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Am J Physiol Gastrointest Liver Physiol 293: G1134-G1146, 2007. First published October 4, 2007; doi:10.1152/ajpgi.00079.2007
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LIVER AND BILIARY TRACT

Obstructive cholestasis induces TNF-{alpha}- and IL-1β-mediated periportal downregulation of Bsep and zonal regulation of Ntcp, Oatp1a4, and Oatp1b2

Markus G. Donner, Stephanie Schumacher, Ulrich Warskulat, Jane Heinemann, and Dieter Häussinger

Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich Heine University, Düsseldorf, Germany

Submitted 13 February 2007 ; accepted in final form 19 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inverse acinar regulation of Mrp2 and 3 represents an adaptive response to hepatocellular cholestatic injury. We studied whether obstructive cholestasis (bile duct ligation) and LPS treatment affect the zonal expression of Bsep (Abcb11), Mrp4 (Abcc4), Ntcp (Slc10a1), and Oatp isoforms (Slco1a1, Slco1a4, and slco1b2) in rat liver, as analyzed by semiquantitative immunofluorescence. Contribution of TNF-{alpha} and IL-1β to transporter zonation in obstructive cholestasis was studied by cytokine inactivation. In normal liver Bsep, Mrp4, Ntcp, and Oatp1a1 were homogeneously distributed in the acinus, whereas Oatp1a4 and Oatp1b2 expression increased from zone 1 to 3. Glutamine synthetase-positive pericentral hepatocytes exhibited markedly lower Oatp1a4 expression than the remaining zone 3 hepatocytes. In cholestatic liver Bsep and Ntcp immunofluorescence in periportal hepatocytes significantly decreased to 66 ± 4% (P < 0.01) and 67 ± 7% (P < 0.05), whereas it was not altered in pericentral hepatocytes. Oatp1a4 was significantly induced in hepatocytes with a primarily low expression, i.e., in periportal hepatocytes and in glutamine synthetase-positive pericentral hepatocytes. Likewise, Oatp1b2 was upregulated in periportal hepatocytes. Mrp4 zonal induction was homogeneous. Inactivation of TNF-{alpha} and IL-1β prevented periportal downregulation of Bsep. Recruitment of neutrophils and polymorphonuclear cells mainly occurred in the periportal zone. Likewise, IL-1β induction was largely found periportally. No significant transporter zonation was seen following LPS treatment. In conclusion, zonal downregulation of Bsep in obstructive cholestasis is associated with portal inflammation and is mediated by TNF-{alpha} and IL-1β. Periportal downregulation of Ntcp and induction of Oatp1a4 and Oatp1b2 may represent adaptive mechanisms to reduce cholestatic injury in hepatocytes with profound downregulation of Bsep and Mrp2.

hepatobiliary transport; bile salts; cytokines; acinar regulation; organic anion transporters

METABOLIC HETEROGENEITY OF different hepatocyte populations has been described for a variety of key enzymes of carbohydrate, lipid, and ammonia metabolism. This heterogeneous enzyme activity in pericentral and periportal hepatocytes is determined by exogenous factors such as the direction of sinusoidal blood stream, metabolite, hormone, and oxygen concentration in the portal blood as well as by intrinsic hepatocellular factors and has led to the concept of metabolic zonation (23, 28, 29). The compartmental organization of metabolic pathways is dynamic and can efficiently adapt to changing nutritional and metabolic requirements. As exemplified by key enzymes of bile acid synthesis, cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase, expressed mainly in pericentral hepatocytes, can be recruited to periportal hepatocytes following interruption of the enterohepatic circulation with colestid (52).

Furthermore, steps of bile formation are zonated. The activity of glucuronidation (14, 15) and glutathione conjugation (31, 40, 53) prevails in pericentral hepatocytes. Expression and activity of different sulfotransferase isoforms display a heterogeneous zonal pattern. Although hydroxysteroid sulfotransferase responsible for the sulfation of bile salts is more prevalent in periportal hepatocytes, expression of estrogen sulfotransferase prevails in perivenous hepatocytes (8, 26, 51). Phenol sulfotransferase shows an even acinar distribution (51). Hepatocellular uptake of taurocholate is similar in periportal and pericentral hepatocytes (20). Various studies provide indirect and direct evidence that zone 1 and zone 3 hepatocytes are important for bile salt secretion (1, 20, 27, 32). Data from various studies on the zonal excretion of Mrp2 substrates yielded conflicting results (1, 11, 21, 22, 50), which has been attributed to different substrates, overlapping transport characteristics of further (e.g., basolateral) transporters, substrate concentrations, hepatocellular metabolism, and flow rate during liver perfusion (1, 44). Thus functional studies make it difficult to assess the contribution of the different steps of uptake, metabolism, intracellular transport, and secretion to hepatobiliary transport of cholephilic substances. Furthermore, there are no data on the zonation of hepatobiliary transport processes in cholestasis.

