Defective hepatocyte aquaporin-8 expression and reduced canalicular membrane water permeability in estrogen-induced cholestasis

Abstract

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...

Aquaporins: Their role in cholestatic liver disease

“…Experimental cholestasis induced by 17α-ethinylestradiol (EE) has been widely used to investigate in vivo alterations in the expression of hepatocyte membrane transporters in this pathological condition. In a recent work, we found that the protein expression of hepatocyte AQP8 is downregulated in estrogen-induced cholestasis possibly by posttranscriptional mechanisms, without significant changes in the sinusoidal AQP9 [59] . In fact, complementary studies in primary cultured rat hepatocytes with protease inhibitors indicated that estrogen-induced AQP8 downregulation was mediated by increased lysosomal degradation.…”

Expression of Aquaporin 9 in Rat Liver and Efferent Ducts of the Male Reproductive System After Neonatal Diethylstilbestrol Exposure

“…In a recent study by Carreras et al (2007), adult male Wistar rats were administered 5 mg ethinylestradiol/kg BW/day for 5 days to induce intrahepatic cholestasis. Treated animals had decreased BW and increased liver weight indicating a general toxic response, whereas ethinylestradiol-induced cholestasis did not seem to change the protein level or localization of AQP9.…”

“…Indeed, the total level of AQP9 expression in the liver is higher in male than in female rats, also having a more localized periacinous expression (Nicchia et al 2001). Nevertheless, in adult male rats, ethinyl estradiol at a dose sufficiently high to cause cholestasis had no effect on AQP9 expression (Carreras et al 2007). …”

Aquaporin gene therapy for cholestasis

“…In line with this is the fact that different animal models of cholestasis show defective canalicular AQP protein expression. (9)(10)(11)(12) In this context, it is conceivable that AQP gene transfer may help to improve certain cholestatic disorders.…”

“…In line with this is the fact that different animal models of cholestasis show defective canalicular AQP protein expression. (9)(10)(11)(12) In this context, it is conceivable that AQP gene transfer may help to improve certain cholestatic disorders.According to the editorial, studies in AQP8 knockout mice provide evidence against a role of canalicular AQP8 in bile secretion. (8) In that study, bile was not collected, and the conclusion was based on the fact that AQP8 knockout mice show only mild dietary fat misprocessing.…”