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Am J Physiol Gastrointest Liver Physiol 293: G391-G402, 2007. First published April 26, 2007; doi:10.1152/ajpgi.00496.2006
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Disruption of function and localization of tight junctional structures and Mrp2 in sustained estradiol-17beta-D-glucuronide-induced cholestasis

Aldo D. Mottino,1,2 Tim Hoffman,1 Fernando A. Crocenzi,2 Enrique J. Sánchez Pozzi,2 Marcelo G. Roma,2 and Mary Vore1

1Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky; and 2Institute of Experimental Physiology, National University of Rosario, Rosario, Argentina

Submitted 24 October 2006 ; accepted in final form 25 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Estradiol-17beta-D-glucuronide (E217G) induces immediate and profound but transient cholestasis in rats when administered as a single bolus dose. Here, we examined the consequence of sustained E217G cholestasis and assessed the function and localization of the tight junctional proteins zonula occludens-1 (ZO-1) and occludin and of the canalicular transporter multidrug resistance-associated protein-2 (Mrp2). An initial dose of E217G (15 µmol/kg iv) followed by five subsequent doses of 7.5 µmol/kg from 60 to 240 min induced a sustained 40–70% decrease in bile flow. Following their biliary retrograde administration, cholera toxin B subunit-FITC or horseradish peroxidase were detected at the sinusoidal domain, indicating opening of the paracellular route; this occurred as early as 15 min after the first dose as well as 15 min after the last dose of E217G, but not following the administration of vehicle in controls. Localization of ZO-1 and occludin was only slightly affected under acute cholestatic conditions but was severely disrupted under sustained cholestasis, with their appearance suggesting a fragmented structure. Endocytic internalization of Mrp2 to the pericanalicular region was apparent 20 min after a single E217G administration; however, Mrp2 was found more deeply internalized and partially redistributed to the basolateral membrane under sustained cholestasis. In conclusion, acute E217G-induced cholestasis increased permeability of the tight junction, while sustained cholestasis provoked a significant redistribution of ZO-1, occludin, and Mrp2 in addition to increased permeability of the tight junction. Altered tight junction integrity likely contributes to impaired bile secretion and may be causally related to changes in Mrp2 localization.

bile secretion; endocytic compartment; estrogen; paracellular permeability

ESTRADIOL-17beta-D-GLUCURONIDE (E217G) is an endogenous metabolite of estradiol belonging to the family of glucuronide conjugates of the estrogen D-ring with cholestatic properties. E217G decreases bile flow and bile salt secretion in the rat in a reversible, dose-dependent manner (16). The mechanism by which E217G induces cholestasis is multifactorial. E217G induces endocytic internalization of the bile salt export pump (Bsep; Abcb11) (3) and of multidrug resistance-associated protein 2 (Mrp2; Abcc2) (21), thus abrogating their function. Bsep mediates the concentrative transport of bile salts across the canalicular membrane (9), thus generating the bile salt-dependent component of bile flow, while Mrp2 mediates the transport of glutathione and of numerous glutathione and glucuronide conjugates into bile and is thus considered crucial for the generation of bile salt-independent bile flow (12, 22). Trans-inhibition of Bsep-mediated transport of bile salts by E217G has also been proposed as a mechanism by which it induces cholestasis (31). Finally, Kan et al. (10) proposed an increase in paracellular permeability as a contributor to cholestasis, based on an increase in the early biliary secretion peak of the paracellular marker horseradish peroxidase (HRP) in response to E217G. Tight junction disruption is expected to impair formation of bile flow, both by dissipating bile-to-plasma osmotic gradients (1) and by disturbing the apical-basolateral intramembrane diffusion barrier, which is critical for maintenance of proper localization of apical versus basolateral transporters (17).

When administered intravenously as a bolus, the cholestatic effect of E217G is virtually instantaneous, unlike that induced by nonconjugated estrogens, such as ethinylestradiol, which appear to require metabolic activation by glucuronidation to exert their cholestatic potential (4, 28). This rapid mechanism makes E217G a unique tool to discriminate initial events of the cholestatic phenomenon from long-lasting, secondary effects due to chronic accumulation of potentially toxic biliary compounds retained as a consequence of bile secretory failure, such as bile salts and bilirubin, that are cholestatic themselves (5, 33). The acute E217G cholestatic model, however, has the limitation of its rapid recovery, which occurs soon after the minimum bile flow value is reached, i.e., 15–20 min after E217G administration, consistent with temporal overlap between cholestatic and recovery mechanisms. A single intravenous dose of E217G to rats promotes partial endocytic internalization of Bsep and Mrp2, with concomitant loss of their secretory activity (3, 21), as well as of the canalicular marker dipeptidyl peptidase IV (DPPIV) (20). This phenomenon is readily apparent as early as 20 min after E217G administration but is reversed within 90–120 min by reinsertion of the transporters in a microtubule-dependent manner (20). Therefore, this model does not permit characterization of late events of E217G-induced cholestasis affecting normal function and localization of canalicular transporters. Similarly, functional and structural alterations of the tight junctional barrier could be critically dependent on the duration of the cholestatic challenge. Indeed, no substantial alteration in the pattern of staining of the tight junctional protein zonula occludens 1 (ZO-1) was noted 20 min after an acute dose of E217G to rats, despite the fact that the maximal decrease in bile flow had been reached (19–21). This differs from what is observed in more chronic models of estrogen-induced cholestasis, such as that induced by ethinylestradiol, where a decrease in the length and depth of tight junctions, as well in the number of sealing elements (strands), is observed (24).