In the past years cloning, localization, and functional characterization of a number of hepatobiliary transport proteins have permitted us to characterize the different steps of bile formation on the molecular level. An even acinar distribution was described for the sodium-dependent taurocholate cotransporting polypeptide (Ntcp, Slc10a1), the organic anion transporting protein 1 (Oatp1, Slco1a1), and Mrp2 (3, 37). The bile salt export pump (Bsep, Abcb11) and two isoforms of the Oatp family, namely Oatp1a4 (Slco1a4) and Oatp1b2 (Slco1b2), show a "gradient-type" zonation with predominant expression in perivenous hepatocytes (3, 7, 41). Differentially zonated Oatp isoforms may serve as "backup" systems for taurocholate uptake at increased sinusoidal bile acid concentrations (43). The multidrug resistance protein 3 (Mrp3, Abcc3) is only expressed in a small number of perivenous hepatocytes (12, 46) and may represent an overflow system in hepatocytes with a higher activity of key enzymes of bile acid synthesis and biotransformation (13).

Regulation of hepatobiliary transport proteins in various models of cholestasis leads to reduced canalicular excretion and promotes alternative basolateral excretion of biliary compounds, thereby minimizing cholestatic injury. In obstructive cholestasis, the downregulation of Mrp2 starts in periportal hepatocytes and extends toward pericentral hepatocytes sparing a zone of pericentral hepatocytes (37). Conversely, Mrp3 extends from pericentral to periportal hepatocytes in biliary obstruction (12, 46). In GY/TR– rats lacking Mrp2 there was an even distribution of basolateral Mrp3 (12). Thus the reciprocal regulation of Mrp2 and Mrp3 can be regarded as a paradigm of differential lobular regulation of hepatobiliary transport in conditions of altered canalicular excretion. There are no data on the lobular regulation of further bile salt and organic anion transporters in cholestasis. In the present study we found an acinar regulation of Ntcp, Bsep, Oatp1a4, and Oatp1b2 in obstructive cholestasis. The periportal downregulation of Bsep was attributed to the action of TNF-{alpha} and IL-1β. Investigating acinar regulation of transporters identified several mechanisms of adaptive regulation not obvious from studying transporter expression in whole liver.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and antibodies. Acrylamide was from AppliChem (Darmstadt, Germany). Protease inhibitor cocktail was obtained from Roche (Mannheim, Germany). LPS was purchased from Sigma (Deisenhofen, Germany). All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany) or Sigma. The TNF-{alpha} receptor fusion protein etanercept (Enbrel) was kindly supplied by Wyeth (Münster, Germany). The IL-1β antagonist anakinra (Kineret) was gratefully provided by Amgen (Thousand Oaks, CA). The rabbit polyclonal antibodies K4 against Ntcp, K44 against Bsep, K10 against Oatp1a1, K15 against Oatp1a4, and K22 against Oatp1b2 were generously provided by Dr. B. Stieger (Zürich, Switzerland). The rabbit polyclonal antibody SNG used for Mrp4 immunofluorescence (42) was a donation from Dr. D. Keppler (Heidelberg, Germany). The rabbit polyclonal antibody against IL-1β was purchased from Biosource (Camarillo, CA). The antibody ED2 against Kupffer cells was obtained from Serotec (Düsseldorf, Germany), the antibody against was from DAKO (Hamburg, Germany). The mouse monoclonal antibody against glutamine synthetase was purchased from BD Biosciences Pharmingen (Heidelberg, Germany). Horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody was obtained from Amersham Biosciences (Little Chalfont, UK). Cy3-conjugated goat anti-rabbit, Cy3-conjugated goat anti-mouse, FITC-conjugated goat anti-mouse, and FITC-conjugated goat anti-rabbit secondary antibodies were from Dianova (Hamburg, Germany).

Animals and animal treatment. Male Sprague-Dawley rats (weighing 200–250 g) were kept with free access to stock diet and water. The animals received care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" (NIH publication 86-23, revised 1985; and APS guidelines). The study protocol was approved by the animal welfare committee at the Regierungspräsidium Düsseldorf. Bile duct ligation (BDL) was performed as described before (12). For cytokine inhibition studies etanercept (8 mg/kg) or anakinra (100 mg/kg) was applied intraperitoneally at the time of laparotomy. Subsequently, etanercept was injected every 48 h, anakinra every 24 h intraperitoneally. Livers were harvested after 3 or 7 days. LPS application and harvesting of the livers was performed as described before (13). To exclude a dose effect of LPS on transporter zonation one group of animals received 2 mg/kg LPS. Livers were harvested 24 h after LPS application.

Real-time PCR. Total RNA extraction, reverse transcription of mRNA, and real-time SYBR green PCR were performed as described elsewhere (13). The primers used for PCR were 5'-GTCCAACCTCTTCACCCTGG-3' and 5'-ACGATGCTGAGGTTCATGTCC-3' for Ntcp, 5'-CAGAATCCAGAGAGCCTCTTTTACA-3' and 5'-GCCCTTACCCAGCTGCTGA-3' for Mrp4, 5'-GCCGACCAGTTGGCTCAT-3'and 5'-CCCACACATAGCCGTCTAC-3' for Bsep, 5'-GGAGAGAAGGAAAGCGAGCA-3' and 5'-CTCGACCTGAGGACTTCCATG-3' for Oatp1a1, 5'-TTCCAGTGGCAGGCTTAACAA-3' and 5'-ATAGTTGGTGCTGAACCCCTTC-3' for Oatp1a4, 5'-TTCACCTTGTCTGGCAGGATG-3' and 5'-TTCGGCTTCTTATCACCACGA-3' for Oatp1b2, and 5'-TTGAATCATGTTTGTGTCATCAGC-3' and 5'-GGCTTTGTACTTGGCTTTTCCAC-3' for Hprt (hypoxanthine-guanine phosphoribosyl transferase) as internal standard. Briefly, PCR conditions were as follows: one cycle at 50°C for 2 min and 95°C for 10 min, and 40 cycles at 95°C for 15 s and 60°C for 1 min. The sample volume was 25 µl for each well with a final primer concentration of 300 nM. PCR reactions were done in triplicate.

