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Am J Physiol Gastrointest Liver Physiol 292: G905-G912, 2007. First published November 16, 2006; doi:10.1152/ajpgi.00386.2006
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LIVER AND BILIARY TRACT

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

Flavia I. Carreras,1 Guillermo L. Lehmann,1 Domenico Ferri,2 Mariana F. Tioni,3 Giuseppe Calamita,4 and Raúl A. Marinelli1

1Instituto de Fisiología Experimental and 3Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina; 2Dipartimento di Zoologia and 4Dipartimento di Fisiologia Generale ed Ambientale, Università degli Studi di Bari, Bari, Italy

Submitted 18 August 2006 ; accepted in final form 13 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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{alpha}-ethinylestradiol administration (5 mg·kg body wt–1·day–1 for 5 days) to rats, the bile flow was reduced by 58% (P < 0.05). 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, 17{alpha}-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 (Pf) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (73 ± 1 vs. 57 ± 2 µm/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 estrogen-induced 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 solutes (14, 21). AQP0 is localized intracellularly, and its significance is currently unclear (21).

Bile secretion results from the coordinated interactions of several solute membrane-transport systems and involves the movement of water into the biliary space in response to transient osmotic gradients generated by active solute transport (31). There is recent evidence suggesting that AQP8 facilitates the osmotically induced canalicular membrane water transport (28) and bile formation by rat hepatocytes (21). Thus it is conceivable that defective AQP membrane expression may lead to alterations of normal bile physiology. Currently, there is no conclusive evidence indicating that derangements of normal AQP function are causative of bile secretory dysfunction. Nevertheless, we found a downregulated expression of rat hepatocyte AQP8 in obstructive extrahepatic cholestasis, a pathological condition in which altered bile secretion occurs (8).

Estrogens are well known to cause intrahepatic cholestasis, whose clinical manifestations are oral contraceptive-induced cholestasis and cholestasis associated with pregnancy or postmenopausal replacement therapy (22). Experimental cholestasis induced by estrogen administration to rodents, mainly 17{alpha}-ethinylestradiol (EE), is an established experimental model to assess the mechanisms of estrogen-induced cholestasis (22). This model has been widely used to investigate alterations in the expression of hepatocyte membrane transporters associated with intrahepatic cholestasis (23, 37, 39). The aim of this study was to test whether a condition with compromised canalicular bile secretion, such as the estrogen-induced intrahepatic cholestasis, displays a defective hepatocyte AQP8 functional expression.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and treatments. Adult male Wistar rats were maintained on a standard diet and water ad libitum and housed in a temperature- and humidity-controlled environment under a constant 12-h light-dark cycle, according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). EE (Sigma-Aldrich, St. Louis, MO) (5 mg/kg body wt sc) was administered daily, for 5 consecutive days. Under these conditions, EE was found to induce cholestasis in rats (23). Control rats received only the EE vehicle (propylene glycol). Because EE treatment in rats is known to induce a reduction in food intake (around 25% in our laboratory), we performed additional pair-feeding experiments, i.e., we restricted the food intake of controls to the amount ingested by the EE-treated rats. After treatments, rats were anesthetized with a single dose of pentobarbital sodium (50 mg/kg body wt ip) and maintained under this condition during bile collection. For this purpose, a middle abdominal incision was made and the common bile duct was cannulated with a PE-10 polyethylene tubing (Intramedic, Clay Adams, Parsippany, NJ). Twenty minutes later, a 30-min period of bile collection was carried out. Bile flow was determined by gravimetry, assuming a density of the bile of 1.0 g/ml (38). At the end of that period, the animals were killed and the livers were harvested for evaluation.

