Endotoxin increases paracellular permeability of isolated rat hepatocyte couplets

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  • Hepatology Research 20 (2001) 144154

    Endotoxin increases paracellular permeability ofisolated rat hepatocyte couplets

    Takumi Kawaguchi a, Shotaro Sakisaka a, Masaru Harada a,*,Shinichiro Hanada a, Eitaro Taniguchi a, Hironori Koga a,

    Kurumi Sasatomi a, Kyuichi Tanikawa b, Michio Sata aa Second Department of Medicine, Kurume Uni6ersity School of Medicine, 67 Asahi-machi,

    Kurume 830-0011, Japanb International Institute for Li6er Research, Kurume Research Center, Kurume, Japan

    Received 2 June 2000; received in revised form 21 September 2000; accepted 2 October 2000


    Hyperbilirubinemia is frequently associated with endotoxemia. Regurgitation of bileconstituents including bilirubin into the sinusoidal space is prevented by tight junctionswhich maintain paracellular permeability between hepatocytes. To investigate the mechanismof endotoxin-associated hyperbilirubinemia, we have studied the changes in paracellularpermeability of primary hepatocyte couplets treated with endotoxin. In addition, we exam-ined the effects of ursodeoxycholic acid (UDCA), which has been widely used for variousliver diseases, on endotoxin-associated changes in paracellular permeability. The paracellularpermeability of hepatocyte couplets was evaluated by paracellular penetration of fluoresceinisothiocyanate (FITC)-dextran with molecular weights of 3, 10 and 70K using confocal laserscanning microscopy. Endotoxin increased the paracellular penetration of FITC-dextran 3and 10K. These changes were prevented by treatment with UDCA. There was littleparacellular penetration of FITC-dextran 70K under any conditions. These results suggestedthat endotoxin increased the paracellular permeability of hepatocyte couplets and thesechanges were prevented by treatment with UDCA. Furthermore, bile regurgitation throughthe paracellular route is involved in endotoxin-associated hyperbilirubinemia, and UDCAmight be a potential therapeutic agent for endotoxin-associated hyperbilirubinemia. 2001Elsevier Science Ireland Ltd. All rights reserved.


    * Corresponding author. Tel.: 81-942-317561; fax: 81-942-342623.E-mail address: harada@med.kurume-u.ac.jp (M. Harada).

    1386-6346:01:$ - see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved.

    PII: S1386 -6346 (00 )00129 -7

  • T. Kawaguchi et al. : Hepatology Research 20 (2001) 144154 145

    Keywords: Endotoxin; Isolated rat hepatocyte couplets; Paracellular permeability; Ursodeoxycholic acid.

    1. Introduction

    Hyperbilirubinemia is frequently seen in patients with endotoxemia [1,2]. Variousfactors such as the impaired hepatocyte transport of organic anions [35] areinvolved in cholestasis. Recently, we demonstrated that endotoxin-associatedcholestasis is caused by change in structure and function of hepatocyte tightjunctions, which regulate the paracellular permeability of hepatocytes, in experi-mental rat model [5].

    Multidrug resistance protein 2 (mrp2) is a canalicular multispecific organic aniontransport protein and excretes conjugated bilirubin into the bile canaliculi [6].Endotoxin-induced cholestasis involves a retrieval of mrp2 from the canalicularmembrane to subcanalicular regions and a down-regulation of mrp2 expression [7].Thus, impaired function of mrp2 is one of the causative factors of hyperbilirubine-mia [8].

    The paracellular permeability of hepatocytes is regulated by tight junctions, theonly intercellular barrier between the sinusoidal and the canalicular spaces [5,9].Increased paracellular permeability of hepatocytes allows paracellular reflux of bileconstituents including conjugated bilirubin into the sinusoidal spaces [3]. In sometypes of hyperbilirubinemia, the paracellular permeability of hepatocytes is in-creased [5,9], however, little is known about the paracellular permeability ofhepatocytes in endotoxin-induced hyperbilirubinemia.

    Ursodeoxycholic acid (UDCA) is used to treat some types of cholestasis [10].Although effects of UDCA on hepatocyte function have been reported [10,11],those on the paracellular permeability of hepatocytes have never been evaluated.

    The goals of this study are to investigate the effect of endotoxin on paracellularpermeability and to examine the effect of UDCA on endotoxin-induced changes ofparacellular permeability in isolated rat hepatocyte couplets, which possess cellpolarity and function in bile formation.

    2. Materials and methods

    2.1. Materials

    All reagents were purchased from Wako Pure Chemical Industries (Osaka,Japan) unless otherwise indicated.

    2.2. Animals

    Male Wistar rats, each weighing 250 g were maintained on a standard diet andwater ad libitum. All rat experiments were conducted in accordance with the USNational Institutes of Health Guidelines for the Care and Use of Laboratory

  • T. Kawaguchi et al. : Hepatology Research 20 (2001) 144154146

    Animals and were approved by the Kurume University Institutional Animal Careand Use Committee. Five rats were used in each experiment.

