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Although bacterial lipopolysaccharides (LPS) are known to cause cholestasis in sepsis, the molecular mechanisms accounting for this effect are only partially known. Because aquaporin-8 (AQP8) seems to facilitate the canalicular osmotic water movement during hepatocyte bile formation, we studied its gene and functional expression in LPS-induced cholestasis. By subcellular fractionation and immunoblotting analysis, we found that 34-kDa AQP8 was significantly decreased by 70% in plasma (canalicular) and intracellular (vesicular) liver membranes. However, expression and subcellular localization of hepatocyte sinusoidal AQP9 were unaffected. Immunohistochemistry for liver AQPs confirmed these observations. Osmotic water permeability (P(f)) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (65 +/- 1 vs. 49 +/- 1 microm/s) by LPS, consistent with defective canalicular AQP8 functional expression. By Northern blot analysis, we found that 1.5-kb AQP8 mRNA expression was increased by 80%, suggesting a posttranscriptional mechanism of protein reduction. The tumor necrosis factor-alpha (TNF-alpha) receptor fusion protein TNFp75:Fc prevented the LPS-induced impairment of AQP8 expression and bile flow, suggesting the cytokine TNF-alpha as a major mediator of LPS effect. Accordingly, studies in hepatocyte primary cultures indicated that recombinant TNF-alpha downregulated AQP8. The effect of TNF-alpha was prevented by the lysosomal protease inhibitors leupeptin or chloroquine or by the proteasome inhibitors MG132 or lactacystin, suggesting a cytokine-induced AQP8 proteolysis. In conclusion, our data suggest that LPS induces the TNF-alpha-mediated posttranscriptional downregulation of AQP8 functional expression in hepatocytes, a mechanism potentially relevant to the molecular pathogenesis of sepsis-associated cholestasis.

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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 (1120%, 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 (290%, P < 0.05). In vivo studies in rats showed that under glucagoninduced ureagenesis, hepatic mtAQP8 protein expression was markedly up-regulated (1160%, P < 0.05). Moreover, transport studies in liver IMM vesicles showed that glucagon increased the diffusional permeability to the ammonia analog [ 14 C]methylamine (180%, 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. (HEPATOLOGY 2013;57:2061-2071 B y means of the urea cycle, hepatocytes metabolize waste nitrogen and prevent the deleterious consequences of hyperammonemia and hepatic encephalopathy. The initial and rate-controlling step in the urea cycle is the mitochondrial synthesis of carbamoyl-phosphate from ammonia by carbamyl phosphate synthetase 1 (CPS1), 1 a process that involves the mitochondrial uptake of ammonia and the metabolization of ammonia from amino acids such as glutamine and alanine. 2 However, the mechanisms involved in the mitochondrial entry of ammonia are still unknown (Fig. 1). Aquaporin-8 (AQP8) is a member of a family of homologous membrane channel proteins that facilitates the movement of water and certain small solutes across biological membranes. 3 Hepatocyte AQP8 is localized as a glycosylated 34-kDa protein in the canalicular plasma membrane domain and intracellular vesicles. 4-7 Canalicular AQP8 works as a water channel facilitating the movement of water molecules coupled to osmotic gradients, 8,9 a process that may play a role in hepatocyte bile formation and cholestasis. 7,10-13 A nonglycosylated 28-kDa form of the AQP8 protei...

Ammonia is a potent neurotoxin that is detoxified mainly by the urea cycle in the liver. Hyperammonemia is a common complication of a wide variety of both inherited and acquired liver diseases. If not treated early and thoroughly, it results in encephalopathy and death. Here, we found that hepatic autophagy is critically involved in systemic ammonia homeostasis by providing key urea-cycle intermediates and ATP. Hepatic autophagy is triggered in vivo by hyperammonemia through an α-ketoglutarate-dependent inhibition of the mammalian target of rapamycin complex 1, and deficiency of autophagy impairs ammonia detoxification. In contrast, autophagy enhancement by means of hepatic gene transfer of the master regulator of autophagy transcription factor EB or treatments with the autophagy enhancers rapamycin and Tat-Beclin-1 increased ureagenesis and protected against hyperammonemia in a variety of acute and chronic hyperammonemia animal models, including acute liver failure and ornithine transcarbamylase deficiency, the most frequent urea-cycle disorder. In conclusion, hepatic autophagy is an important mechanism for ammonia detoxification because of its support of urea synthesis, and its enhancement has potential for therapy of both primary and secondary causes of hyperammonemia.

Estrogens can cause liver cholestatic disease. As downregulation of hepatocyte canalicular aquaporin-8 (AQP8) water channels has been involved in estrogen-induced bile secretory failure, we tested whether the archetypal water channel AQP1 improves 17α-ethinylestradiol (EE)-induced cholestasis. EE administration to rats reduced bile flow by 50%. A recombinant adenoviral (Ad) vector encoding human AQP1 (hAQP1), AdhAQP1, or a control vector was administered by retrograde bile ductal infusion. Hepatocyte canalicular hAQP1 expression was confirmed by liver immunostaining and immunoblotting in purified membrane fractions. Accordingly, canalicular osmotic water permeability was markedly increased. Bile flow, either basal or bile salt-stimulated was significantly augmented by over 50%. The choleretic efficiency of endogenous bile salts (that is, volume of bile per μmol of excreted bile salt) was significantly increased by 45% without changes in the biliary bile salt composition. Our data suggest that the adenoviral transfer of hAQP1 gene to the livers of EE-induced cholestatic rats improves bile flow by enhancing the AQP-mediated bile salt-induced canalicular water secretion. This novel finding might have potential therapeutic implications for cholestatic diseases.