To better address the events leading to the cholestatic manifestations in estrogen-induced cholestasis, we repeatedly administered E217G intravenously so as to reach a constant 40–70% decrease in bile flow. The localization and function of Mrp2 and the impairment in both barrier function and distribution of tight junctional-associated proteins (ZO-1 and occludin) were studied both acutely as soon as bile flow reached its minimum value and after 4 h of sustained cholestasis (Fig. 1A). E217G markedly increased tight junctional permeability both acutely and after sustained cholestasis. Furthermore, when administered in a sustained manner, E217G provoked profound changes in the localization pattern of tight junctional components and Mrp2 relative to that seen acutely. Furthermore, changes in Mrp2 localization included its abnormal expression in the lateral membrane, a finding not observed in the acute cholestatic model.


Figure 1
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  		Fig. 1. Diagram of experimental protocols (A) and detection of cholera toxin B subunit (B) and horseradish peroxidase (HRP; C) localization after their biliary retrograde administration. A: after a 20-min postsurgery stabilization period, estradiol-17beta-D-glucuronide (E217G; 15 µmol/kg) or its solvent were administered intravenously (single injection protocol) at time 0, and cholera toxin B-FITC subunit or HRP were administered through the bile duct catheter at 5–15 min (horizontal black arrow). At 15 min, the liver was removed (arrowhead A) and used in the detection of cholera toxin by confocal microscopy or HRP by optical and electron microscopy. In a different set of animals, identically treated with E217G but without receiving retrograde infusion, the liver was removed at 20 min (arrowhead B) and used in confocal immunofluorescent microscopy of multidrug resistance-associated protein-2 (Mrp2), dipeptidyl peptidase IV (DPPIV), zonula occludens-1 (ZO-1), occludin, and organic anion transporter polypeptide 1a4 (Oatp1a4). In the sustained cholestatic protocol, E217G (15 µmol/kg iv) was administered followed by the repeated administration of 7.5 µmol/kg iv at the times indicated, leading to a sustained 40–70% decrease in bile flow relative to solvent controls (see the bile flow monitoring curve). Cholera toxin B subunit-FITC was administered at 245–255 min (horizontal black arrow); at 255 min, the liver was removed (arrowhead C) and used in its detection by confocal microscopy. In a different set of animals, identically treated with E217G but without receiving retrograde infusion, the liver was removed at 260 min (arrowhead D) and used for confocal immunofluorescent microscopy of Mrp2, DPPIV, ZO-1, occludin, and Oatp1a4 and for Western blot immunodetection of Mrp2, ZO-1, and Oatp1a4 in plasma membranes. Bile flow was monitored in 10-min periods throughout the experiment and was significantly lower in both acute and sustained cholestatic conditions (P < 0.05, n = 3). Only the data at the time of E217G injections, or shortly after that, are shown in Fig 1A, bottom. Total bilirubin biliary excretion was determined at 10–20 and 250–260 min as an estimation of Mrp2 activity (for details, see Ref. 26). B: green fluorescent cholera toxin B subunit-FITC was retrogradely administered after single (2) or repeated (3) injections of E217G and detected by confocal microscopy in the periportal region of the liver and in rats treated with a single dose of solvent (1). Under conditions of sustained cholestasis, cholera toxin B subunit-FITC was detected at the apical domain (arrows in 4) or at the basolateral membrane (arrows in 5) of the hepatocyte, as demonstrated by simultaneous detection of red fluorescent Mrp2 or Oatp1a4, respectively. Pictures are representative images from 3 independent experiments per group. A similar pattern of localization was observed under conditions of acute cholestasis (images not shown). C: optical microscopy detection of HRP after its retrograde administration under acute cholestatic conditions. HRP was detected in periportal regions of the liver from animals treated with E217G (2) but not in controls (1). At x100 magnification, HRP was detected at the periphery of cells (white circles in the inset). Pictures are representative images from 3 independent experiments per group.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. Leupeptin, aprotinin, PMSF, pepstatin A, E217G, HRP (type II), cholera toxin B subunit-FITC, methylbutane (isopentane), BSA, and DMSO were from Sigma Chemical (St. Louis, MO). All other chemicals were of analytical grade purity and used as supplied.

In vivo experiments. Female Sprague-Dawley rats (180–210 g, Harlan Industries, Indianapolis, IN) were used throughout. Rats had free access to food and water and were maintained on a 12-h, automatically timed light-dark cycle. All procedures involving animals were conducted in accordance with National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Kentucky.

Rats were anesthetized with urethane (1,000 mg/kg ip) and maintained thus throughout. Body temperature was measured with a rectal probe and maintained at 37°C with a heating pad connected to a temperature regulator (model 73A, Yellow Springs Instruments, Yellow Springs, OH). The femoral vein and common bile duct were cannulated with polyethylene (PE) tubing (PE-50 and PE-10, respectively). Saline was administered intravenously throughout the experiment to replenish body fluids. Bile was collected at 10-min intervals to monitor bile flow, which was determined gravimetrically, assuming a density of 1 g/ml. An E217G stock solution (100 mM in DMSO) was diluted 1:25 in 10% BSA in saline to a final concentration of 4.25 mM. E217G (15 µmol/kg) or solvent was administered through the femoral vein after a 20-min period of basal bile collection, as shown in Fig. 1A. For sustained cholestasis, E217G (7.5 µmol/kg) or solvent was administered every 45 min five more times (Fig. 1A). This dosage regimen was based on data demonstrating that ~50% of a cholestatic dose of [3H]E217G is excreted in bile within 1 h (16) and was designed to maintain an average steady-state concentration (Css) of E217G in the liver to compensate for its clearance by biliary and urinary excretion and metabolism to noncholestatic E217G-3-sulfate, according to the following relationship: Css = dose per unit time/clearance.

