Comparative Hepatology - Latest Articles

Severe diabetes and leptin resistance cause differential hepatic and renal transporter expression in mice

Vijay More — 2012-04-23T00:00:00Z

Background: Type-2 Diabetes is a major health concern in the United States and other Westernized countries, with prevalence increasing yearly. There is a need to better model and predict adverse drug reactions, drug-induced liver injury, and drug efficacy in this population. Because transporters significantly contribute to drug clearance and disposition, it is highly significant to determine whether a severe diabetes phenotype alters drug transporter expression, and whether diabetic mouse models have altered disposition of acetaminophen (APAP) metabolites. Results: Transporter mRNA and protein expression were quantified in livers and kidneys of adult C57BKS and db/db mice, which have a severe diabetes phenotype due to a lack of a functional leptin receptor. The urinary excretion of acetaminophen-glucuronide, a substrate for multidrug resistance-associated proteins transporters was also determined. The mRNA expression of major uptake transporters, such as organic anion transporting polypeptide Slco1a1 in liver and kidney, 1a4 in liver, and Slc22a7 in kidney was decreased in db/db mice. In contrast, Abcc3 and 4 mRNA and protein expression was more than 2 fold higher in db/db male mouse livers as compared to C57BKS controls. Urine levels of APAP-glucuronide, -sulfate, and N-acetyl cysteine metabolites were higher in db/db mice. Conclusion: A severe diabetes phenotype/presentation significantly altered drug transporter expression in liver and kidney, which corresponded with urinary APAP metabolite levels.

Protection against Fas-induced fulminant hepatic failure in liver specific integrin linked kinase knockout mice

Shashikiran Donthamsetty — 2011-11-21T00:00:00Z

Background: Programmed cell death or apoptosis is an essential process for tissue homeostasis. Hepatocyte apoptosis is a common mechanism to many forms of liver disease. This study was undertaken to test the role of ILK in hepatocyte survival and response to injury using a Jo-2-induced apoptosis model. Methods: For survival experiments, ILK KO and WT mice received a single intraperitoneal injection of the agonistic anti-Fas monoclonal antibody Jo-2 at the lethal dose (0.4 μg/g body weight) or sublethal dose (0.16 μg/g body weight). For further mechanistic studies sublethal dose of Fas monoclonal antibody was chosen. Results: There was 100% mortality in the WT mice as compared to 50% in the KO mice. We also found that hepatocyte specific ILK KO mice (integrin linked kinase) died much later than WT mice after challenge with a lethal dose of Fas agonist Jo-2. At sublethal dose of Jo-2, there was 20% mortality in KO mice with minimal apoptosis whereas WT mice developed extensive apoptosis and liver injury leading to 70% mortality due to liver failure at 12 h. Proteins known to be associated with cell survival/death were differentially expressed in the 2 groups. In ILK KO mice there was downregulation of proapoptotic genes and upregulation of antiapoptotic genes. Conclusions: Mechanistic insights revealed that pro-survival pathways such as Akt, ERK1/2, and NFkB signaling were upregulated in the ILK KO mice. Inhibition of only NFkB and ERK1/2 signaling led to an increase in the susceptibility of ILK KO hepatocytes to Jo-2-induced apoptosis. These studies suggest that ILK elimination from hepatocytes protects against Jo-2 induced apoptosis by upregulating survival pathways. FAK decrease may also play a role in this process. The results presented show that the signaling effects of ILK related to these functions are mediated in part mediated through NFkB and ERK1/2 signaling.

The effect of cocoa supplementation on hepatic steatosis, reactive oxygen species and LFABP in a rat model of NASH