The adenoviral gene transfer of human aquaporin-1 (hAQP1) water channels to the liver of 17a-ethinylestradiol-induced cholestatic rats improves bile flow, in part by enhancing canalicular hAQP1-mediated osmotic water secretion. To gain insight into the mechanisms of 17a-ethinylestradiol cholestasis improvement, we studied the biliary output of bile salts (BS) and the functional expression of the canalicular BS export pump (BSEP; ABCB11). Adenovector encoding hAQP1 (AdhAQP1) or control vector was administered by retrograde intrabiliary infusion. AdhAQP1-transduced cholestatic rats increased the biliary output of major endogenous BS (50%-80%, P < 0.05) as well as that of taurocholate administered in choleretic or trace radiolabel amounts (around 60%, P < 0.05). Moreover, liver transduction with AdhAQP1 normalized serum BS levels, otherwise markedly elevated in cholestatic animals. AdhAQP1 treatment was unable to improve BSEP protein expression in cholestasis; however, its transport activity, assessed by adenosine triphosphate-dependent taurocholate transport in canalicular membrane vesicles, was induced by 90% (P < 0.05). AdhAQP1 administration in noncholestatic rats induced no significant changes in either biliary BS output or BSEP activity. Canalicular BSEP, mostly present in raft (high cholesterol) microdomains in control rats, was largely found in nonraft (low cholesterol) microdomains in cholestasis. Considering that BSEP activity directly depends on canalicular membrane cholesterol content, decreased BSEP presence in rafts may contribute to BSEP activity decline in 17a-ethinylestradiol cholestasis. In AdhAQP1-transduced cholestatic rats, BSEP showed a canalicular microdomain distribution similar to that of control rats, which provides an explanation for the improved BSEP activity. Conclusion: Hepatocyte canalicular expression of hAQP1 through adenoviral gene transfer promotes biliary BS output by modulating BSEP activity in estrogen-induced cholestasis, a novel finding that might help us to better understand and treat cholestatic disorders. (HEPATOLOGY 2016;64:535-548) SEE EDITORIAL ON PAGE 344 C holestatic liver diseases result in systemic and intrahepatic accumulation of endogenous bile salts (BS) that causes liver injury, ultimately leading to fibrosis and cirrhosis. (1) The canalicular BS export pump (BSEP; ABCB11) is the primary hepatocyte transporter for biliary BS secretion, i.e., the rate-limiting step in the enterohepatic circulation of BS. (2) Impairment of expression and/or function of BSEP has been directly linked to inherited and acquired cholestatic liver diseases. (3)(4)(5) Estrogens and Abbreviations: AdhAQP1, adenovector encoding human aquaporin-1; AQP8, aquaporin-8; BS, bile salts; BSEP, bile salt export pump/ABCB11; EE, 17a-ethinylestradiol; hAQP1, human aquaporin-1; TC, taurocholate.

Larocca MC, Soria LR, Espelt MV, Lehmann GL, Marinelli RA. Knockdown of hepatocyte aquaporin-8 by RNA interference induces defective bile canalicular water transport. Am J Physiol Gastrointest Liver Physiol 296: G93-G100, 2009. First published October 23, 2008 doi:10.1152/ajpgi.90410.2008 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 ϳ70% (P Ͻ 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 (Ϫ65%, P Ͻ 0.05) or to the bile secretory agonist dibutyryl cAMP (Ϫ80%, P Ͻ 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.HepG2; human liver; bile secretion; dibutyryl cAMP BILE IS COMPOSED OF 98% WATER, which is mainly secreted by hepatocytes at bile canaliculi. Water transport at this level is driven by transient osmotic gradients generated across the hepatocyte membrane by active solute transport (1). Aquaporins (AQPs) are a family of integral membrane proteins that facilitate the osmotically induced water transport through cell membranes (18). At least 13 mammalian aquaporins have been identified in diverse human and animal cells. Rat hepatocytes express four AQPs, i.e., AQP8 (9, 13, 16), AQP9 (10, 16), AQP11 (14), and AQP0 (16). AQP8 is localized, as a glycosylated 34-kDa protein, in intracellular vesicles and at the canalicular plasma membrane (9, 13, 16). There is experimental evidence suggesting that AQP8 facilitates the canalicular water transport during hepatocyte bile formation (16) and that the defective expression of hepatocyte AQP8 may contribute to bile secretory dysfunction in cholestasis (4,5,20). Nevertheless, conclusive evidence for AQP8 involvement in bile canalicular water transport should come from studies performed in hepatocytes lacking AQP8 expression. Recently, it has been described that hepatocytes from AQP8-null mice show simila...