Evaluation of tight junctional integrity. The paracellular passage of both HRP and cholera toxin was assessed by their retrograde biliary administration, a method that avoids basolateral-to-canalicular transcytosis as a confounding factor, which may be the case upon their anterograde administration. After a 20-min period of basal bile collection, E217G or solvent was injected intravenously according to the administration protocol shown in Fig. 1A. HRP (type II), cholera toxin B subunit-FITC (10 and 1 mg/ml, respectively, in PBS), or PBS alone in controls was infused retrogradely through the bile duct catheter 5 min after the initial dose of E217G at a rate of 50 µl·min–1·kg–1 over 10 min (horizontal black arrow in Fig. 1A, left). In separate experiments, cholera toxin was similarly administered at 245–255 min (horizontal black arrow in Fig. 1A, right). The average flow rate of retrograde administration of HRP or cholera toxin was similar to that reported by Rahner et al. (24), who demonstrated unaltered integrity of tight junctions under such conditions. Immediately following these administration periods (arrowheads in Fig. 1, A and C), the liver was perfused in situ with PBS for 30 s to remove blood. For HRP detection, PBS was replaced with perfusion of 2% paraformaldehyde-2.5% glutaraldehyde in 0.1 mol/l Na+-cacodylate (pH 7.2) for 5 min. Livers were then immersed in this fixative for 2 h at 4°C and used for slice preparation. For cholera toxin detection, livers were perfused with PBS for 30 s, immediately frozen in liquid nitrogen-precooled isopentane, and kept at –80°C until used in slice preparation.

Localization of Mrp2, DPPIV, ZO-1, occludin, and organic anion transporter polypeptide 1a4. Mrp2, DPPIV, ZO-1, occludin, and organic anion transporter polypeptide 1a4 (Oatp1a4) proteins were detected by confocal immunofluorescent microscopy both at 20 min after 15 µmol/kg E217G (arrowhead B in Fig. 1A) and under conditions of sustained cholestasis at 260 min (arrowhead D in Fig. 1A). For confocal localization of Mrp2, DPPIV, ZO-1, and occludin as well as of Oatp1a4, a basolateral membrane marker, livers were removed, rinsed with PBS at the times indicated above, gently frozen in isopentane precooled in liquid nitrogen, and stored at –80°C until used in slice preparations. The transport activity of Mrp2 was monitored in vivo during both acute and sustained cholestasis by assessment of total bilirubin in bile at 10- to 20-min and 250- to 260-min bile collection periods, as previously described (20).

In a separate group of animals with sustained E217G cholestasis, the expression of Mrp2, ZO-1, and Oatp1a4 was evaluated in basolateral and canalicular membranes by Western blot analysis. For this purpose, the whole liver was removed (arrowhead D in Fig. 1A) and immediately used in the preparation of membranes.

To control for deleterious effects produced by the accumulation of potentially toxic biliary compounds on Mrp2 and ZO-1 localization during sustained conditions of cholestasis, additional animals were subjected to bile duct ligation (BDL) under urethane anesthesia as previously described (34). After 260 min, the liver was removed, and localization of Mrp2 and ZO-1 was determined by confocal microscopy.

Preparation of liver membranes and Western blot experiments. Membrane fractions enriched in canalicular and basolateral plasma membranes were prepared as previously described (15). Protein concentration was measured by the method of Lowry et al. (24). Immunoblotting was performed using a monoclonal antibody to human Mrp2 (1:2,000, M2 III-6, Alexis Biochemicals, Carlsbad, CA), a polyclonal antibody directed to human ZO-1 (1:1,000, Zymed Laboratories, San Francisco, CA), and a rabbit anti-rat Oatp1a4 Ab (1:1,000, a generous gift from Drs. Bruno Stieger and Peter Meier, Department of Internal Medicine, University Hospital, Zürich, Switzerland), as previously described in detail (20, 21). Subsequent densitometry was performed using Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD) software.

Microscopy experiments. Liver samples fixed in paraformaldehyde-glutaraldehyde were processed as previously described (24) for further detection of HRP. For both optical and electron microscopy, HRP was visualized by detection of its peroxidase activity using 3,3'-diaminobenzidine as a substrate (2) as modified by Rahner et al. (30). Electron microscopy detection of vesicular structures containing HRP was performed in a Tecnar 12 electron microscope (Phillips Electron Optics B.V., Eindhoven, The Netherlands).

For confocal immunofluorescence microscopy of Mrp2, DPPIV, ZO-1, occludin, and Oatp1a4, liver samples were sectioned and fixed as previously described (21). Mrp2, ZO-1, and Oatp1a4 were labeled with the indicated anti-Mrp2, ZO-1, and Oatp1a4 primary antibodies, whereas DPPIV was detected with a monoclonal antibody against rat DPPIV (a generous gift from Dr. Werner Reutter, Institut für Molekularbiologie und Biochemie, Charité-Universitätsmedizin Berlin, Berlin, Germany), and occludin was labeled with a monoclonal antibody directed to human occludin (Zymed Laboratories), followed by treatment with the appropriate Cy2- or Cy3-conjugated donkey anti-IgGs (Jackson ImmunoResearch Laboratory, West Grove, PA). For detection of cholera toxin B subunit-FITC, liver samples were cut in 5-µm sections, fixed in methanol (–20°C) for 10 min, mounted, and used in confocal microscopy analysis. Mrp2 or Oatp1a4 was detected by indirect immunofluorescence as described above, as markers of apical or basolateral domains, respectively, together with cholera toxin detection. All confocal experiments were performed in a True Confocal Scanner Leica TCS SP II microscope (Heidelberg, Germany). To ensure comparable staining and image capture performance for the different groups belonging to the same experimental protocol, liver slices were prepared on the same day, mounted on the same glass slide in a single well, and subjected to the staining procedure and confocal microscopy analysis simultaneously.