Isolation and culture of hepatocytes. Hepatocytes were isolated from normal livers of male Wistar rats by collagenase perfusion and mechanical disruption (16). Cell viability (assessed by Trypan blue exclusion) was greater than 86%. Freshly isolated hepatocytes were plated onto collagen-coated glass plates at 3.8 x 104 cells/cm2, containing DMEM supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) (Invitrogen, San Diego, CA). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 3 h, allowing cells attachment to plates. After that time, medium was changed, and hepatocytes were incubated in the presence or absence of the estrogen estradiol-17beta-D-glucuronide (E17G) (10 µM) (Sigma, St. Louis, MO) for 20 h. Under these conditions, E17G was able to decrease hepatocyte AQP8 protein expression. Alternatively, some groups were preincubated for 30 min in the presence of either the lysosomal inhibitor leupeptin (250 µM) (Chemicon International, Temecula, CA) (2) or the proteasome inhibitor MG132 (10 µM) (Sigma) (41). At the end of the experiment, cells were washed and sonicated in 0.3 M sucrose. Total hepatocyte membranes were prepared for immunoblotting studies. EE (10 µM) was also able to decrease hepatocyte AQP8 expression but only after 48 h of culture when the AQP8 expression was not stably maintained (data not shown).

Preparations of hepatic subcellular membrane fractions. Livers were homogenized by 15 up-down strokes with a loose fitting Dounce homogenizer in four volumes of 0.3 M sucrose, containing 0.1 mM phenylmethylsulfonyl fluoride (Sigma) and 0.1 mM leupeptin. Liver homogenates (or sonicated cultured hepatocytes) were subjected to low-speed centrifugation to obtain postnuclear supernatants and then centrifuged at 200,000 g for 60 min, yielding the total liver or hepatocyte membrane fractions (8). Fractions enriched in plasma or intracellular microsomal membranes were prepared from liver homogenates by centrifugation at 200,000 g for 60 min on a discontinuous 1.3 M sucrose gradient as previously described (8). The plasma membrane band was removed, diluted to 0.3 M, and centrifuged at 200,000 g for 60 min to yield the plasma membrane fraction. The gradient remainder was sonicated, diluted to 0.3 M, and centrifuged at 17,000 g for 30 min. The resulting supernatant was centrifuged at 200,000 g for 60 min to yield the intracellular microsomal membrane fraction. Proteins in membrane fractions were determined according to Lowry et al. (25) by using bovine serum albumin as standard. The purity of the plasma and intracellular membranes were assessed by enzymatic assays for hepatic subcellular markers. 5'-Nucleotidase (plasma membrane), acid phosphatase (lysosomes), and aspartate aminotransferase (mitochondria) were assessed with commercial kits (Wiener Lab, Rosario, Argentina). Microsomal glucose-6-phosphatase was determined as described (8). The data obtained are summarized in Table 1. The enrichment and purity of membrane fractions were comparable to those reported previously (8) and similar between EE and control rats. Besides, EE administration did not alter the yield of total proteins in membrane fractions (Table 1).


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  		Table 1. Assessment of plasma and microsomal membrane purity

 
Isolation of canalicular liver plasma membranes. Canalicular plasma membrane fractions were prepared as previously (28). Briefly, a purified plasma membrane fraction was obtained by centrifugations on sucrose gradients and then further subfractionated by high-speed centrifugation through a discontinuous sucrose gradient to obtain highly enriched canalicular plasma membranes. Livers from EE-treated rats and corresponding controls were processed in parallel; the protein membrane yields did not differ between control and EE-treated groups (0.38 ± 0.06 vs. 0.33 ± 0.09 mg protein/g liver). The relative enrichment (membrane fraction/homogenate enzyme specific activity) of the specific canalicular marker 5'-nucleotidase in these membrane preparations was similar between control and EE-treated groups (19.9 ± 3.1 vs. 22.1 ± 5.7 µmol product formed·mg protein–1·h–1).