Mile Janevski — 2011-11-14T00:00:00Z

Background: Non alcoholic steatohepatitis is hypothesised to develop via a mechanism involving fat accumulation and oxidative stress. The current study aimed to investigate if an increase in oxidative stress was associated with changes in the expression of liver fatty acid binding protein in a rat model of non alcoholic steatohepatitis and whether cocoa supplementation attenuated those changes. Methods: Female Sprague Dawley rats were fed a high fat control diet, a high fat methionine choline deficient diet, or one of four 12.5% cocoa supplementation regimes in combination with the high fat methionine choline deficient diet. Results: Liver fatty acid binding protein mRNA and protein levels were reduced in the liver of animals with fatty liver disease when compared to controls. Increased hepatic fat content was accompanied by higher levels of oxidative stress in animals with fatty liver disease when compared to controls. An inverse association was found between the levels of hepatic liver fatty acid binding protein and the level of hepatic oxidative stress in fatty liver disease. Elevated NADPH oxidase protein levels were detected in the liver of animals with increased severity in inflammation and fibrosis. Cocoa supplementation was associated with partial attenuation of these pathological changes, although the severity of liver disease induced by the methionine choline deficient diet prevented complete reversal of any disease associated changes. Red blood cell glutathione was increased by cocoa supplementation, whereas liver glutathione was reduced by cocoa compared to methionine choline deficient diet fed animals. Conclusion: These findings suggest a potential role for liver fatty acid binding protein and NADPH oxidase in the development of non alcoholic steatohepatitis. Furthermore, cocoa supplementation may have be of therapeutic benefit in less sever forms of NASH.

Autoimmune liver disease - Are there spectra that we do not know?

Hind Fallatah — 2011-09-12T00:00:00Z

Autoimmune liver diseases (AILDs) are common leading causes for liver cirrhosis and terminal stage of liver disease. They have variable prevalence among patients with liver disease and have two major clinical and biochemical presentations. Autoimmune hepatitis (AIH) is the typical example of hepatocellular AILD, but it can also be presented under a cholestatic pattern. AIH has a scoring diagnostic system and respond in most cases to the treatment with prednisolone and azathioprine. Primary biliary cirrhosis (PBC) is the second most common AILD, with a cholestatic presentation and characterized by positive antimitochondrial antibody (AMA). It has an excellent response and long term outcome with the administration of ursodeoxycholic acid (UDCA). Another AILD that is thought to be a variant of PBC is the autoimmune cholangitis, being a disease that has biochemical and histological features similar to PBC; but the AMA is negative. Primary sclerosing cholangitis (PSC) is a rare entity of AILD that has a cholestatic presentation and respond poorly to the treatment, with the ultimate progression to advance liver cirrhosis in most patients. Other forms of AILD include the overlap syndromes (OS), which are diseases with mixed immunological and histological patterns of two AILD; the most commonly recognized one is AIH-PBC overlap (AIH-PSC overlap is less common). The treatment of OS involves the trial of UDCA and different immunosuppressants. Here we present three case reports of unusual forms of chronic liver diseases that most likely represent AILD. The first two patients had a cholestatic picture, whereas the third one had a hepatocellular picture at presentation. We discussed their biochemical, immunological and histological features as well as their response to treatment and their outcomes. Then, we compared them with other forms of AILD.

Evaluation of the effects of a VEGFR-2 inhibitor compound on alanine aminotransferase gene expression and enzymatic activity in the rat liver

Carmen Fuentealba — 2011-08-17T00:00:00Z

Background: Traditional assessment of drug-induced hepatotoxicity includes morphological examination of the liver and evaluation of liver enzyme activity in serum. The objective of the study was to determine the origin of drug-related elevation in serum alanine aminotransferase (ALT) activity in the absence of morphologic changes in the liver by utilizing molecular and immunohistochemical techniques. Methods: Sixteen female Sprague-Dawley rats were divided into 2 groups (control and treated, n = 4 per group) and treated rats were dosed orally twice daily (400 mg/kg/day) for 7 days with a VEGFR-2 compound (AG28262), which in a previous study caused ALT elevation without morphological changes. Serum of both treated and control animals were evaluated on day 3 of treatment and at day 8. Three separate liver lobes (caudate, right medial, and left lateral) were examined for determination of ALT tissue activity, ALT gene expression and morphological changes. Results: ALT activity was significantly (p < 0.01) elevated on day 3 and further increased on day 8. Histologic changes or increase in TUNEL and caspase3 positive cells were not observed in the liver lobes examined. ALT gene expression in the caudate lobe was significantly up-regulated by 63%. ALT expression in the left lateral lobe was not significantly affected. Statistically significant increased liver ALT enzymatic activity occurred in the caudate (96%) and right medial (41%) lobes but not in the left lateral lobe. Conclusions: AG28262, a VEFG-r2 inhibitor, causes an increase in serum ALT, due in part to both gene up-regulation. Differences between liver lobes may be attributable to differential distribution of blood from portal circulation. Incorporation of molecular data, such as gene and protein expression, and sampling multiple liver lobes may shed mechanistic insight to the evaluation of hepatotoxicity.