Membrane preparation and immunoblot analysis. Crude membrane-containing homogenates were subjected to SDS-PAGE and immunoblotting as described before (13). Autoradiography films were scanned and quantification of blots was performed with the TotalLab imaging software (TotalLab TL 100, version 2006, Nonlinear Dynamics, Newcastle upon Tyne, UK). The following antibody concentrations were used: K4 at 1:2,000; K44 at 1:5,000; K10, K15, and K22 at 1:1,000; horseradish peroxidase-conjugated goat anti-rabbit antibody at 1:7,500.

Indirect immunofluorescence and confocal laser scanning microscopy. Preparation of cryosections, immunostaining, and imaging was carried out as described elsewhere (6, 12). The following antibody concentrations were used: K4, K44, K10, K15, and K22 at 1:200, {alpha}-glutamine synthetase and {alpha}-MPO at 1:200, {alpha}-IL-1β at 1:50, {alpha}-ED2 and SNG at 1:25, Cy3-conjugated goat anti-rabbit and goat anti-mouse IgG at 1:500, FITC-conjugated goat anti-mouse IgG at 1:100, FITC-conjugated donkey anti-goat IgG at 1:100. The stained cryosections were visualized with a confocal laser scanning microscope (Zeiss LSM 510 Meta, Carl Zeiss, Jena, Germany) and imaging software (version 3.2, SP2). The excitation wavelengths were 488 and 543 nm. For better comparison, treated samples and respective controls were prepared and analyzed on the same slide.

Quantification of immunofluorescence membrane stainings. Zonation of the transporters studied was analyzed by densitometry of the peak immunofluorescent membrane signals in periportal and pericentral hepatocytes following 7 days of BDL. Quantification of the immunofluorescent images was carried out as described by Kubitz et al. (31) with minor modifications. The cryosections of treated and control animals were prepared and stained on the same slide to minimize errors due to the experimental procedure. Subsequently, low-magnification images containing a portal field and a central vein were taken of control and treated animals under identical conditions for exposure. The pericentral region was identified by costaining with an antibody against glutamine synthetase. Peak immunofluorescent membrane signals were obtained by placing a cursor across the membrane signal and reading the maximal signal intensity. The residual cytoplasmic staining was defined as background staining and was subtracted from the peak membrane signal. Quantification was carried out with the LSM imaging software. Ten periportal and 10 pericentral peak immunofluorescent membrane signals of one cryosection were recorded. In the case of Oatp1b2 we separately quantified central (i.e., glutamine synthetase-positive hepatocytes) membrane signals. The data set obtained from one animal was compared with the respective control animal for statistical significance by applying the t-test. Subsequently the mean values of the central, pericentral, and periportal peak immunofluorescent membrane data set of each animal were used to calculate data sets giving means ± SE of four animals per group. For the expression in relative units, the mean peak immunofluorescence in pericentral hepatocytes of each control was set to 100%. The data are given as the relative peak immunofluorescence compared with the peak immunofluorescence of pericentral hepatocytes of controls. The statistical significance shown represents the P value of the data sets of the pair of animals (or perivenous vs. periportal membrane stainings) with the least difference.

Statistics. For real-time PCR and densitometry of immunoblots data are expressed as means ± SE. The experiments were carried out in three to four animals per group. Statistic significance was examined by means of the Student's t-test. A P value of less than 0.5 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Real-time PCR analysis of Bsep, Ntcp, and Oatp isoforms. Relative mRNA expression levels of bile duct-ligated animals compared with controls are given in Fig. 1A. One day after BDL none of the transporters studied significantly differed from the controls. Three days after BDL Mrp4 mRNA was 58 ± 3%, Ntcp mRNA was 15 ± 2%, Oatp1a1 mRNA was 32 ± 2%, and Oatp1b2 mRNA was 23 ± 1% of sham-treated controls (P < 0.05). Bsep mRNA and Oatp1a4 mRNA were not significantly different from the controls. Seven days after BDL relative mRNA expression was 78 ± 19% for Bsep, 63 ± 6% for Mrp4, 54 ± 6% for Ntcp, 72 ± 16% for Oatp1a1, 119 ± 19% for Oatp1a4, and 46 ± 15% for Oatp1b2 [not significant (n.s.)]. LPS treatment did not significantly change Mrp4 mRNA but deceased mRNA levels of the remaining transporters studied after 24 h (Fig. 1B).