Confocal image analysis. The extent of colocalization of Mrp2 and Oatp1a4 or of DPPIV and Oatp1a4 was analyzed in confocal images using Image J 1.34m software (NIH) with the Mander's coefficients Plugin. This Plugin calculates Pearson's and Manders' coefficients for two 8- or 16-bit images or stacks (14). Colocalization coefficients were then compared by ANOVA followed by the Newman-Keuls test.

To estimate the area corresponding to green fluorescent cholera toxin in the retrograde perfusion experiments, confocal images were analyzed by densitometry using Image J 1.34m software. Six to eight images from each group were randomly captured and analyzed. Green fluorescence was considered as a positive toxin signal only when the intensity was higher than 25, from a maximum of 255. Cholera toxin area was expressed as the percentage of green fluorescence relative to the total unit area examined. Differences among groups were analyzed by ANOVA followed by the Newman-Keuls test.

Statistical analysis. Data are expressed as means ± SD. Statistical analysis was carried out using Student's t-test unless otherwise stated. Values of P < 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Disrupted tight junctional integrity in E217G-induced cholestasis. In agreement with our previous studies (16, 21), a single dose of E217G produced a maximal decrease in bile flow at 20 min, followed by a slow, spontaneous recovery (Fig. 1A). Under conditions of sustained cholestasis, bile flow exhibited some variability (40–70% of that of solvent-treated animals; Fig. 1A), with minimal values obtained 10–30 min after each E217G dose. Figure 1B shows representative images of confocal detection of cholera toxin B subunit-FITC following its retrograde administration at 5–15 or 245–255 min (horizontal black arrows in Fig. 1A). Very low levels of green fluorescence were detected in livers from rats treated with a single (Fig. 1B,1) or repeated doses of solvent (image not shown). In contrast, a strong fluorescence signal was detected in samples either after single (Fig. 1B,2) or repeated administration (Fig. 1B,3) of E217G and was associated with periportal regions of the liver (arrows). Densitometry of the corresponding areas confirmed a significantly (P < 0.05) increased accumulation of cholera toxin after E217G administration either as a single (6.37 ± 3.96%, n = 6) or repeated administration (10.09 ± 3.68%, n = 8) with respect to solvent controls (1.43 ± 0.46% for single and repeated administration of solvent, n = 3 each). Although there was a clear trend toward increased accumulation of toxin after repeated versus single treatment with E217G, this did not reach statistical significance. Under conditions of sustained cholestasis, costaining of cholera toxin with red fluorescent Mrp2, as a marker of the canalicular membrane, was minimal (yellow fluorescence, arrows in Fig. 1B,4) and was restricted to the vicinity of the canaliculus. A major proportion of toxin was, however, preferentially localized to the basolateral region of the hepatocyte, at the sinusoidal space, as detected by colocalization with red fluorescent Oatp1a4 (yellow fluorescence, arrows in Fig. 1B,5). Since cholera toxin was mainly found in the sinusoidal region of periportal hepatocytes and only partially colocalized with Mrp2 or Oatp1a4, the possibility that Kupffer or endothelial cells also bound or internalized the marker could not be excluded. A similar extent of colocalization of cholera toxin with Mrp2 or Oatp14 was observed in liver slices under conditions of acute cholestasis (images not shown).

Optical microscopy detection of HRP by the diaminobenzidine technique following its retrograde administration is shown in Fig. 1C. Livers from solvent-treated rats did not exhibit any brown staining corresponding to HRP (Fig. 1C,1). In contrast, livers from E217G-treated rats showed strong HRP signals around the periportal regions of the liver (black arrows in Fig. 1C,2), which were mainly detected at the cell periphery (white circles in the inset of Fig. 1C,2), although it was not possible to discriminate between apical or basolateral localization. Further electron microscopy analysis of these same samples gave information on the precise localization of HRP. In the perivenous region of an E217G liver retrogradely perfused with HRP, the image of the intracanalicular space was partially occupied by microvilli (Fig. 2A, 1), whereas in the periportal region of this same liver, the area inside the canaliculus was relatively enlarged due to the retrograde perfusion. This was noted in livers from solvent- or E217G-treated rats perfused with either HRP or PBS. Electron micrography of a solvent-treated rat liver receiving PBS retrogradely showed the absence of HRP-positive vesicles around the canaliculus (Fig. 2A,2). In contrast, HRP-positive vesicular structures were present close to the canalicular membrane (arrows) in solvent-treated (Fig. 2A,3) or E217G-treated (Fig. 2A,4) rats further perfused with HRP. The size of these vesicles was ~80 nm, in agreement with a report (24) showing HRP-positive vesicles associated with fluid-phase endocytosis in the normal rat liver.