Assessment and histological analysis of the IPRL technique for sequential in situ liver biopsy

Anthony Rowe — 2011-08-08T00:00:00Z

Background: The isolated perfused rat liver (IPRL) is a technique used in a wide range of liver studies. Typically livers are assessed at treatment end point. Techniques have been described to biopsy liver in the live rat and post-hepatectomy. Results: This paper describes a technique for obtaining two full and one partial lobe biopsies from the liver in situ during an IPRL experiment. Our approach of retaining the liver in situ assists in minimising liver capsule damage, and consequent leakage of perfusate, maintains the normal anatomical position of the liver during perfusion and helps to keep the liver warm and moist. Histological results from sequential lobe biopsies in control perfusions show that cytoplasmic vacuolation of hepatocytes is a sign of liver deterioration, and when it occurs it commences as a diffuse pattern which tends to develop a circumscribed, centrilobular pattern as perfusion progresses. Conclusions: Liver lobe biopsies obtained using this method can be used to study temporal effects of drug treatments and are suitable for light and electron microscopy, and biochemical analyses.

Hepatic expression of multidrug resistance protein 2 in biliary atresia

Keita Terui — 2011-08-03T00:00:00Z

Background: Biliary atresia (BA) is an idiopathic inflammatory obliterative cholangiopathy of neonates, leading to progressive biliary cirrhosis. Hepatoportoenterostomy (Kasai procedure) can cure jaundice in 30% to 80% of patients. Postoperative clearance of jaundice is one of the most important factors influencing long-term outcomes of BA patients. Multidrug resistance protein 2 (MRP2) is one of the canalicular export pumps located in hepatocytes; it exports organic anions and their conjugates (e.g., bilirubin) into bile canaliculus. Although MRP2 is an essential transporter for the excretion of bilirubin, its role in the clinical course of BA patients is unclear. The present study investigated the relationship between hepatic MRP2 expression and clinical course in BA patients, with particular emphasis in curing jaundice after hepatoportoenterostomy. Results: No significant differences in hepatic MRP2 expression level were observed between BA and controls groups. There was no correlation between MRP2 expression and age at time of surgery in BA and control groups. In BA patients, MRP2 expression level in the jaundice and jaundice-free group did not differ significantly (2.0 × 10-4 vs 3.1 × 10-4, p = 0.094). Although the serum level of total bilirubin just before surgery did not correlate with MRP2 expression level (rs = 0.031, p = 0.914), the serum level of total bilirubin measured at 2 weeks (rs = -0.569, p = 0.034) and 4 weeks after surgery (rs = -0.620, p = 0.018) were significantly correlated with MRP2 expression level. Furthermore, MRP2 expression level was inversely correlated with ratio of change in serum total bilirubin level over 4 weeks (rs = -0.676, p = 0.008), which represents the serum bilirubin level measured at 4 weeks after surgery divided by value just before surgery. There was no correlation between expression level of MRP2 and nuclear receptors, such as retinoid × receptor α, farnesoid × receptor, pregnane × receptor, or constitutive androstane receptor. Conclusions: Hepatic MRP2 expression level was associated with postoperative clearance of jaundice in BA patients, at least within 1 month after hepatoportoenterostomy. This finding suggests that not only morphological appearance of the liver tissue but also the biological status of hepatocytes is important for BA pathophysiology.

