The data provide proof of concept that renal dysfunction in patients with cirrhosis with superimposed inflammation is associated with significant tubular injury and apoptosis and with increased renal expression and urinary excretion of the TLR4, suggesting a potential role of TLR4 as mediator of renal injury.

Background and Aims Portal hypertension is characterized by reduced hepatic eNOS activity. Asymmetric-dimethylarginine (ADMA), an eNOS inhibitor, is elevated in cirrhosis and correlates with severity of portal hypertension. Dimethylargininedimethylaminohydrolase-1 (DDAH-1) is the key enzyme metabolizing hepatic ADMA. This study characterized DDAH-1 in cirrhosis, and explored hepatic DDAH-1 reconstitution through FXR agonism and DDAH-1 gene therapy. Methods DDAH-1 Immunohistochemistry was conducted on human cirrhosis and healthy liver tissue. Subsequently, sham-operated or bile-duct-ligated (BDL) cirrhosis rats were treated with FXR agonist Obeticholic acid (OA, 5mg/kg) or vehicle for 5 days. Further animals underwent hydrodynamic injection with DDAH-1-expressing plasmid or saline control. Groups: Sham+saline, BDL+saline, BDL+DDAH-1-plasmid. Portal pressure (PP) measurements were performed. Plasma ALT was measured by Cobas-Integra; DDAH-1 expression by qPCR and Western blot; eNOS activity by radiometric assay. Results Immunohistochemistry and Western-blotting confirmed hepatic DDAH-1 was restricted to hepatocytes, and expression decreased significantly in cirrhosis. In BDL rats, reduced DDAH-1 expression was associated with elevated hepatic ADMA, reduced eNOS activity and high PP. OA treatment significantly increased DDAH-1 expression, reduced hepatic tissue ADMA, and increased liver NO generation. PP was significantly reduced in BDL+OA vs. BDL+vehicle (8±1 vs. 13.5±0.6 mmHg; p<0.01) with no change in MAP. Similarly, DDAH-1 hydrodynamic injection significantly increased hepatic DDAH-1 gene and protein expression, and significantly reduced PP in BDL+DDAH-1 vs. BDL+ saline (p<0.01). Conclusion This study demonstrates DDAH-1 is a specific molecular target for portal pressure reduction, through actions on ADMA-mediated regulation of eNOS activity. Our data support translational studies targeting DDAH-1 in cirrhosis and portal hypertension.

Background & Aims: 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. Methods: Tissue microarrays containing liver samples from patients with cirrhosis, viral hepatitis and HCC were examined for expression of TLR7 and TLR9 and the data obtained was validated in liver specimens from the hospital archives. Proliferation of human HCC cell lines was studied following stimulation of TLR7 and TLR9 using agonists (imiquimod and CpG-ODN respectively) and inhibition with a specific antagonist (IRS-954) or chloroquine. The effect of these interventions was confirmed in a xenograft model and diethylnitrosamine (DEN)/nitrosomorpholine (NMOR)-induced model of HCC. Results: TLR7 and TLR9 expression was up-regulated in human HCC tissue. Proliferation of HuH7 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 and TLR9 inhibition using IRS-954 or chloroquine significantly reduced HuH7 cell proliferation in vitro and inhibited tumour growth in the mouse xenograft model. HCC development in the DEN/NMOR rat model was also significantly inhibited by chloroquine (P < 0.001). Conclusion: The data suggest that inhibiting TLR7 and TLR9 with IRS-954 or chloroquine could potentially be used as a novel therapeutic approach for preventing HCC development and/or progression in susceptible patients.