Figure 2
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  		Fig. 2. Electron microscopy of canaliculus and HRP localization. The liver was analyzed 15 min after a single dose of E217G or solvent and intrabiliary retrograde infusion of HRP or PBS. A: canalicular regions in perivenous hepatocytes in E217G-treated rats receiving HRP (1) versus periportal hepatocytes of rats receiving E217G and either PBS (2) or HRP (3 and 4). HRP-positive vesicles were detected in the pericanalicular region of periportal hepatocytes, both in solvent-treated rats (arrows in 3) and E217G-treated rats (arrows in 4). B: HRP-positive vesicles at the sinusoidal domain of the periportal region were detected only in E217G-treated rats and were mainly associated with endocytosis by endothelial and Kupffer cells (arrows in inset). Pictures are representative images from 3 independent experiments per group. HRP activity was detected using the diaminobenzidine method as described in MATERIALS AND METHODS.

 
To evaluate the importance of the fluid phase endocytic process in the internalization of Mrp2 and Bsep induced by E217G, we quantified the numbers of vesicles in control and E217G livers. There was no difference in the density of vesicles in a band 1,000 nm wide surrounding the canaliculus between solvent and E217G treatment (1.00 ± 1.11 vs. 1.22 ± 1.09 vesicles/canaliculus, respectively, n = 9). In contrast, a very strong signal was found in the sinusoidal region of periportal hepatocytes in E217G-treated (Fig. 2B) but not solvent-treated rats (image not shown), which was associated with the presence of a significant number of vesicles detected mainly in endothelial and Kupffer cells (see arrows in higher-magnification micrograph in the inset of Fig. 2B). Thus, electron microscopy results were consistent with access of HRP to the sinusoidal domain of the periportal region of the liver in the E217G group, in agreement with cholera toxin data. These data did not support a role for fluid phase endocytosis in the internalization of Mrp2 and Bsep.

Localization of Mrp2 and tight junctional structures and Mrp2 activity in E217G-induced cholestasis. Changes in Mrp2 localization (red fluorescence in Fig. 3A) were detected by using ZO-1 as a marker of the border of the canaliculus (green or yellow fluorescence). Control livers from rats given repeated doses of solvent did not exhibit any significant changes in the pattern of Mrp2 and ZO-1 staining, detected at 260 min, with respect to that receiving a single dose of the solvent, detected at 20 min, and thus only a control liver from the single dose protocol is shown in Fig. 3A,1. As we (22) previously reported, 20 min after the administration of E217G (Fig. 3A,2), Mrp2 exhibited a pattern of staining different from that of the solvent group (Fig. 3A,1). This was particularly evident in some areas of the liver, where endocytic retrieval of Mrp2 outside the limits of the canaliculus could be observed (arrows in the inset of Fig. 3A,2). At 260 min, under conditions of sustained cholestasis (Fig. 3A,3), the Mrp2 staining pattern was substantially different from controls and also from the single-dose cholestatic group. In response to repeated E217G administration, Mrp2 was detected in some areas as a uniform intracellular staining, whereas in others, it was detected as more deeply internalized vesicles (arrows in the inset of Fig. 3A,3).


Figure 3
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  		Fig. 3. Confocal immunofluorescent microscopy of Mrp2 and ZO-1 (A) or ZO-1 or occludin (B) in response to single versus repeated administration of E217G. A: confocal immunofluorescent microscopy of Mrp2 (red) and ZO-1 (green) in response to solvent (20 min; 1) or single (20 min; 2) or repeated (260 min; 3) administration of E217G. Animals given single or repeated doses of solvent did not differ for either Mrp2 or ZO-1 localization (images not shown). Arrows in the inset of 2 indicate endocytic internalization of Mrp2 to pericanalicular vesicles, next to the limits of the canalicular domain, at 20 min. After repeated administration, Mrp2 was found more deeply internalized into the hepatocyte (arrows in the inset of 3). 4 shows localization of Mrp2 and ZO-1 in the liver 260 min after bile duct ligation (BDL). B: pattern of localization of ZO-1 and occludin. ZO-1 green fluorescence is shown for solvent controls at 20 min (1) and for E217G either at 20 (2) or 260 min (3). Occludin red fluorescence is shown for controls at 20 min (4) and for E217G either at 20 (5) or 260 min (6). Insets from 3 (ZO-1) and 6 (occludin) represent higher-magnification images showing details of altered localization under sustained cholestasis. Pictures in A and B are representative images from at least 3 independent experiments per group.

 
The staining pattern of ZO-1 and occludin was only slightly affected under conditions of acute cholestasis but was substantially altered after the repeated administration of E217G. Twenty minutes after a single E217G dose (Fig. 3B,2 and 5), the junctional proteins presented some degree of distortion compared with controls (Fig. 3B,1 and 4), which was more evident for occludin, although both still appeared as parallel lines. After repeated administration of E217G leading to sustained cholestasis, this pattern was significantly altered, giving the canaliculus the appearance of a highly fragmented structure (Fig. 3B,3 and 6, and the high-magnification images in the insets).

Confocal analysis of Mrp2 and ZO-1 in BDL animals was used as a control for the potentially deleterious effect of biliary compounds retained during sustained cholestasis. No internalization of Mrp2 was detected 260 min after BDL (Fig. 3A,4). The limits of the canaliculus exhibited only a minimal distortion as demonstrated by ZO-1 staining, with preservation of the parallel line appearance, in the periportal region (inset in Fig. 4), where changes in localization of tight junctional and canalicular proteins were expected to occur initially (11, 23).


Figure 4
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  		Fig. 4. Bile flow and biliary excretion of total bilirubin in response to single versus repeated administration of E217G. Bile flow and total bilirubin concentration in bile samples were determined at 10–20 and 250–260 min. Data are expressed as percentages of the respective basal values, determined at –10 to 0 min, and represent means ± SD of 3 rats/group. *Significantly different from solvent-treated rats (P < 0.05).