Figure 1
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  		Fig. 1. Real-time PCR of Bsep, Mrp4, Ntcp, and Oatp isoforms following bile duct ligation (BDL), LPS treatment, and obstructive cholestasis inhibited by TNF-{alpha} and IL-1β blockade. Total RNA from rat liver samples was isolated and reverse transcribed. The level of gene expression was measured by real-time PCR. Hypoxanthine-guanine phosphoribosyl transferase (Hprt) was used as an internal standard. Data were expressed as the ratio of mRNA copies to Hprt mRNA copies and represent means ± SE of 3–4 animals per group (*P < 0.05 compared with sham-treated animals). Transporter mRNA expression following BDL (A), LPS treatment (B), and obstructive cholestasis inhibited by TNF-{alpha} and IL-1β blockade (C) is shown.

 
Immunoblot analysis of Bsep, Ntcp, and Oatp isoforms. Immunoblot analysis and densitometry results of the BDL model are given in Fig. 2, A and B. One and 3 days after BDL there was a significant increase in Bsep protein level to 142 ± 9 and 123 ± 5%, respectively (P < 0.05). Ntcp protein levels progressively decreased to 32 ± 4 and 33 ± 11% 3 and 7 days after BDL, respectively (P < 0.05). Oatp1a1 protein levels significantly decreased to 50 ± 15% 7 days after BDL. Protein expression of Bsep, Oatp1a4, and Oatp1b2 was not different from sham-treated animals following 7 days of BDL. Results of transporter expression following LPS treatment are given in Fig. 2, C and D. Twelve hours following LPS treatment Ntcp, Oatp1a1, and Oatp1a4 protein levels significantly decreased to 67 ± 4, 76 ± 4, and 65 ± 8%, respectively (P < 0.05). Twenty-four hours after LPS treatment Ntcp, Oatp1a1, Oatp1a4, and Oatp1b2 protein levels were 58 ± 6, 66 ± 6, 49 ± 9, and 37 ± 7% of controls, respectively (P < 0.05). Bsep protein levels showed a nonsignificant decrease to 77 ± 5%.


Figure 2
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  		Fig. 2. Immunoblot analysis of Bsep, Ntcp, and Oatp isoforms following BDL and LPS treatment. Rats were subjected to BDL or injected with LPS (4 mg/kg body wt ip). Controls received sham operation or were injected with normal saline. Crude membrane-containing liver homogenates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with respective antibodies. Representative immunoblots of Bsep, Ntcp, Oatp1a1, Oatp1a4, and Oatp1b2 after various periods of BDL (A) or LPS treatment (C) are shown. Densitometric analysis immunoblots following BDL (B) and LPS treatment (D) are given. Data represent means ± SE of 3–4 animals per group (*P < 0.05 compared with sham-treated animals).

 
Confocal laser scanning microscopy of Bsep, Mrp4, Ntcp, and Oatp isoforms. Double-label immunofluorescence microscopy and densitometry of membrane signals in controls showed that Bsep, Ntcp, and Oatp1a1 were equally expressed in the plasma membrane of pericentral and periportal hepatocytes (Figs. 3, A–C; Go5, A–C; and Go7, A–C). Mrp4 immunofluorescence was too weak to be quantified. Mrp4 membrane staining appeared to be equally distributed in pericentral and periportal hepatocytes (Fig. 6, A–C). Oatp1a4 and Oatp1b2 showed a gradient-type zonation with a high expression in zone 3 hepatocytes, which gradually diminished in zones 2 and 1 (Figs. 4, A–C, and 5, G–I). Relative periportal peak immunofluorescence of Bsep, Ntcp, Oatp1a1, Oatp1a4, and Oatp1b2 was 106 ± 5, 104 ± 6, 95 ± 7, 48 ± 6, and 61 ± 5% of the relative peak immunofluorescence in pericentral hepatocytes, respectively (Fig. 7, A–E, P < 0.001 for Oatp1a4 and Oatp1b2). A small group of pericentral hepatocytes, i.e., hepatocytes expressing glutamine synthetase, had a markedly lower relative Oatp1a4 peak immunofluorescence than the surrounding pericentral hepatocytes (39 ± 2%, P < 0.001).


Figure 3
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  		Fig. 3. Double-label immunofluorescence and confocal laser scanning microscopy of Bsep with glutamine synthetase (GS) following BDL and inhibitory effects of etanercept and anakinra treatment. Cryosections were stained with a polyclonal antibody against rat Bsep and with a monoclonal antibody detecting GS as marker for pericentral hepatocytes. Representative images of 4 livers per group are shown. In normal rat liver Bsep was equally distributed in the pericentral and periportal area of the liver lobule (A, magnification in B and C). Seven days after BDL there was a selective diminution of the Bsep immunostaining in periportal hepatocytes (D, magnification in E and F). Broadening of the membrane staining and increasing intracellular Bsep-positive vesicles were suggestive of selective periportal endocytic retrieval (F). Etanercept (G–I) and anakinra treatment (J–L) in cholestatic rats largely abolished the selective periportal downregulation of Bsep. pc, Pericentral area; pp, periportal area. Bar in A, D, G, J = 50 µm; bar in B, C, E, F, H, I, K, L = 10 µm.

