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We recently reported that hepatocyte mitochondrial aquaporin-8 (mtAQP8) channels facilitate the uptake of ammonia and its metabolism into urea. Here we studied the effect of bacterial lipopolysaccharides (LPS) on ammonia-derived ureagenesis. In LPS-treated rats, hepatic mtAQP8 protein expression and diffusional ammonia permeability (measured utilizing ammonia analogues) of liver inner mitochondrial membranes were downregulated. NMR studies using 15N-labeled ammonia indicated that basal and glucagon-induced ureagenesis from ammonia were significantly reduced in hepatocytes from LPS-treated rats. Our data suggest that hepatocyte mtAQP8-mediated ammonia removal via ureagenesis is impaired by LPS, a mechanism potentially relevant to the molecular pathogenesis of defective hepatic ammonia detoxification in sepsis., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Unlabelled: Hepatocyte mitochondrial ammonia detoxification via ureagenesis is critical for the prevention of hyperammonemia and hepatic encephalopathy. Aquaporin-8 (AQP8) channels facilitate the membrane transport of ammonia. Because AQP8 is expressed in hepatocyte inner mitochondrial membranes (IMMs), we studied whether mitochondrial AQP8 (mtAQP8) plays a role in ureagenesis from ammonia. Primary cultured rat hepatocytes were transfected with small interfering RNAs (siRNAs) targeting two different regions of the rat AQP8 molecule or with scrambled control siRNA. After 48 hours, the levels of mtAQP8 protein decreased by approximately 80% (P < 0.05) without affecting cell viability. mtAQP8 knockdown cells in the presence of ammonium chloride showed a decrease in ureagenesis of approximately 30% (P < 0.05). Glucagon strongly stimulated ureagenesis in control hepatocytes (+120%, P < 0.05) but induced no significant stimulation in mtAQP8 knockdown cells. Contrarily, mtAQP8 silencing induced no significant change in basal and glucagon-induced ureagenesis when glutamine or alanine was used as a source of nitrogen. Nuclear magnetic resonance studies using 15N-labeled ammonia confirmed that glucagon-induced 15N-labeled urea synthesis was markedly reduced in mtAQP8 knockdown hepatocytes (-90%, P < 0.05). In vivo studies in rats showed that under glucagon-induced ureagenesis, hepatic mtAQP8 protein expression was markedly up-regulated (+160%, P < 0.05). Moreover, transport studies in liver IMM vesicles showed that glucagon increased the diffusional permeability to the ammonia analog [(14) C]methylamine (+80%, P < 0.05)., Conclusion: Hepatocyte mtAQP8 channels facilitate the mitochondrial uptake of ammonia and its metabolism into urea, mainly under glucagon stimulation. This mechanism may be relevant to hepatic ammonia detoxification and in turn, avoid the deleterious effects of hyperammonemia., (Copyright © 2013 American Association for the Study of Liver Diseases.)

Bile formation by hepatocytes is an osmotic secretory process that is ultimately dependent on the biliary secretion of osmotically-active solutes (mainly bile salts) via specialized canalicular transporters as well as on the water permeability of the canalicular plasma membrane domain. Hepatocytes express aquaporins, a family of membrane channel proteins that facilitate the osmotically-driven movement of water molecules. Aquaporin-8 (AQP8), localized to canalicular membranes, modulates membrane water permeability providing a molecular mechanism for the osmotically-coupled transport of solute and water during bile formation. There is experimental evidence suggesting that defective hepatocyte AQP8 expression leads to alterations in normal bile physiology. Thus, AQP8 protein is downregulated (and canalicular water permeability decreased), in established rat models of cholestasis, such as sepsis-associated cholestasis, estrogen-induced cholestasis and extrahepatic obstructive cholestasis. Moreover, AQP8 gene silencing in the human hepatocyte-derived cell line HepG2 inhibits canalicular water secretion. Based on current knowledge, it is conceivable that cholestasis results from a mutual occurrence of impaired solute transport and AQP8-mediated decrease of canalicular water permeability.

Glucagon stimulates the vesicle trafficking of aquaporin-8 (AQP8) water channels to the rat hepatocyte canalicular membranes, a process thought to be relevant to glucagon-induced bile secretion. In this study, we investigated whether glucagon is able to modulate the gene expression of hepatocyte AQP8. Glucagon was administered to rats at 0.2 mg/100 g body wt ip in 2, 3, or 6 equally spaced doses for 8, 16, and 36 h, respectively. Immunoblotting analysis showed that hepatic 34-kDa AQP8 was significantly increased by 79 and 107% at 16 and 36 h, respectively. Hepatic AQP9 protein expression remained unaltered. AQP8 mRNA expression, assessed by real-time PCR, was not modified over time, suggesting a posttranscriptional mechanism of AQP8 protein increase. Glucagon effects on AQP8 were directly studied in primary cultured rat hepatocytes. Immunoblotting and confocal immunofluorescence microscopy confirmed the specific glucagon-induced AQP8 upregulation. The RNA polymerase II inhibitor actinomycin D was unable to prevent glucagon effect, providing additional support to the nontranscriptional upregulation of AQP8. Cycloheximide also showed no effect, suggesting that glucagon-induced AQP8 expression does not depend on protein synthesis but rather on protein degradation. Inhibitory experiments suggest that a reduced calpain-mediated AQP8 proteolysis could be involved. The action of glucagon on hepatocyte AQP8 was mimicked by dibutyryl cAMP and suppressed by PKA or phosphatidylinositol-3-kinase (PI3K) inhibitors. In conclusion, our data suggest that glucagon induces the gene expression of rat hepatocyte AQP8 by reducing its degradation, a process that involves cAMP-PKA and PI3K signal pathways.

Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bile canalicular membranes, are involved in water transport during bile formation. Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion into the bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencing by RNA interference inhibits canalicular water secretion in the human hepatocyte-derived cell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cells express AQP8 and by confocal immunofluorescence microscopy that it is localized intracellularly and on the canalicular membrane, as described in rat hepatocytes. We also verified the expression of AQP8 in normal human liver. Forty-eight hours after transfection of HepG2 cells with RNA duplexes targeting two different regions of human AQP8 molecule, the levels of AQP8 protein specifically decreased by 60-70%. We found that AQP8 knockdown cells showed a significant decline in the canalicular volume of approximately 70% (P < 0.01), suggesting an impairment in the basal (nonstimulated) canalicular water movement. We also found that the decreased AQP8 expression inhibited the canalicular water transport in response either to an inward osmotic gradient (-65%, P < 0.05) or to the bile secretory agonist dibutyryl cAMP (-80%, P < 0.05). Our data suggest that AQP8 plays a major role in water transport across canalicular membrane of HepG2 cells and support the notion that defective expression of AQP8 causes bile secretory dysfunction in human hepatocytes.