 
Data on Mrp2 transport activity are shown in Fig. 4. Bile flow and the biliary excretory rate of conjugated bilirubin, an Mrp2 substrate, were impaired by E217G to a similar extent at 20 and 260 min compared with the respective controls, suggesting that altered function of Mrp2 contributes to impaired bile flow formation similarly under both conditions.

Basolateral detection of Mrp2 in E217G-induced sustained cholestasis. Relocalization of canalicular transporters to the basolateral membrane and the consequent reversal of the direction of biliary secretion have been postulated as a potential cause of cholestasis (13). We therefore examined this possibility following acute and sustained cholestasis. Mrp2 (red fluorescence) did not colocalize with Oatp1a4 (green fluorescence) either in the solvent group or in the group with acute cholestasis (Fig. 5A, 1 and 2, respectively). In contrast, at 260 min, some colocalization was detected in E217G-treated livers (arrow in Fig. 5A,3) as isolated yellow areas, under higher magnification, were shown to be in the vicinity of the canaliculi (arrows in Fig. 5A,4). Simultaneous detection of DPPIV and Oatp1a4 showed similar results (Fig. 5A,5 and 6). Densitometric analysis confirmed increased overlapping of Oatp1a4 with both Mrp2 and DPPIV. Comparison of Pearson's and Mander's correlation coefficients between solvent and both acute and sustained E217G cholestatic groups demonstrated a significant increase in colocalization of the apical and basolateral proteins at 260 min following sustained cholestasis relative to the solvent and acute E217G cholestasis group (P < 0.05). Figure 5A,3 also shows that Mrp2 was internalized more deeply under conditions of sustained cholestasis, which was detected as a homogeneous (asterisk) or a vesicular (arrowhead) intracellular staining pattern. To confirm that a fraction of Mrp2 was present in the basolateral membrane, we performed Western blot experiments of Mrp2 and Oatp1a4 in canalicular and basolateral membranes from the control and sustained cholestatic groups. Expression of Mrp2 and Oatp1a4 was preserved in apical membranes from E217G-treated livers (Fig. 5B). Detection of some expression of Oatp1a4 in apical membranes likely represents contamination with basolateral membranes, consistent with the isolation methodology (15). In sustained E217G cholestasis, detection of Mrp2 in basolateral membranes was increased, whereas that of Oatp1a4 remained unchanged with respect to the solvent group. A change in the degree of contamination between both membrane fractions is unlikely to account for this result, since the tight junctional-associated protein ZO-1 was primarily recovered with the canalicular membrane fraction and, in contrast to Mrp2, did not increase in basolateral membrane fractions after repeated E217G administration (data not shown). These results are therefore consistent with relocalization of Mrp2 to the lateral membrane near the apical domain of the hepatocyte in the E217G group. The densitometry data show that expression of Mrp2 at the canalicular membrane was not affected despite the profound internalization of Mrp2 detected by confocal microscopy under conditions of sustained cholestasis.


Figure 5
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  		Fig. 5. Confocal immunofluorescent microscopy of Mrp2, DPPIV, and Oatp1a4 (A) and Western blot analysis of Mrp2 and Oatp1a4 (B) in response to E217G. A: Mrp2 (red) and Oatp1a4 (green) localizations were distinct in animals treated with solvent at 20 min (1) or 260 min (image not shown) or with E217G at 20 min (2). After repeated administration of E217G, colocalization (arrow in 3) of Mrp2 and Oatp1a4 was detected, as shown in detail at higher magnification (arrows in 4). Sustained cholestasis also led to deeper internalization of Mrp2 vesicles (arrowhead in 3) or to homogeneous intracellular distribution of Mrp2 (asterisk in 3). Similarly, DPPIV (red) and Oatp1a4 (green) localizations were distinct in animals treated with solvent at 20 min (5). Some colocalization was detected at 260 min (arrows in 6). B: representative Western blot analysis of Mrp2 in canalicular (5 µg) and basolateral (20 µg) plasma membranes and of Oatp1a4 in canalicular (20 µg) and basolateral (5 µg) membranes at 260 min. Additional experiments were included for final statistical analysis of densitometry (n = 3). Data in the E217G group are expressed as means ± SD of densitometry relative to the respective control.*Significantly different from solvent-treated rats (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Bile is generated by the transport of osmotically active solutes into the confined space of the canaliculus, followed by the passive movement of water and counterions until osmotic equilibrium is achieved. Essential components of this process include Bsep and Mrp2, which mediate the ATP-dependent transport of these key solutes, i.e., bile acids and glutathione, respectively, into the canaliculus, and the tight junctions, which permit movement of water and small ions (e.g., Na+ and Cl–), but not of larger solutes, between bile and plasma (1). The present study supports the hypothesis that loss of both of these key components contributes to the profound impairment of bile flow induced by E217G. E217G rapidly decreases bile flow, but its cholestatic effect is transient following a single dose, as recovery mechanisms occur almost simultaneously. These self-limiting mechanisms prevent the extrapolation of results from the acute model to the more common, long-lasting, clinical situations in which the liver is progressively and continuously exposed to cholestatic estrogen glucuronides, as is thought to occur in pregnancy and ethinylestradiol-induced cholestasis (4, 28). We therefore developed an alternative cholestatic model in which decreased bile flow was sustained by repeated E217G administration. Using this approach, we found both structural and functional changes in tight junctional integrity and in the activity and localization of Mrp2.

