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.

Hepatocytes express the water channel aquaporin-8 (AQP8), which is mainly localized in intracellular vesicles, and its adenosine 3 ,5 -cyclic monophosphate (cAMP)-induced translocation to the plasma membrane facilitates osmotic water movement during canalicular bile secretion. Thus, defective expression of AQP8 may be associated with secretory dysfunction of hepatocytes caused by extrahepatic cholestasis. We studied the effect of 1, 3, and 7 days of bile duct ligation (BDL) on protein expression, subcellular localization, and messenger RNA (mRNA) levels of AQP8; this was determined in rat livers by immunoblotting in subcellular membranes, light immunohistochemistry, immunogold electron microscopy, and Northern blotting. One day of BDL did not affect expression or subcellular localization of AQP8. Three days of BDL reduced the amount of intracellular AQP8 (75%; P < .001) without affecting its plasma membrane expression. Seven days after BDL, AQP8 was markedly decreased in intracellular (67%; P < .05) and plasma (56%; P < .05) membranes. Dibutyryl cAMP failed to increase AQP8 in plasma membranes from liver slices, suggesting a defective translocation of AQP8 in 7-day BDL rats. Immunohistochemistry and immunoelectron microscopy in liver sections confirmed the BDLinduced decreased expression of hepatocyte AQP8 in intracellular vesicles and canalicular membranes. AQP8 mRNA expression was unaffected by 1-day BDL but was significantly increased by about 200% in 3-and 7-day BDL rats, indicating a posttranscriptional mechanism for protein level reduction. In conclusion, BDL-induced extrahepatic cholestasis caused posttranscriptional downregulation of hepatocyte AQP8 protein expression. Defective expression of AQP8 water channels may contribute to bile secretory dysfunction of cholestatic hepatocytes.

Carreras FI, Lehmann GL, Ferri D, Tioni MF, Calamita G, Marinelli RA. Defective hepatocyte aquaporin-8 expression and reduced canalicular membrane water permeability in estrogeninduced cholestasis. Am J Physiol Gastrointest Liver Physiol 292: G905-G912, 2007. First published November 16, 2006; doi:10.1152/ajpgi.00386.2006.-Our previous work supports a role for aquaporin-8 (AQP8) water channels in rat hepatocyte bile formation mainly by facilitating the osmotically driven canalicular secretion of water. In this study, we tested whether a condition with compromised canalicular bile secretion, i.e., the estrogen-induced intrahepatic cholestasis, displays defective hepatocyte AQP8 functional expression. After 17␣-ethinylestradiol administration (5 mg ⅐ kg body wt Ϫ1 ⅐ day Ϫ1 for 5 days) to rats, the bile flow was reduced by 58% (P Ͻ 0.05). By subcellular fractionation and immunoblotting analysis, we found that 34 kDa AQP8 was significantly decreased by ϳ70% in plasma (canalicular) and intracellular (vesicular) liver membranes. However, 17␣-ethinylestradiol-induced cholestasis did not significantly affect the protein level or the subcellular localization of sinusoidal AQP9. Immunohistochemistry for liver AQPs confirmed these observations. Osmotic water permeability (P f) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (73 Ϯ 1 vs. 57 Ϯ 2 m/s) in cholestasis, consistent with defective canalicular AQP8 functional expression. By Northern blotting, we found that AQP8 mRNA expression was increased by 115% in cholestasis, suggesting a posttranscriptional mechanism of protein level reduction. Accordingly, studies in primary cultured rat hepatocytes indicated that the lysosomal protease inhibitor leupeptin prevented the estrogen-induced AQP8 downregulation. In conclusion, hepatocyte AQP8 protein expression is downregulated in estrogeninduced intrahepatic cholestasis, presumably by lysosomal-mediated degradation. Reduced canalicular membrane AQP8 expression is associated with impaired osmotic membrane water permeability. Our data support the novel notion that a defective expression of canalicular AQP8 contributes as a mechanism for bile secretory dysfunction of cholestatic hepatocytes.aquaporins; intrahepatic cholestasis; water transport; liver AQUAPORIN (AQP) water channels are a family of integral membrane proteins known to facilitate the osmotic water movement across the cellular membranes (3). Three members of the AQP family are expressed in rat hepatocytes: AQP8 (7, 13, 16, 21), AQP9 (14, 21, 40), and AQP0 (21). AQP8 is localized in the canalicular plasma membrane domain (7,13,16,21) as well as in intracellular vesicles (7,13,16,21) and mitochondria (6). Its trafficking from a vesicular compartment to the canalicular membrane can be induced by a choleretic stimulus, such as dibutyryl-cAMP (16,21) or the hormone glucagon (18,19,30). AQP9 resides exclusively on the sinusoidal plasma membranes of hepatocytes and thereby may facilitate the movement of water and certain small so...