 

Figure 4
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  		Fig. 4. Double-label immunofluorescence and confocal laser scanning microscopy of Oatp1a4 with GS in obstructive cholestasis. Cryosections were stained with a polyclonal antibody against rat Oatp1a4 and with a monoclonal antibody detecting GS as marker for pericentral hepatocytes. Representative images of 4 livers are shown. In normal rat Oatp1a4 immunostaining gradually decreased from pericentral to periportal hepatocytes (A–C). GS-positive hepatocytes had a markedly lower Oatp1a4 membrane staining (G–I). In cholestatic rat liver Oatp1a4 was induced in pericentral GS-positive hepatocytes and in periportal hepatocytes, resulting in an even acinar expression (D–F, J–L). Bar in A, D, G, J = 50 µm; bar in B, C, E, F, H, I, K, L = 10 µm.

 

Figure 5
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  		Fig. 5. Double-label immunofluorescence and confocal laser scanning microscopy of Ntcp and Oatp1b2 with GS following BDL. Cryosections were stained with polyclonal antibodies against rat Ntcp and Oatp1b2 and with a monoclonal antibody detecting GS as marker for pericentral hepatocytes. Representative images of 4 livers per group are shown. In normal rat liver Ntcp was equally distributed in the pericentral and periportal area of the liver lobule (A, magnification in B and C). Seven days after BDL there was a selective diminution of the Ntcp immunostaining in periportal hepatocytes (D, magnification in E and F). Broadening of the membrane staining and increasing intracellular Ntcp-positive vesicles were suggestive of selective periportal endocytic retrieval (F). In normal rat liver Oatp1a4 immunostaining showed a "gradient-type" zonation with decreasing expression from pericentral to periportal hepatocytes (G–I). In cholestatic rat liver Oatp1a4 was induced in periportal hepatocytes (D, L). Bar in A, D, G, J = 50 µm; bar in B, C, E, F, H, I, K, L = 10 µm.

 

Figure 6
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  		Fig. 6. Double-label immunofluorescence and confocal laser scanning microscopy of Mrp4 with GS following BDL. Cryosections were stained with a polyclonal antibody against Mrp4 and with a monoclonal antibody detecting GS as marker for pericentral hepatocytes. Representative images of 4 livers per group are shown. In normal rat liver Mrp4 was equally distributed in the pericentral and periportal area of the liver lobule (A, magnification in B and C). In obstructive cholestasis induction of Mrp4 occurred in pericentral and periportal hepatocytes (D, magnification in E and F). Bar in A, D = 50 µm; bar in B, C, E, F = 10 µm.

 

Figure 7
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  		Fig. 7. Densitometry of the peak immunofluorescence signals of Bsep, Ntcp, Oatp1a1, Oatp1a4, and Oatp1b2 in obstructive cholestasis and following LPS treatment. Peak immunofluorescence signals of Bsep, Ntcp, Oatp1a1, Oatp1a4, and Oatp1b2 in periportal and pericentral hepatocytes were studied by densitometry. Data are expressed as the relative peak immunofluorescence compared with pericentral hepatocytes of controls. Results of periportal and pericentral densitometry of the transporters studied after 7 days of BDL (A–E) or 24 h of LPS treatment, 2 and 4 mg/kg (F–J) are shown. pc (GS+), pericentral, GS-positive hepatocytes; pc, pericentral area; pp, periportal area. Data represent means ± SE of 4 animals per group (*P < 0.05, **P < 0.01, ***P < 0.001 compared with sham-treated animals). n.s., Not significant.

 
In bile duct-ligated rats Bsep and Ntcp immunofluorescence in periportal hepatocytes progressively decreased from 3 days after BDL reaching a maximum after 7 days (Figs. 3, D–F, and 5, D–F). After 7 days of obstructive cholestasis Bsep and Ntcp peak immunofluorescence in periportal hepatocytes had decreased to 66 ± 4 and 67 ± 7% of pericentral hepatocytes of controls, respectively (Fig. 7, A–B, P < 0.01). Peak immunofluorescence in pericentral hepatocytes was not significantly altered. Periportal hepatocytes showed a fuzzy Bsep and Ntcp membrane staining and increasing intracellular Bsep- and Ntcp-positive vesicles. Oatp1a1 immunofluorescence equally declined in pericentral and periportal hepatocytes starting after 3 days of BDL and reaching a maximum after 7 days (66 ± 8 and 57 ± 6% of pericentral hepatocytes of controls, respectively; Fig. 7C). Oatp1a4 was induced in hepatocytes with a primarily low expression level, i.e., in periportal and in glutamine synthetase-positive pericentral hepatocytes. Induction began after 3 days after BDL reaching a maximum after 7 days (Fig. 4, D–L). Oatp1a4 immunofluorescence intensity in periportal and glutamine synthetase-positive hepatocytes amounted 94 ± 3 and 93 ± 11% of pericentral hepatocytes of controls, respectively (Fig. 7D). Likewise, Oatp1b2 was induced in periportal hepatocytes (Fig. 5, J–L), reaching 92 ± 5% peak immunofluorescence of pericentral hepatocytes of controls (Fig. 7E). The magnitude of induction of Mrp4 was comparable in pericentral and periportal hepatocytes (Fig. 6, D–F). The intensity of membrane signals even within one cryosection displayed considerable variation (Fig. 6, D–F) and therefore did not permit a reliable quantitative analysis. A summary of zonal transporter regulation following BDL is given in Table 1.


