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 BDL-induced 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.
Bibliographical noteFunding Information:
Supported by grant PICT 05-03589 (to R.A.M.) from Agencia Nacional de Promoción Científica y Tecnológica, CONICET; grant PRIN 2001, MURST ex-40% (to G.C.) from Italian Ministero della Ricerca Scientifica e Tecnologica; and grant DK24031 (to N.F.L.) from the National Institutes of Health. F.I.C. was supported by a fellowship from the Ministerio de Salud (Argentina).