Kan et al. (10) showed an increase in the early biliary secretion peak of HRP in response to E217G in the isolated perfused rat liver model, suggesting increased paracellular permeability. The present study clearly demonstrates that cholera toxin and HRP are both able to penetrate the tight junction after their retrograde intrabiliary administration in rats that have been given E217G intravenously, both following a single acute dose and after repeated administration, causing sustained cholestasis. Both markers were found primarily in the periportal, sinusoidal region of the liver acinus. HRP was found mainly endocytosed by endothelial and Kupffer cells, consistent with the high rate of fluid phase endocytosis exhibited by these cells (24). Cholera toxin-associated green fluorescence was increased in periportal hepatocytes of E217G-treated rats and was at least partially localized to the sinusoidal domain. Cholera toxin normally binds to the sinusoidal membrane of the hepatocyte and is endocytosed in part by a caveolin-mediated mechanism (18). This distribution pattern of HRP and cholera toxin indicates significant paracellular penetration of the markers, followed by internalization by endothelial or Kupffer cells or by hepatocytes, from the space of Disse. Importantly, this paracellular penetration occurred as early as 5–15 min after E217G administration, i.e., simultaneously with the decrease in bile flow. These data support the hypothesis that loss of integrity of the tight junction is an additional contributor to the dramatic decrease in bile flow occurring immediately after E217G administration. Thus, a single dose of E217G (15 µmol/kg) causes a striking (85–90%), rapid decrease in bile flow in rats. Neither trans-inhibition of Bsep by E217G (31) nor its partial retrieval from the canalicular membrane (3) can fully account for this decrease in bile flow, since bile salt-dependent bile flow represents only 50% of the total bile flow in the rat (6). In line with this, we have shown that retrieval of Bsep by E217G is almost completely prevented by pretreatment with dibutyryl-cAMP, whereas the decrease in bile flow is only partially prevented (3). Similarly, endocytic internalization of Mrp2, shown to occur simultaneously with Bsep (21), is unlikely to account entirely for the remaining decrease in bile flow because of the partial role of Mrp2 in the excretion of substrates determining the complementary, bile salt-independent fraction of the bile flow (35). Therefore, factors other than impaired activity of Bsep and localization of Bsep/Mrp2 are required in the dramatic decrease in bile flow occurring in acute E217G-induced cholestasis. The present study indicates that increased tight junctional permeability is a likely significant contributor.

Despite clear evidence of increased paracellular permeability to large molecules such as HRP and cholera toxin, localization of ZO-1 and occludin showed only minor changes during acute E217G cholestasis. These data indicate that functional changes in tight junction permeability precede structural changes detectable by the confocal immunofluorescent methodology. However, disruption of these tight junction-associated proteins became very apparent following sustained E217G cholestasis; confocal immunofluorescent microscopy of ZO-1 and occludin revealed a highly fragmented appearance and loss of the double line representing the border of the normal canaliculus, changes that were uniformly distributed throughout the liver lobule. The appearance of detectable structural distortion of these tight junction proteins did not correlate with changes in bile flow, so that despite visibly increased disruption of the tight junction, bile flow did not decline further but remained stable. This suggests that early changes in tight junction permeability are sufficient to dissipate the bile-to-plasma osmotic gradient of small molecules, e.g., bile salts and GSH, solutes that are essential for the generation of bile flow. The fragmentation of the tight junction structural proteins ZO-1 and occludin, which became detectable by confocal immunofluorescence, apparently did not further disrupt this bile-to-plasma osmotic gradient.

Only minor modifications in the normal pattern of staining of ZO-1 were detected 260 min after BDL, where biliary constituents would be expected to accumulate in the liver to a greater extent than after the E217G-induced 40–70% sustained cholestasis. Consequently, the substantial changes in ZO-1 localization detected after sustained E217G exposure are mainly attributable to a direct action of E217G rather than to deleterious effects of the accumulation of bile salts or other endogenous biliary anions that can affect paracellular permeability (25, 26). BDL alters the localization and expression of ZO-1, occludin, and the phosphoprotein 7H6 as well as the localization and expression of Mrp2 and P-glycoprotein in rats (7, 8, 11, 23). However, these effects are observed 2 days after BDL or later, and no studies have reported such changes at earlier times after BDL. Consequently, whereas in obstructive cholestasis decreased integrity of the tight junction is likely secondary to increased intrabiliary hydrostatic pressure and/or chronic intrahepatic accumulation of toxins, tight junctional disruption appears to be a primary event in E217G-induced cholestasis and causally linked with biliary secretory failure.

After its retrograde administration, HRP was found not only in endothelial and Kupffer cells but also in the pericanalicular region of the hepatocyte. Pericanalicular localization of retrogradely administered HRP in normal rats is consistent with results of Rahner et al. (24), demonstrating that retrograde HRP administration leads to the formation of HRP-positive pericanalicular vesicles attributable to fluid phase endocytosis, a normal event at the canalicular membrane. We did not observe, however, any differences in the extent of formation of intracellular HRP vesicles in the pericanalicular region of hepatocytes between control and E217G-treated rats. This finding excludes exacerbation of fluid phase endocytosis as a mechanism by which E217G induces endocytic internalization of Bsep and Mrp2 (3, 21). On the other hand, only a minor proportion of cholera toxin was detected at the canalicular membrane following E217G. Because colocalization of cholera toxin and Mrp2 was detected adjacent to the canaliculus (Fig. 1B,4), it is not possible to distinguish between apical endocytosis of cholera toxin and redistribution of Mrp2 to the lateral domain of the membrane, as shown in Fig. 5.