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  		Table 1. Zonal regulation of hepatocellular bile salt and organic anion transporters in obstructive cholestasis

 
In rats treated with 4 mg/kg LPS we found a significant downregulation of Bsep in pericentral and periportal hepatocytes, amounting 74 ± 4% (P < 0.03) and 54 ± 2% (P < 0.001) of pericentral hepatocytes of controls (Fig. 7F). Similarly, Ntcp immunostaining equally decreased in pericentral (48 ± 4%, P < 0.001) and periportal (48 ± 4%, P < 0.001) hepatocytes 24 h after LPS treatment (Fig. 7G). Downregulation of Oatp1a1 equally occurred in pericentral and periportal hepatocytes (Fig. 7H). The gradient-type zonation of Oatp1a4 and 1b2 was preserved during downregulation following LPS treatment. Peak immunofluorescence of Oatp1a4 in pericentral, pericentral glutamine synthetase-positive, and periportal hepatocytes was 78 ± 3% (P < 0.01), 30 ± 2% (P < 0.001), and 46 ± 1% (P < 0.001) 24 h after LPS treatment, respectively (Fig. 7I). Oatp1b2 immunofluorescence amounted 52 ± 3% (P < 0.001) and 29 ± 1% (P < 0.01) in pericentral and periportal hepatocytes 24 h after LPS treatment, respectively (Fig. 7J). Mrp4 immunofluorescence after LPS treatment did not differ from controls (data not shown). Treating animals with a reduced dose of LPS (2 mg/kg) did not change the zonation pattern of the transporters investigated (Fig. 7, F–J).

Periportal downregulation of Bsep in obstructive cholestasis is reversed by TNF-{alpha} and IL-1β blockade. To investigate the mechanism underlying zonal transporter regulation, rats undergoing BDL were treated with the TNF-{alpha} antagonist etanercept or with the IL-1β antagonist anakinra. Following 3 days of BDL relative hepatic mRNA expression levels in animals treated with etanercept were 113 ± 9% for Bsep, 90 ± 42% for Mrp4, 40 ± 14% for Ntcp, 22 ± 11% for Oatp1a1 (P < 0.05 vs. controls), 63 ± 30% for Oatp1a4, and 54 ± 13% for Oatp1b2 (P < 0.05 vs. controls, Fig. 1C). Relative hepatic mRNA expression levels in animals treated with anakinra were 85 ± 49% for Bsep, 48 ± 39% for Mrp4, 33 ± 16% for Ntcp, 36 ± 16% for Oatp1a1 (P < 0.05 vs. controls), 87 ± 34% for Oatp1a4, and 36 ± 6% for Oatp1b2 (P < 0.05 vs. controls, Fig. 1C). In animals cholestatic for 7 days treated with etanercept or anakinra, mRNA of the transporters investigated was detectable but could not be quantified owing to regulation of several housekeeping genes (beta glucuronidase, Hprt). Relative protein expression levels of bile duct-ligated animals treated with etanercept for 7 days were 83 ± 5% for Bsep (n.s.), 22 ± 10% for Ntcp (P < 0.05 vs. controls), 48 ± 9% for Oatp1a1 (P < 0.05 vs. controls), 129 ± 16% for Oatp1a4 (n.s.), and 95 ± 13% for Oatp1b2 (n.s.) (Figs. 8, A and B). Cholestatic rats treated with anakinra for 7 days showed relative protein expression of 110 ± 9% for Bsep (n.s.), 11 ± 2% for Ntcp (P < 0.05 vs. controls), 54 ± 13% for Oatp1a1 (P < 0.05 vs. controls), 91 ± 6% for Oatp1a4 (n.s.), and 99 ± 10% for Oatp1b2 (n.s.) (Fig. 8, A and B). In bile duct-ligated rats treated with etanercept or anakinra downregulation of Bsep in periportal hepatocytes was largely abolished. Relative peak immunofluorescence in periportal hepatocytes amounted to 96 ± 5 and 99 ± 3% of the immunofluorescence in pericentral hepatocytes of controls (etanercept: <0.001 compared with BDL, P = 0.30 compared with controls; anakinra: P < 0.001 compared with BDL, P = 0.56 compared with controls, Figs. 3, J–L, and 8C). Periportal downregulation of Ntcp was not inhibited by etanercept or anakinra treatment (Fig. 8D). TNF-{alpha} or IL-1β blockade had no effect on the downregulation of Oatp1a1 (Fig. 8E), on the induction of Oatp1a4 in pericentral glutamine synthetase-positive and periportal hepatocytes (Fig. 8F), or on the induction of Oatp1b2 in periportal hepatocytes (Fig. 8G). Mrp4 zonal induction was not affected by TNF-{alpha} or IL-1β blockade (data not shown).


Figure 8
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  		Fig. 8. Periportal downregulation of Bsep in obstructive cholestasis is inhibited by TNF-{alpha} and IL-1β blockade. Rats were subjected to BDL and TNF-{alpha} blockade by etanercept (Eta) or IL-1β blockade by anakinra (Ana). Crude membrane-containing liver homogenates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with respective antibodies. A: representative immunoblots of Bsep, Ntcp, Oatp1a1, Oatp1a4, and Oatp1b2 after BDL and etanercept or anakinra treatment. B: densitometric analysis of immunoblots. Peak immunofluorescence signals of Bsep, Ntcp, Oatp1a1, Oatp1a4, and Oatp1b2 in periportal and pericentral hepatocytes were studied by densitometry. Data are expressed as the relative peak immunofluorescence compared with pericentral hepatocytes of controls (C–G). Data represent means ± SE of 4 animals per group (*P < 0.05, **P < 0.01, ***P < 0.001 compared with sham-treated animals).