Vesicle size measurements. To determine the size of vesicles, electron micrographs were taken on a ME 10 Zeiss electron microscope. Pellets of membrane vesicles were fixed with 2.5% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.2), treated with 1% OsO4, washed with the same phosphate buffer, and then treated with uranyl acetate. After dehydration and embedding in the resin Durcupan, thin sections were stained with lead citrate. Size measurements were made on micrographs at x30,000 magnification using the software Image-Pro Plus (Media Cybernetics, Silver Spring, MD). The software was set to select objects (i.e., vesicular structures) in the range of 50 to 500 nm (28).

Stopped-flow measurements. The time course of vesicular volume was followed from changes in intensity of scattered light at the wavelength of 450 nm by using a SX.18MVR stopped-flow spectrometer (Applied Photophysics, Surrey, UK), which has a 1.3-ms dead time and 99% mixing efficiency in <1 ms. The sample temperature (20°C) was controlled by a circulating water bath. To perform the experiments, 50 µl of a concentrated vesicle suspension were diluted into 450 µl of 50 mM sucrose and 5 mM HEPES-Tris, pH 7.4. One of the syringes of the stopped-flow apparatus was filled with the membrane suspension while the other was filled with the same buffer containing sucrose to establish a hypertonic gradient of 300 mosM upon mixing. The final protein concentration after mixing was 150 µg/ml. Immediately after application of a hypertonic gradient, water outflow occurs and the vesicles shrink, causing an increase in scattered light intensity. The data were fitted to single exponential functions. The osmotic water permeability coefficient (Pf) was calculated as previously (28), using the equation: Pf = Kexp·Vo·Av–1·Vw–1·{Delta}C–1, where Kexp is the fitted exponential rate constant, Vo is the initial mean vesicle volume, Av is the mean vesicle surface, Vw is the molar volume of water, and {Delta}C is the osmotic gradient. Vo and Av were calculated by using the mean canalicular vesicle diameter assessed by morphometric analysis of electron micrographs.

Immunoblotting. Solubilized membrane fractions were subjected to 12% SDS-PAGE and transferred to polyvinyl difluoride membranes (NEN Life Science Products, Boston, MA). After blocking and washing, blots were incubated overnight at 4°C with rabbit affinity-purified antibodies against AQP8 and AQP9 (1 µg/ml; Alpha Diagnostics International, San Antonio, TX) or with monoclonal antibody to human Mrp2 (M2III-6; Alexis Biochemicals, Carlsbad, CA). The blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (DakoCytomation, Glostrup, Denmark). Protein bands were detected by enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech). Autoradiographs were obtained by exposing the membranes to Kodak XAR films (Eastman Kodak, Rochester, NY), and the bands were evaluated by densitometry using Gel-Pro32 software (Gel-Pro Analyzer, Media Cybernetics).

Immunohistochemistry. Livers from control and EE-treated rats were perfused with phosphate-buffered saline to eliminate the blood and then sliced and fixed by immersion with 4% paraformaldehyde. The samples were incubated overnight in phosphate-buffered saline with 6.8% sucrose, dehydrated with acetone, and embedded in the resin Technovit 8100 (Heraeus-Kulzer, Wehrheim, Germany) at 4°C. Before staining, semithin sections were incubated for 5 min at 37°C in 0.01% trypsin/0.1% CaCl2 (pH 7.8). Sections were incubated for 5 h at 37°C with AQP8 affinity-purified antibodies (5 µg/ml) and then treated for 1 h with corresponding secondary antibodies (Sigma), followed by an incubation with peroxidase-antiperoxidase at a dilution of 1:100 for 1 h at 37°C. Finally, the immunolabeling was visualized by incubation with 3,3'-diaminobenzidine-H2O2 medium for 10 min at room temperature. Controls were performed by omitting the primary antibodies. Images were captured with an E 600 photomicroscope equipped with a DMX 1200 digital camera (Nikon, Kawasaki, Japan).

