DOES NOT PREVENT DEVELOPMENT OF HEPATOCELLULAR CARCINOMA (HCC)

Introduction Recent studies suggest that TLR4 inhibition may prevent fibrosis in murine models. Chronic antigenic stimulation due to increased bacterial gut translocation leads to upregulation of hepatic TLR4 and this may lead to fibrosis, cirrhosis and HCC. The aims of this study were to determine whether gut decontamination with Norfloxacin prevents cirrhosis and HCC in rodent model of cirrhosis and HCC. Methods 18 Fisher rats divided into three groups; 1st treated with DEN and NMOR (carcinogens). 2nd was treated with the same carcinogens + Norfloxacin from the beginning to 14 weeks (end of experiment). 3rd group was control. H&E, reticulin and Immunohistochemistry were done also liver function and TNF-α were measured. Results All the rats in 1st and 2nd groups developed HCC, the severity of which was not different between groups. With reticulin stain there was significantly reduced fibrosis in the group treated with Norfloxacin (score: 1 (0–2)) compared with the untreated group (score: 4 (3–5)) (p ** ). No difference inTLR4 expression was observed in the HCC nodules in both groups. There was a reduction of ALT (p Conclusion The results of this study suggest that selective decontamination of the gut may be a novel strategy to prevent cirrhosis probably by inhibiting hepatic TLR4 expression. In this model of cirrhosis and HCC, reduction of hepatic TLR4 does not prevent development of HCC suggesting that the mechanisms of its development are unrelated to severity of fibrosis and TLR4 related mechanisms. Competing interests None declared.

577 ALBUMIN IS CENTRAL IN MEDIATING THE CARDIO-RENAL DYSFUNCTION OF CIRRHOSIS: A STUDY IN ANALBUMINAEMIC RATS

Introduction Albumin is a multifunctional protein which is reduced in concentration and function in liver disease. Albumin infusion prevents and improves renal dysfunction in patients with advanced liver failure but the mechanisms of its beneficial properties are unclear. We hypothesised that albumin is central in the maintenance of cardio-renal function in cirrhosis and albumin impairment worsens outcome. In order to answer this question we investigated analbuminaemic rats, characterised by lack of albumin but with normal protein concentration, following induction of cirrhosis with bile duct ligation (BDL). Methods Male Sprague-Dawley (SD) and Nagase analbuminaemic (NAR) rats were studied 6 weeks after BDL or sham surgery (n=6 sham-SD, 8 sham-NAR, 7 SD-BDL, 7 NAR-BDL). Rats underwent systemic mean arterial pressure (MAP) and portal pressure (PP) assessment under terminal anaesthesia. Plasma and urine were collected for measurements of renal function and protein profile. Plasma renin activity (PRA) was measured as a marker of cardio-renal dysfunction. Urinary neopterin, a marker of macrophage activation was assessed. Results NARs showed plasma total protein concentration similar to SD despite lack of albumin before (72±15 vs 82±8) and after BDL (67±7 vs 75±22). After BDL both groups of animals showed histological evidence of severe liver damage, though the NARs showed a significantly worse systemic haemodynamics with lower MAP (p=0.01), evidence of renal dysfunction indicated by higher plasma creatinine and higher PRA (p Conclusion A lack of albumin was associated with a raised PRA and a marked deterioration in systemic haemodynamics and renal function after liver injury (BDL), despite normal total plasma protein concentration. This worsened outcome in the absence of albumin strongly supports the central role of albumin in maintenance of cardio-renal function in liver failure and may indicate that albumin plays a crucial role in moderating inflammation. Competing interests None declared.