 
Periportal downregulation of Bsep in obstructive cholestasis is associated with periportal infiltration of neutrophils and polymorphonuclear cells and induction of IL-1β. TNF-{alpha} and IL-1β are known to be crucial factors of the inflammatory response following LPS and are capable of downregulating Bsep in rat liver (18). We therefore hypothesized that the zonal pattern of Bsep downregulation in obstructive cholestasis was due to local secretion of proinflammatory cytokines by inflammatory cells proliferating periportally. In LPS-treated and bile duct-ligated cholestatic rat liver the acinar distribution of Kupffer cells was preserved, showing a preponderance in the periportal area (Fig. 9, B and C). Proliferation of neutrophils and polymorphonuclear cells was largely confined to the periportal fields in obstructive cholestasis, whereas it did not show a zonal pattern following LPS treatment (Fig. 9, A–C). Similarly, induction of IL-1β was predominantly found in the periportal area in obstructive cholestasis (Fig. 9, G and I). LPS treatment resulted in a homogeneous staining for IL-1β throughout the liver acinus (Fig. 9H). Unfortunately, there was no antibody suitable for TNF-{alpha} immunofluorescence in rat liver.


Figure 9
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  		Fig. 9. Periportal downregulation of Bsep in obstructive cholestasis is associated with periportal proliferation of neutrophils and polymorphonuclear cells and induction of IL-1β. Cryosections were double stained with polyclonal antibodies against Kupffer cells, myeloperoxidase-positive cells (neutrophils and polymorphonuclear cells), or IL-1β and with a monoclonal antibody detecting GS as marker for pericentral hepatocytes. Representative images of 4 livers per group are shown. In normal rat liver Kupffer cells showed a preponderance in the periportal area (A) and virtually no myeloperoxidase-positive cells were detected (D). The pattern of Kupffer cell proliferation in LPS-treated rat liver and in obstructive cholestasis was characterized by a preponderance in periportal hepatocytes (B–C). In LPS-treated rat liver the number of neutrophils and polymorphonuclear cells (MPO-positive cells) evenly increased in the periportal and the pericentral area (E), whereas there was predominantly portal proliferation of MPO-positive cells in obstructive cholestasis (F). There was an even distribution of IL-1β in LPS-treated rat liver (H). In obstructive cholestasis IL-1β was predominantly found in the periportal area (I). Bar = 100 µm.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The inverse regulation of Mrp2 and 3 in cholestasis provided the first evidence of a heterogeneous response of different hepatocyte populations to cholestatic stimuli (12, 13, 37, 46). In the present study we found that a number of other bile salt and organic anion transporters undergo coordinated zonation. Zonal regulation was, in part, dependent on proinflammatory cytokines.

The expression of Bsep was reported to be relatively maintained in obstructive cholestasis, thereby preserving a route of additional hepatocellular bile salt export (33). Bile salt feeding even induced hepatocellular Bsep expression in rodent liver and was mediated by farnesoid X receptor (FXR) activation (54). Our data suggest the existence of two compartments with different regulation of Bsep. The periportal hepatocyte population largely looses canalicular Bsep expression. The presence of Bsep-positive intracellular vesicles in periportal hepatocytes and the stable mRNA expression suggested that this regulation may in part be posttranscriptional and mediated by endocytic retrieval as also shown for hyperosmolality (45) and oxidative stress (38). The quantity of Bsep downregulation in Western blot may be underestimated, since analysis of crude membrane preparations used in this and other papers (33) includes the endosomal compartment. Retrieved transporter molecules may still be contained in this preparation. Downregulation of Bsep was previously shown following LPS (35), TNF-{alpha}, and IL-1β treatment (18). We suggested that these proinflammatory cytokines also released in obstructive cholestasis (24) could mediate selective periportal downregulation. Indeed, inhibition experiments with etanercept and anakinra could largely restore the canalicular Bsep staining. The negative effects of TNF-{alpha} and IL-1β have been shown to be mediated by decreased binding of the heterodimer farnesoid X receptor:retinoid X receptor (FXR:RXR) to the IR-1 element of the Bsep promoter (18). The current study suggests that TNF-{alpha} and IL-1β counteract bile salt-mediated FXR activation and Bsep expression in cholestatic periportal hepatocytes. Furthermore, downregulation of Bsep by TNF-{alpha} and IL-1β was shown to be regulated by posttranscriptional mechanisms (16), underlining our hypothesis that zonal downregulation may, in part, be due to endocytic retrieval. Induction of Mrp4 in periportal and pericentral hepatocytes occurred to a similar extent and was not dependent on TNF-{alpha} or IL-1β. Transcriptional activation of the Mrp4 promoter by the constitutive androstane receptor (2) makes it likely that Mrp4 upregulation in obstructive cholestasis is governed by this bilirubin-activated nuclear receptor.