Northern blotting. Total RNA from livers of control and EE-treated rats was isolated by the TRIzol reagent (Invitrogen) following the manufacturer's protocol. RNA samples (20 µg per lane) were electrophoresed through 1.2% agarose-formaldehyde gels and transferred to nylon membranes (Hybond-N+, Amersham Biosciences). Hybridization was performed in 50% formamide, 5x saline-sodium citrate, 5x Denhardt's solution, and 0.1% SDS at 42°C for 20 h, with a specific rat AQP8 complementary DNA probe labeled with [{alpha}-32P]deoxycytidine 5'-triphosphate. The membranes were washed and autoradiographed with intensifying screens for 5 days. Expression of the AQP8 mRNA was normalized against the expression of the 28S rRNA which was not altered with the treatment.

Statistical analysis. Data are expressed as means ± SE. Significance was determined by Student's t-test or the 1-way ANOVA, Tukey's test; P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Administration of EE to rats daily during 5 days resulted in intrahepatic cholestasis as indicated by a decrease in bile flow and both a significant decrease in body weight and increase in liver weight, in agreement with results reported elsewhere (11). Bile flow was reduced by 58% (P < 0.05) from 1.9 ± 0.3 µl·min–1·g liver–1 in control rats to 0.8 ± 0.2 µl·min–1·g liver–1 in EE-treated rats.

Expression of AQP proteins in EE-induced cholestatic liver. To study the AQP protein expression and subcellular localization in EE-induced cholestasis, we initially performed immunoblotting on liver membrane fractions. Compared with controls, AQP8 protein levels were significantly reduced in all membrane fractions, i.e., 81% in total membranes, 87% in plasma membranes, and 76% in intracellular membranes (Fig. 1A). EE treatment did not significantly affect AQP9 protein levels in total or plasma membranes (Fig. 1B). Consistent with the predominant hepatocyte surface localization of AQP9 (21), intracellular membranes showed no detectable signal (data not shown).


Figure 1
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  		Fig. 1. Immunoblotting for aquaporins AQP8 and AQP9 in cholestatic liver. Cholestasis was induced by ethinylestradiol (EE; 5 mg·kg body wt–1·day–1 for 5 days). Liver membrane fractions were isolated from control and EE-treated rats and subjected to immunoblotting as described in MATERIALS AND METHODS. A: anti-AQP8 immunoblots of total (TM), plasma (PM), and intracellular microsomal membranes (IM) (30 µg protein/lane) with corresponding densitometric analysis (n = 5). B: anti-AQP9 immunoblots of TM and PM (30 µg protein/lane) with corresponding densitometric analysis (n = 5). IM did not show any signal for AQP9. Data (means ± SE) are expressed as percentage of control livers. *P < 0.05.

 
Immunohistochemical studies in serial liver sections were performed to confirm the expression and subcellular localization of hepatocyte AQPs 8 and 9 in EE-induced cholestasis (Fig. 2). Immunohistochemical data were in agreement with the quantitative immunoblotting analysis. As previously observed (7, 8, 21), AQP8 in control rat livers was found to be predominantly intracellular in hepatocytes, although some immunoreactivity was observed on the plasma membrane (Fig. 2A). After EE administration, AQP8 staining was markedly decreased (Fig. 2B). The staining pattern for AQP9 in control livers was in agreement with previous results (21), i.e., AQP9 was mainly observed on the hepatocyte sinusoidal plasma membranes (Fig. 2D). AQP9 staining remained unaltered in EE-treated rat livers (Fig. 2E). Immunohistochemical controls showed no labeling when the corresponding primary antibody was omitted (Fig. 2, C and F). Similar immunostaining patterns were observed in three separate control and EE-treated rats.