Rat hepatocytes express aquaporin-9 (AQP9), a basolateral channel permeable to water, glycerol, and other small neutral solutes. Although liver AQP9 is known for mediating the uptake of sinusoidal blood glycerol, its relevance in bile secretion physiology and pathophysiology remains elusive. Here, we evaluated whether defective expression of AQP9 is associated to secretory dysfunction of rat hepatocytes following bile duct ligation (BDL). By immunoblotting, 1-day BDL resulted in a slight decrease of AQP9 protein in basolateral membranes and a simultaneous increase of AQP9 in intracellular membranes. This pattern was steadily accentuated in the subsequent days of BDL since at 7 days BDL basolateral membrane AQP9 decreased by 85% whereas intracellular AQP9 increased by 115%. However, the AQP9 immunoreactivity of the total liver membranes from day 7 of BDL rats was reduced by 49% compared with the sham counterpart. Results were confirmed by immunofluorescence and immunogold electron microscopy and consistent with biophysical studies showing considerable decrease of the basolateral membrane water and glycerol permeabilities of cholestatic hepatocytes. The AQP9 mRNA was slightly reduced only at day 7 of BDL, indicating that the dysregulation was mainly occurring at a posttranslational level. The altered expression of liver AQP9 during BDL was not dependent on insulin, a hormone known to negatively regulate AQP9 at a transcriptional level, since insulinemia was unchanged in 7-day BDL rats. Overall, these results suggest that extrahepatic cholestasis leads to downregulation of AQP9 in the hepatocyte basolateral plasma membrane and dysregulated aquaporin channels contribute to bile flow dysfunction of cholestatic hepatocyte.

Background information. PI3K (phosphoinositide 3-kinase) mediates several signal transduction pathways in hepatocytes, including some involved in the regulation of vesicle trafficking. Hepatocytes express the water channel AQP8 (aquaporin-8) predominantly in an intracellular location, and it redistributes to the canalicular membrane, upon stimulation with the hormone glucagon, by a cAMP/protein kinase A-dependent mechanism. Since glucagon is capable of stimulating PI3K activity in hepatocytes and a cross talk between cAMP and PI3K has been suggested, in the present study, we examine whether PI3K activation is involved in the glucagon-induced translocation of AQP8.Results. By quantitative immunoblotting of purified hepatocyte plasma membranes, we found that the preincubation of cells with two structurally different PI3K inhibitors, wortmannin or LY294002, prevented the glucagon-induced translocation of AQP8 to hepatocyte plasma membrane. Confocal immunofluorescence microscopy in cultured hepatocytes confirmed the dependence of the hormone-induced redistribution of AQP8 on PI3K activity. Functional studies showed that the PI3K inhibitors were also capable of preventing the glucagon-induced increase in hepatocyte osmotic membrane water permeability. Conclusions.Our results suggest that PI3K activation is involved in the glucagon-dependent signal transduction pathways leading to hepatocyte AQP8 translocation.

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Hepatocytes express aquaporin‐9 (AQP9), a basolateral channel permeable to water, glycerol and other neutral solutes. While liver AQP9 is known for mediating the uptake of glycerol its relevance in bile secretion remains elusive. Here, we evaluated whether defective expression of AQP9 is associated to secretory dysfunction of rat hepatocytes following bile duct ligation (BDL). By immunoblotting, 1‐day BDL resulted in a slight decrease of AQP9 protein in basolateral membranes and a simultaneous increase of AQP9 in intracellular membranes. This pattern was steadily accentuated in the subsequent days of BDL as at 7‐days BDL basolateral membrane AQP9 decreased by 85% whereas intracellular AQP9 increased by 115%. However, the total amount of AQP9 in the livers from day 7 of BDL rats was reduced by 49% compared to the sham counterpart. Results were confirmed by immunocytochemistry and consistent with biophysical studies showing considerable decrease of the basolateral membrane water and glycerol permeabilities of cholestatic hepatocytes. The AQP9 mRNA was slightly reduced only at day 7 of BDL indicating that the dysregulation was mainly occurring at a posttranslational level. The altered expression of liver AQP9 during BDL was not dependent on insulin, a hormone known to downregulate AQP9, since insulinemia was unchanged in 7‐days BDL rats. These results suggest that extrahepatic cholestasis leads to downregulation of liver AQP9 and dysregulated AQP channels contribute to bile flow dysfunction of cholestatic hepatocyte.