    2.3. Preparation of isolated rat hepatocyte couplets

    Isolated rat primary hepatocyte couplets were obtained from rat livers by thecollagenase perfusion technique [12] with some modification. The liver was firstpre-perfused with Ca2-free Hanks solution supplemented with 5 mM EDTA and5 mM glucose at 30 ml:min for three different times; 5, 10 and 15 min, followed by0.0125% collagenase solution. After 7 min of collagenase perfusion at 30 ml:min,each liver was excised, dispersed in Hanks solution, and the resulting cell suspen-sion was filtered through 300-gauge mesh. Cells (5105) were seeded into 35-mmculture dishes (Becton Dickinson and Company, NJ), and were grown in Williamsmedium E (Flow Laboratories, Irvine, Scotland) with 0.1 mg:ml streptomycin(Streptomycin Sulfate, Meiji Seika Kaisya, Tokyo, Japan), 100 IU:ml penicillin(Crystalline Penicillin G Meiji, Meiji Seika Kaisya), and 10% fetal calf serum. Cellswere incubated at 37C. Four hours after plating, cell viability examined by trypanblue exclusion exceeded 95%.

    2.4. Cell culture

    Isolated rat hepatocyte couplets, pre-perfused for 5 min, were used for evaluationin effects of endotoxin, endotoxin and anti-rat TNF-a antibody, or endotoxin andUDCA administrations on paracellular permeability. After 2 h incubation, endo-toxin (lipopolysaccharide from Escherichia coli 0111: B4, 100 mg:ml; Sigma, St.Louis, MO) alone, endotoxin and anti-rat TNF-a antibody (4 mg:ml; Genzyme,Cambridge, MA), or endotoxin and UDCA (50 mg:ml) were added. After 2 hincubation under each of four conditions, cell viability examined by trypan blueexclusion exceeded 95% and paracellular permeability of hepatocyte couplets wasexamined by paracellular penetration assay [13]. In addition, the medium level oflactate dehydrogenase (LDH) was measured in each condition.

    2.5. Measurement of paracellular permeability of hepatocyte couplets

    Isolated rat hepatocyte couplets with expanded canalicular spaces were identifieda priori. Then, each medium was replaced with medium containing fluoresceinisothiocyanate (FITC)-dextran with a molecular weight of 3, 10 or 70K (0.5 mg:ml;Molecular Probe, Eugene, OR). Paracellular penetration of FITC-dextran into thebile canalicular space was examined in 3 min using a confocal laser-scanningmicroscope (LSM-GB200; Olympus, Tokyo, Japan). When hepatocyte coupletsmaintained paracellular permeability, the canalicular space was absent with FITC-dextran (Fig. 1A). FITC-dextran was seen within the canalicular space, when theparacellular permeability of hepatocyte couplets increased (Fig. 1C). The rate ofcanalicular accumulation of FITC-dextran was expressed as the percentage ofhepatocyte couplets, having canalicular accumulation of FITC-dextran, of the total

  • T. Kawaguchi et al. : Hepatology Research 20 (2001) 144154 147

    counted hepatocyte couplets. Fifty hepatocyte couplets from five rats were analyzedin each experiment. The gain level was adjusted to an undetectable level ofautofluorescence of hepatocytes.

    2.6. Statistical Analysis

    All data were expressed as the mean9S.E. Comparisons among multiple groupswere analyzed using the KruskalWallis analysis of variance. Differences betweentwo groups were analyzed by the MannWhitney U test. A P value B0.05 wasconsidered statistically significant.

    3. Results

    3.1. Effect of pre-perfusion time on paracellular permeability of isolated rathepatocyte couplets

    Canalicular accumulation rates of FITC-dextran 3 (Fig. 2A) and 10K (Fig. 2B)

    Fig. 1. Representative photograph of maintained and increased paracellular permeability in isolated rathepatocyte couplets incubated with FITC-dextran for 3 min. (A) FITC-dextran was absent in the bilecanaliculus, when paracellular permeability was maintained. (B) Transmission image of the couplet seenin (A). (C) FITC-dextran was present in the bile canaliculus (arrow), when paracellular permeabilityincreased. (D) Transmission image of the couplet seen in (C).

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    Fig. 2. Effect of pre-perfusion time on paracellular permeability of hepatocyte couplets. (A) Paracellularpermeability to FITC-dextran 3K. (B) Paracellular permeability to FITC-dextran 10K. (C) Paracellularpermeability to FITC-dextran 70K. Values are expressed as mean9S.E. (n50 hepatocyte coupletsfrom five rats for each group). 5 min, 5 min pre-perfused hepatocyte couplets; 10 min, 10 minpre-perfused hepatocyte couplets; 15 min, 15 min pre-perfused hepatocyte couplets. * PB0.01.

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    were significantly increased in 10 and 15 min compared with 5 min pre-perfusedhepatocyte couplets. The canalicular accumulation rate of FITC-dextran 70K wasless than 10% in an


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