Figure 2
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  		Fig. 2. Immunohistochemistry for AQP8 and AQP9 in cholestatic liver. Cholestasis was induced by EE (5 mg·kg body wt–1·day–1 for 5 days). Immunohistochemistry was performed as described in MATERIALS AND METHODS. A: AQP8 in normal rats. Immunoreactivity (brown staining) was seen on the canalicular plasma membrane (arrows; inset) as well as in intracellular membrane compartments of hepatocytes. B: AQP8 in cholestatic rats. Labeling was uniformly decreased. D: AQP9 in normal rats. Immunoreactivity (brown staining) was mainly seen on the sinusoidal plasma membranes of hepatocytes (arrows). E: AQP9 in cholestatic rats. Labeling was not significantly changed. C and F: negative controls. No staining was observed in experiments omitting the AQP8 (C) or AQP9 (F) primary antibody. Magnification x400.

 
The pair-feeding experiments indicated that EE reduced AQP8 protein expression in total liver membranes (assessed by immunoblotting) by 78% (P < 0.001) and bile flow by 58% (P < 0.05). AQP9 protein expression was found not to be altered. These data did not differ from those obtained with rats allowed to have free access to food.

Water permeability of canalicular membranes from EE-induced cholestatic liver. To study whether EE-induced AQP8 downregulation caused a reduction in canalicular water permeability, we assessed the osmotic water permeability by a stopped-flow method in which vesicles were subjected rapidly (1 ms) to a hypertonic osmotic gradient. The time course of vesicle volume was followed from the change in scattered light. Figure 3A shows the typical tracings of a time course of scattered light intensity (water transport) in canalicular plasma membrane vesicles from normal and EE-cholestatic livers in response to a 300 mosM hypertonic sucrose gradient. No change in scattered light was observed when vesicles were mixed with isosmotic buffer, showing an absence of mixing artifacts. Data fit well to a single exponential function indicating the presence of functional homogenous populations of canalicular vesicles. The corresponding calculated Pf values are shown in Fig. 3B. The canalicular Pf value for control rats was 73 ± 1 µm/s, comparable to that reported previously (28). Canalicular Pf for EE-cholestatic rats was significantly reduced (57 ± 2 µm/s, P < 0.05). Figure 3C shows, consistent with data from Fig. 1A, a significant reduction of canalicular AQP8 protein levels in EE cholestasis.


Figure 3
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  		Fig. 3. Water permeability of canalicular membranes from cholestatic liver. Cholestasis was induced by EE (5 mg·kg body wt–1·day–1 for 5 days). Canalicular liver plasma membranes were isolated as described in MATERIALS AND METHODS. A: typical tracings of a time course of scattered light intensity (osmotic water transport), along with single exponential fits, in canalicular plasma membrane vesicles from control and cholestatic livers in response to a 300 mosM hypertonic sucrose gradient. No change in scattered light was observed when vesicles were mixed with isosmotic buffer (isosmolar). B: osmotic membrane water permeability (Pf) values calculated from the rate constant of the single-exponential fits, as described in MATERIALS AND METHODS. Data are means ± SE from 3 independent vesicle preparations. *P < 0.05. C: anti-AQP8 immunoblot of the canalicular plasma membranes used for Pf assessment (15 µg protein/lane) with corresponding densitometric analysis (n = 3). Data (means ± SE) are expressed as percentage of controls. *P < 0.05.

 
The size of control and EE canalicular plasma membrane vesicles was not significantly different. Mean canalicular vesicle diameter from control liver was 176 ± 7 nm (n = 1,102), and mean canalicular vesicle diameter from EE-treated liver was 160 ± 9 nm (n = 1,018). Thus the decreased canalicular Pf in EE-treated rats cannot be due to a difference in either the initial vesicle size or the purification of the membranes (see MATERIALS AND METHODS).

Expression of AQP8 mRNA in EE-induced cholestatic liver. To begin to explore the mechanisms of EE-induced AQP8 protein downregulation, we assessed the steady-state mRNA levels by Northern blot analysis. AQP8 mRNA expression was found not to be reduced, actually increased by 115% with the treatment (Fig. 4), suggesting the involvement of posttranscriptional mechanisms.