The amount of Mrp2 present in the canalicular membrane under sustained E217G-cholestatic conditions, as revealed by Western blot experiments, was similar to that in solvent-treated rats, suggesting that only a small fraction of Mrp2 was deeply internalized at 260 min. Indeed, if the magnitude of Mrp2 deeply internalized had been higher, a reduction in canalicular membrane-associated Mrp2 would have been observed by Western blot analysis, since the so-called "canalicular membrane fraction" contains both canalicular membrane and subapical vesicles but not late endosomes or lysosomes (21, 27). The impact of the degree of internalization on transporter function seems to be minimal, as no difference in Mrp2 transport function was recorded between acute and sustained E217G cholestasis. Consequently, sustained cholestasis apparently only increases the depth of transporter internalization rather than the recruitment of new transporters for internalization. In addition, the biliary excretion of total bilirubin, used to assess Mrp2 function, results from a balance between the functional capability of Mrp2, which is lost due to its internalization, and the hepatocyte-to-canalicular concentration gradient, which increases steadily due to the secretory failure; this counterbalance likely limits the extent of the decrease in the bilirubin secretory rate in sustained cholestasis.

Apart from its potential impact on paracellular permeability, altered integrity of tight junctions may be a key contributor to the redistribution of Mrp2 from the canalicular to the apical end of the lateral membrane (Fig. 5). Impairment of the "fence" function of the tight junctional complex may induce a loss of cell polarity by promoting the diffusion of membrane lipids and proteins between the apical and lateral membrane domains (2). Alternatively, this may represent missorting of endocytosed Mrp2-containing vesicles in the pericanalicular area to the apical end of the lateral membrane. In support of this latter possibility, Schmitt et al. (29) demonstrated recently that the formation of belt-like structures positive for occludin preceded the detection of pseudocanalicular MRP2 in HepG2 cells, leading the authors to postulate that formation of the tight junctional barrier is a prerequisite for apical transport proteins to be targeted correctly to their membrane domain. Furthermore, the canalicular protein Ca2+-Mg2+-ATPase was shown to undergo endocytic internalization and selective redistribution to the lateral membrane in chronic, experimental obstructive cholestasis (30), a model that is also accompanied by increased tight junctional permeability (32) and endocytic internalization of Mrp2 (23). Irrespective of the mechanism, endocytic internalization and selective relocalization to the lateral membrane under sustained E217G cholestasis were not restricted to Mrp2, as a similar phenomenon was observed for DPPIV.

The major findings of the current study are shown in Fig. 6, with emphases on the localization of Mrp2, ZO-1, and occludin as well as increased paracellular permeability. In view of the importance of transport of osmotically active solutes into the canaliculus and the integrity of the tight junctions in maintaining bile flow, it is likely that these apparent changes in both the localization of Mrp2 and increased permeability of the tight junctions contribute to E217G-induced cholestasis. Questions that remain, however, include whether the degree of cholestasis, which is known to depend on the dose of E217G (16), is due to a greater number of internalized transporters, the extent of opening of the tight junction, the numbers of canaliculi affected by these processes, or a combination of all of these events. Further studies are needed to address these questions and to determine if increased permeability of the tight junction is linked to endocytic retrieval of Mrp2 and even Bsep. In summary, functional and confocal microscopy experiments clearly demonstrated that acute E217G-induced cholestasis is accompanied by a significant disruption of tight junction barrier properties and by changes in Mrp2 distribution and function. Most of these changes were aggravated when cholestasis was maintained, due to the sustained deleterious effect of E217G itself rather than to retention of potentially toxic biliary metabolites secondary to secretory failure. Furthermore, relocalization of Mrp2 to the lateral domain became apparent under conditions of sustained cholestasis, a finding likely reflecting loss of the capability of tight junctions to maintain the differential integrity of the apical and lateral membrane. Finally, the present study suggests that E217G-induced disruption of tight junctions and changes in Mrp2 localization are causally interrelated.


Figure 6
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  		Fig. 6. Model diagram of the major changes detected in Mrp2, ZO-1, and occludin localization and paracellular permeability in acute versus sustained E217G-induced cholestasis. Mrp2 is internalized from the canalicular to the pericanalicular region of the hepatocyte to a similar extent in acute versus sustained cholestasis. Under sustained cholestasis, a small proportion of pericanalicular Mrp2 is more deeply internalized or relocalized to the lateral membrane, with no further decrease in the amount/functionalilty of Mrp2 that remains at the canaliculus. Permeability of the paracellular route is increased after a single dose of E217G, with significant leakeage of the osmotically active solutes (glutathione and bile acids) essential for bile flow formation and of HRP and cholera toxin B subunit to a lesser extent. ZO-1 and occludin localization, however, remains essentially preserved. These two proteins show a markedly distorted appearance under sustained cholestasis, indicating a significant disruption of tight junction structure that likely increases the permeability of large molecules such as HRP and cholera toxin subunit B-FITC, with no further increased leakage of bile salts and glutathione.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of General Medical Sciences Grant GM-55343 (to M. Vore) and by grants from Agencia Nacional de Promoción Científica y Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas, and Universidad Nacional de Rosario (to A. D. Mottino).


   ACKNOWLEDGMENTS
 
We express our gratitude to Drs. Mary E. Jennes, Mary Gail Engle, and Bruce Maley for the kind help in performing confocal and electron microscopy experiments and to Dr. Paiboon Jungsuwadee for the illustration nicely summarizing the major changes reported here.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Vore, Graduate Center for Toxicology, 306 Health Sciences Research Bldg., Univ. of Kentucky, Lexington, KY 40536-0305 (e-mail: maryv{at}uky.edu)

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
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