Situs inversus totalis and secondary biliary cirrhosis: a case report

Haci Mehmet Sokmen — 2011-08-03T00:00:00Z

Situs inversus totalis is is a congenital anomaly associated with various visceral abnormalities, but there is no data about the relationship between secondary biliary cirrhosis and that condition. We here present a case of a 58 year-old female with situs inversus totalis who was admitted to our clinic with extrahepatic cholestasis. After excluding all potential causes of biliary cirrhosis, secondary biliary cirrhosis was diagnosed based on the patient's history, imaging techniques, clinical and laboratory findings, besides histolopathological findings. After treatment with tauroursodeoxycholic acid, all biochemical parameters, including total/direct bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and gama glutamyl transferase, returned to normal ranges at the second month of the treatment. We think that this is the first case in literature that may indicate the development of secondary biliary cirrhosis in a patient with situs inversus totalis. In conclusion, situs inversus should be considered as a rare cause of biliary cirrhosis in patients with situs inversus totalis which is presented with extrahepatic cholestasis.

Characterization of chronic HCV infection-induced apoptosis

Abdel-Rahman Zekri — 2011-07-23T00:00:00Z

Background: To understand the complex and largely not well-understood apoptotic pathway and immune system evasion mechanisms in hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) and HCV associated chronic hepatitis (CH), we studied the expression patterns of a number of pro-apoptotic and anti-apoptotic genes (Fas, FasL, Bcl-2, Bcl-xL and Bak) in HepG2 cell line harboring HCV- genotype-4 replication. For confirmation, we also assessed the expression levels of the same group of genes in clinical samples obtained from 35 HCC and 34 CH patients. Methods: Viral replication was assessed in the tissue culture medium by RT-PCR, quantitative Real-Time PCR (qRT-PCR); detection of HCV core protein by western blot and inhibition of HCV replication with siRNA. The expression level of Fas, FasL, Bcl-2, Bcl-xL and Bak was assessed by immunohistochemistry and RT-PCR whereas caspases 3, 8 and 9 were assessed by colorimetric assay kits up to 135 days post infection. Results: There was a consistent increase in apoptotic activity for the first 4 weeks post-CV infection followed by a consistent decrease up to the end of the experiment. The concordance between the changes in the expression levels of Fas, FasL, Bcl-2, Bcl-xL and Bak in vitro and in situ was statistically significant (p < 0.05). Fas was highly expressed at early stages of infection in cell lines and in normal control liver tissues followed by a dramatic reduction post-HCV infection and an increase in the expression level of FasL post HCV infection. The effect of HCV infection on other apoptotic proteins started very early post-infection, suggesting that hepatitis C modulating apoptosis by modulating intracellular pro-apoptotic signals. Conclusions: Chronic HCV infection differently modulates the apoptotic machinery during the course of infection, where the virus induces apoptosis early in the course of infection, and as the disease progresses apoptosis is modulated. This study could open a new opportunity for understanding the various signaling of apoptosis and in the developing a targeted therapy to inhibit viral persistence and HCC development.

Effects of ischemic pre- and postconditioning on HIF-1alpha, VEGF and TGF-beta expression after warm ischemia and reperfusion in the rat liver

Anders Knudsen — 2011-07-19T00:00:00Z

Background: Ischemic pre- and postconditioning protects the liver against ischemia/reperfusion injuries. The aim of the present study was to examine how ischemic pre- and postconditioning affects gene expression of hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor A (VEGF-A) and transforming growth factor β (TGF-β) in liver tissue. Methods: 28 rats were randomized into five groups: control; ischemia/reperfusion; ischemic preconditioning (IPC); ischemic postconditioning (IPO); combined IPC and IPO. IPC consisted of 10 min of ischemia and 10 min of reperfusion. IPO consisted of three cycles of 30 sec. reperfusion and 30 sec. of ischemia. Results: HIF-1α mRNA expression was significantly increased after liver ischemia compared to controls (p = 0.010). HIF-1α mRNA expression was significantly lower in groups subjected to IPC or combined IPC and IPO when compared to the ischemia/reperfusion group (p = 0.002). VEGF-A mRNA expression increased in the ischemia/reperfusion or combined IPC and IPO groups when compared to the control group (p < 0.05). Conclusion: Ischemic conditioning seems to prevent HIF-1α mRNA induction in the rat liver after ischemia and reperfusion. This suggests that the protective effects of ischemic conditioning do not involve the HIF-1 system. On the other hand, the magnitude of the HIF-1α response might be a marker for the degree of I/R injuries after liver ischemia. Further studies are needed to clarify this issue.