Figure 4
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  		Fig. 4. Northern blotting for AQP8 mRNA in cholestatic liver. Cholestasis was induced by EE (5 mg·kg body wt–1·day–1 for 5 days). Total liver RNA was isolated and then processed as described under MATERIALS AND METHODS. Autoradiograph for AQP8 mRNA (1.5 kb) corresponding to 3 independent experiments per group. The ethidium bromide staining of the gel indicating the presence of 28S rRNA is shown as a control for equal loading and RNA integrity. Corresponding densitometric analysis is also shown. Data (means ± SE) are expressed as percentage of controls. *P < 0.05.

 
Role of lysosomal and proteasomal proteolytic pathways in estrogen-induced AQP8 downregulation. An in vitro model was established to study whether a posttranscriptional mechanism, such as an increased proteolysis, is involved in the AQP8 protein downregulation observed in estrogen cholestasis. As shown in Fig. 5A, the hepatocyte protein expression of AQPs 8 and 9 (but not that of the organic anion transporter Mrp2) was stably maintained under our culture conditions. This seems to be a particular feature of hepatocyte AQPs, since most of the solute transporters (including Mrp2) are quickly downregulated in primary hepatocytes (35). In agreement with the in vivo data (see Fig. 1), we found that the cholestatic estrogen E17G decreased hepatocyte AQP8 protein expression, whereas that of AQP9 was unaltered (Fig. 5B). The lysosomal protease inhibitor leupeptin, but not the proteasome inhibitor MG132, prevented the estrogen-induced AQP8 downregulation (Fig. 5B). These data suggest a lysosomal-mediated degradation of AQP8 protein in estrogen-induced cholestasis.


Figure 5
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  		Fig. 5. Effect of lysosomal and proteasome inhibitors on estrogen-induced AQP8 downregulation in cultured hepatocytes. Rat hepatocytes were cultured and subjected to subcellular fractionation as described in MATERIALS AND METHODS. A: expression of AQP8, AQP9, and Mrp2 after 3 h of cell attachment (time 0) and after additional 20 h of culture. Representative immunoblots of 3 separate experiments in total hepatocyte membrane fraction (20 µg protein/lane). Expression of AQPs 8 and 9 were not significantly changed, whereas that of Mrp2 decreased by 80% (P < 0.05). B: primary cultured rat hepatocytes were preincubated for 30 min in the presence of the lysosomal protease inhibitor leupeptin (250 µM) or the proteasome inhibitor MG132 (10 µM), and then for additional 20 h in the absence (–) or presence (+) of the estrogen (10 µM; E, estradiol-17beta-D-glucuronide). Anti-AQP8 and anti-AQP9 immunoblots of total hepatocyte membranes (20 µg protein/lane) and corresponding densitometric analysis (n = 4) are shown. Data (means ± SE) are expressed as percentage of controls. *P < 0.05 compared with controls.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our results demonstrate for the first time that the expression of hepatocyte AQP8 water channels as well as the canalicular membrane water permeability is downregulated in estrogen-induced intrahepatic cholestasis. This supports the novel concept that a defective AQP canalicular expression contributes as a mechanism for bile secretory dysfunction of cholestatic hepatocytes.

Bile formation is an osmotic secretory process that involves the secretion of osmotically active solutes, followed by the passive movement of water into the biliary space. Water may move across the hepatic epithelial cells by either of two pathways: a paracellular pathway between the tight junctions of adjacent cells or a transcellular pathway across the cells. Although the quantitative contribution of these two pathways of water transport is still unclear, current experimental evidence appears to favor the transcellular pathway (5, 9, 27–29). AQPs partially account for the water permeability of both hepatocyte plasma membrane domains, AQP9 facilitating the basolateral movement of water and AQP8 modulating its canalicular, rate-limiting, transport (28). Thus the generation of bile flow would be ultimately dependent on the molecular and functional expression of solute transporters in the canalicular plasma membrane domain as well as on the canalicular membrane water permeability determined by the level of AQP expression and the lipid composition. The biliary excretion of bile salts, via the bile salt transporter Bsep, glutathione, via the organic anion transporter Mrp2, and HCO3–, via the Cl–/HCO3– exchanger AE2, are thought to be the major osmotic driving forces for canalicular bile flow (31). Canalicular AQPs would allow the efficient coupling of osmotic solutes and water transport during bile formation.