Similar to Bsep, downregulation of Ntcp mainly occurred in periportal hepatocytes. Although there was significant decrease in mRNA and protein expression, the appearance of cytoplasmic Ntcp-positive vesicles in periportal hepatocytes suggested that also this regulation could, in part, be posttranscriptional. Because TNF-{alpha} and IL-1β blockade had no effect on the downregulation of Ntcp (17) and Ntcp repression was abrogated in FXR–/– mice in obstructive cholestasis (55), it was assumed that Ntcp regulation was mediated by bile salt-induced activation of FXR and subsequent repression of the Ntcp promoter by reduced binding of HNF-1{alpha} (17). In line with these data, TNF-{alpha} and IL-1β blockade in our study did not change the zonal pattern of Ntcp downregulation. The steep periportal downregulation may be explained by the fact that bile salt concentrations are highest in the portal blood passing zone 1 hepatocytes. Persistent Bsep expression and enhanced basolateral bile salt export by induction of Mrp3 (12, 46), Mrp4 (10), and Ost{alpha}/β (5) may be sufficient to prevent FXR-dependent Ntcp downregulation in perivenous hepatocytes. It would be interesting whether FXR activation differs in periportal and pericentral hepatocytes.

Unlike Oatp1a1, Oatp1a4 mRNA in mice was reported to be unchanged or even induced in obstructive cholestasis (48, 49). In the present study we show that Oatp1a4 is induced in cells with a low intrinsic expression (i.e., in glutamine synthetase-positive pericentral hepatocytes and periportal hepatocytes), resulting in an even acinar expression pattern in cholestatic rat liver. Induction in these cell populations was not evident from analyzing mRNA or protein expression in whole liver. Oatp1a4 expression is governed by the activity of pregnane X receptor (PXR). Treating rats with PXR activators such as phenobarbital or pregnenolone-16{alpha}-carbonitrile induced Oatp1a4 (19, 25, 39). PXR activation is also accomplished by lithocholic acid (47), which is accumulated in obstructive cholestasis. Also, feeding of rats with cholic acid resulted in upregulation of Oatp1a4 (43). These data and the fact that Oatp1a4 mRNA induction is abrogated in cholestatic PXR–/– mice make it conceivable that zonal upregulation of this isoform is mediated by PXR. Because Oatp1a4 expression and zonation was not affected by TNF-{alpha} and IL-1β blockade, it is concluded that downregulation of Oatp1a4 by inflammatory cytokines is overruled by the action of PXR on the Oatp1a4 promoter in obstructive cholestasis.

Similarly, Oatp1b2 was induced in periportal hepatocytes of cholestatic rat liver. Less is known about the control of Oatp1b2 transcription. This Oatp isoform was found to be downregulated in liver after feeding of rats with cholic acid (43), virtually excluding bile salts as inductors. It is suggested that a yet-unknown factor mediates the selective periportal induction of Oatp1b2.

There are two possible explanations for the zonal induction of these two Oatp isoforms. Firstly, direction of transport could be reversed under cholestatic conditions. In vitro experiments with Oatp1a4-transfected Xenopus oocytes already suggested that Oatp1a4 could function as an exporter, as shown for glutathione conjugates (34). Compensatory basolateral export of cholephiles by these Oatp isoforms would aid in minimizing cholestatic injury in hepatocytes virtually devoid of Bsep and Mrp2. Secondly, increased expression on zone 1 hepatocytes could serve to increase uptake capacity for bile salt cycling at the basolateral hepatocyte membrane. Bile salts exported basolaterally by Mrp3 and 4 could be taken up to undergo several hepatocellular passages. This may increase residence time and facilitate increased hydroxylation and conjugation making bile salts more amendable for alternative renal excretion in cholestasis.

In conclusion, bile salt and organic anion transporter undergo dynamic zonal changes in obstructive cholestasis. The zonal regulation of Ntcp and Oatp1a4 could be due to an acinar bile salt gradient with peak bile salt concentrations in zone 1. Bile salts may trigger a proinflammatory response in the liver (9) with subsequent periportal secretion of IL-1β and possibly TNF-{alpha} governing local downregulation of Bsep. Zonal regulation of transporters did not change with different doses of LPS, suggesting that indeed the pattern of cytokine secretion (pericentral vs. panacinar) seems to be responsible for the different zonation pattern of Bsep in the two models investigated. In contrast to the uniform transporter regulation following LPS treatment, obstructive cholestasis results in two different hepatocellular compartments with a periportal hepatocyte population exhibiting a cholestatic expression pattern and a perivenous hepatocyte population with a virtually normal expression pattern. This leads to a shift of bile formation to zone 3 and may cause periportal hepatocytes to promote mechanisms of alternative basolateral excretion of cholephiles. The "cholestatic phenotype" of periportal hepatocytes may ameliorate cholangiocyte injury but may promote hepatocyte injury in this cell population. These data further demonstrate that acinar distribution of hepatobiliary transporters should always be analyzed not to miss or misinterpret important means of transporter regulation taking place in different hepatocyte populations.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Forschungskommission of the Medical Faculty, University of Düsseldorf.


   ACKNOWLEDGMENTS
 
The technical assistance of Daniela Stapelkamp and Nicole Eichhorst is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. G. Donner, Dept. of Gastroenterology, Hepatology and Infectious Diseases, Heinrich Heine Univ. Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf, Germany (e-mail: Markus_Donner{at}yahoo.de)

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.


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 REFERENCES
 

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