As observed in this and previous studies (6, 7, 13, 16, 21), rat hepatocyte AQP8 is localized in intracellular compartments as well as on the plasma (canalicular) membrane. Nevertheless, there are conflicting data about AQP8 subcellular localization. Thus, whereas immunohistochemical and immunogold electron microscopy studies showed similar AQP8 localization (i.e., in intracellular compartments and on canalicular membrane) between mouse and rat hepatocytes (15), studies from other investigators showed a predominant plasma membrane (basolateral) localization of mouse AQP8 with no major contribution to the hepatocyte water permeability (42). Studies in AQP8 null mice showed no significant dietary fat misprocessing (42), an observation that suggests no alteration of the biliary excretion of primary bile salts reaching intestine for lipid digestion. Nevertheless, direct studies exploring the mechanisms of bile formation are pending to be able to know whether AQP8-null mice develop cholestasis.

There is experimental evidence suggesting that AQP8 facilitates the osmotically induced canalicular membrane water transport (21, 28) and bile formation by rat hepatocytes (21). Thus defective AQP8 plasma membrane expression might be associated with alterations of normal bile physiology. We have recently found that in other cholestatic condition, such as the obstructive cholestasis by bile duct ligation of rats, there is also an abnormal expression and trafficking of AQP water channels (8). Thus both extrahepatic and EE-induced intrahepatic cholestasis showed considerable downregulation of hepatocyte plasma membrane AQP8 expression.

Estrogens are involved in the pathogenesis of intrahepatic cholestasis developed in susceptible women during pregnancy, after administration of oral contraceptives or during postmenopausal replacement therapy (22). In rats, administration of EE causes a reduction of bile flow and an impairment of various transport mechanisms in both basolateral and canalicular membranes of hepatocytes. As reported here for AQP8, the canalicular transport proteins Bsep and Mrp2 are downregulated (23, 39), although the expression of Bsep is relatively preserved compared with Mrp2. Even though the HCO3– excretion is impaired in EE-treated rats, the functional activity of the Cl–/HCO3– exchanger AE2 was found not to be affected by EE (1). In contrast to the lack of effect of EE-treatment on basolateral AQP9, the expression of the solute basolateral transporters, Na+ taurocholate cotransporting polypeptide Ntcp, and the Na+-independent organic anion transporting polypeptide Oatp were significantly reduced (17, 37). The inhibition of the basic process of canalicular fluid secretion is believed to be the primary event in the development of cholestasis induced by estrogens (22, 31). Thus, EE-induced cholestasis may be ultimately caused by an impairment of the transient osmotic gradients generated by defective canalicular functional expression of solute transporters (i.e., Bsep and Mrp2) together with a reduced canalicular water permeability linked to defective AQP8 expression.

The pair-feeding experiments suggested that EE effects are associated with the estrogen per se and not with the reduced food consumption. This is in agreement with our in vitro data in primary cultured hepatocytes that indicate that the addition of estradiol to the culture media induced downregulation of AQP8 protein (Fig. 5B).

Water transport through the cell membranes may either occur through the lipid bilayer or be channel mediated. We previously provided evidence for the presence of both lipid- and AQP-mediated pathways for water movement across hepatocyte canalicular plasma membranes (28). The lipid membrane pathway can be disturbed by increasing cholesterol content, which is known to reduce water permeability (12). Nevertheless, because canalicular lipid composition (including cholesterol level) is known to be unaltered in EE cholestasis (4), the data strongly suggest a decrease in the fraction of water that moves through AQPs instead of through the lipid bilayer. The AQP-mediated water pathway was roughly estimated to contribute by ~30% to total canalicular water transport under nonstimulated (basal) conditions (28). In agreement with this, present data showed that a decrease of 60% in canalicular AQP8 expression in cholestasis (see Fig. 3C) is associated with a 22% reduction in membrane water permeability. This alteration in canalicular water permeability may be sufficient to impair the efficient coupling between osmotic solutes and water transport during bile formation.

Our data show that steady-state AQP8 mRNA levels are increased in EE-treated rat livers. Similar observations (i.e., decrease protein and increased AQP8 mRNA) were previously observed by us in obstructive extrahepatic cholestasis (8), suggesting a common compensatory mechanism for AQP protein reduction in cholestasis. Besides, as we observed in extrahepatic cholestasis (8), AQP8 downregulation does not seem to be associated with any major alteration of AQP8 mRNA structure, as judged by the Northern blotting studies, or AQP8 molecular mass (34 kDa), which indicates that its glycosylation processed properly. Our data suggest, as previously for both Bsep and Mrp2 (23), the involvement of some posttranscriptional mechanism (e.g., increased protein degradation) for AQP8 protein downregulation in estrogen-induced cholestasis. Other members of the AQP family of proteins (i.e., AQPs 1, 2, 4, and 5), which are not expressed in hepatocytes, have been described to be targeted for proteolysis through the lysosomal and/or the proteasome system (20, 24, 26, 36). Our studies in primary cultured hepatocytes support the notion of an estrogen-increased lysosomal degradation of AQP8, since the lysosomal protease inhibitor leupeptin, but not the proteasome inhibitor MG132, specifically prevented AQP8 downregulation. An increased lysosomal proteolysis has been implicated in the Mrp2 downregulation in obstructive cholestasis (34), but, to our knowledge, degradative pathways have not been explored for the known downregulation of solute transporters in estrogen-induced cholestasis. Our data indicate that estrogen treatment was able to decrease plasma membrane as well as intracellular (vesicular) AQP8 (see Fig. 1A). Whether estrogens derive AQP8 to the lysosomal degradation pathway by causing either mistrafficking of canalicularly targeted AQP8-containing vesicles or endocytic retrieval of canalicular AQP8 needs further investigation. Nevertheless, it is worth mentioning that after short-term treatment, E17G, in contrast to that observed for Mrp2 and Bsep (10, 32), failed to cause endocytic retrieval of canalicular AQP8 (33).

In conclusion, hepatocyte canalicular AQP8 (but not sinusoidal AQP9) protein expression is downregulated in estrogen-induced cholestasis, presumably by increased lysosomal-mediated degradation. Reduced AQP8 expression is associated with impaired canalicular membrane water permeability. Our data support the novel notion that a defective expression of canalicular AQP water channels contributes as a mechanism for bile secretory dysfunction of cholestatic hepatocytes.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Grant PICT 05-10590 (R. A. Marinelli) from Agencia Nacional de Promoción Científica y Tecnológica, by Grant PIP 6440 from Consejo Nacional de Investigaciones Científicas y Técnicas, and by Grant PRIN-Cofin 2004 and FIRB 2001 (G. Calamita) from Italian "Ministero dell'Istruzione, dell'Università e della Ricerca."


   ACKNOWLEDGMENTS
 
We thank Dr. N. F. LaRusso for providing the AQP8 complementary DNA probe, Dr. A. J. Vila for allowing us to make use of the stopped-flow spectrophotometer, Dr. E. J. Sanchez Pozzi for advising in estrogen studies in cultured hepatocytes, and J. E. Ochoa for assistance in cell isolation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Marinelli, Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 570, 2000 Rosario, Santa Fe, Argentina (e-mail: rmarinel{at}unr.edu.ar or rmarinel{at}fbioyf.unr.edu.ar)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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