Basic Research Open Access
Copyright ©2006 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Apr 28, 2006; 12(16): 2536-2548
Published online Apr 28, 2006. doi: 10.3748/wjg.v12.i16.2536
A comparison of gene expression in mouse liver and kidney in obstructive cholestasis utilizing high-density oligonucleotide microarray technology
Gerald U Denk, Shi-Ying Cai, Wen-Sheng Chen, Aiping Lin, Carol J Soroka, James L Boyer
Gerald U Denk, Shi-Ying Cai, Wen-Sheng Chen, Carol J Soroka, James L Boyer, Liver Center, Yale University School of Medicine, New Haven, Connecticut, United States
Aiping Lin, W. M. Keck Biotechnology Resource Laboratory, Yale University School of Medicine, New Haven, Connecticut, United States
Gerald U Denk, Department of Medicine II-Großhadern, Ludwig-Maximilians University, München, Germany
Wen-Sheng Chen, Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Co-first-author: Gerald U Denk and Shi-Ying Cai
Supported by the USPHS grants DK 25636 (J. L. B.), the Yale Liver Center Cellular and Molecular Physiology and Morphology Cores (P30-34989), and the Deutsche Forschungsgemeinschaft Grant DE 872/1-1 (G. U. D.)
Correspondence to: James L Boyer, MD, Ensign Professor of Medicine, Director, Liver Center, Yale University School of Medicine, PO Box 208019, 333 Cedar Street, 1080 LMP, New Haven, Connecticut 06520-8019, United States. james.boyer@yale.edu
Telephone: +1-203-7855279 Fax: +1-203-7857273
Received: June 29, 2005
Revised: December 1, 2005
Accepted: December 7, 2005
Published online: April 28, 2006

Abstract

AIM: To assess the effects of obstructive cholestasis on a wider range of gene expression using microarray technology.

METHODS: Male C57BL/6J mice underwent common bile duct ligation (BDL) and were matched with pair-fed sham-operated controls. After 7 d, the animals were sacrificed and total RNA was isolated from livers and kidneys. Equal amounts of RNA from each tissue were pooled for each group and hybridized to Affymetrix GeneChip®MG-U74Av2 containing a total of 12 488 probe sets. Data analysis was performed using GeneSpring®6.0 software. Northern analysis and immunofluorescence were used for validation.

RESULTS: In sham-operated and BDL mice, 44 and 50% of 12 488 genes were expressed in livers, whereas 49 and 51% were expressed in kidneys, respectively. Seven days after BDL, 265 liver and 112 kidney genes with GeneOntology annotation were up-regulated and 113 liver and 36 kidney genes were down-regulated in comparison with sham-operated controls. Many genes were commonly regulated in both tissues and metabolism-related genes represented the largest functional group.

CONCLUSION: Following BDL, microarray analysis reveals a broad range of gene alterations in both liver and kidney.

Key Words: Bile duct ligation, Cholestasis, Kidney, Liver, Microarray



INTRODUCTION

Cholestasis, defined as impairment of bile secretion, is a feature of many hepatic disorders and systemic diseases. The recent cloning and functional characterization of different transport proteins for bile acids, organic anions and cations in hepatocytes and cholangiocytes have provided new insights into the molecular biology and physiology of bile formation and have increased understanding of the pathophysiology of cholestatic disorders[1]. Thus it is now established that a number of transport proteins in the basolateral and canalicular hepatocyte membrane undergo adaptive regulation in response to cholestatic liver injury to minimize the hepatic accumulation of toxic substances, such as hydrophobic bile acids[2-4]. Previous studies have indicated that in addition to the liver, adaptive regulation of these transporters in cholestasis also occurs in extrahepatic tissues, including the kidney[5] and the intestine[6]. Other alterations in cholestasis affect hepatic signal transduction[7,8], vesicular transport[7], apoptosis[9,10], metabolism[11], and the structure of the extracellular matrix[12,13].

Given the wide range of signaling, regulatory, and metabolic pathways, structural elements, and transport proteins which may be affected in cholestasis, much further research will be necessary to more fully understand the extent of these adaptations. High-density DNA microarrays containing thousands of DNA fragments and oligonucleotides are a potentially promising approach to identify additional genes of interest that play a role in this pathophysiologic process. Based on their ability to monitor large numbers of genes at a time, high-density DNA microarrays are a sensitive, time-saving, and efficient tool in determining gene expression and finding regulatory pathways[14].

In the present study, we, therefore, have utilized high-density oligonucleotide microarray technology to screen for gene alterations in the liver and kidney following bile duct ligation (BDL) in mice, an established model of obstructive cholestasis. This study has allowed a comprehensive gene expression profile to be obtained in cholestatic mouse liver and kidney as well as it has highlighted a number of genes whose expression is particularly altered by this process.

MATERIALS AND METHODS
Animals and animal treatment

Male C57BL/6J mice (8-12-wk-old) purchased from Jackson Lab (Bar Harbor, ME) underwent BDL or sham-surgery as previously described[15]. The common bile duct was identified, ligated twice close to the liver hilum immediately below the cystic duct, and then divided between the ligatures. Control mice underwent sham-surgery in which the common bile duct was exposed but not ligated. Since sham-operated mice tend to consume more food than BDL mice and the expression of some genes may be affected by caloric intake, food intake of BDL mice was monitored daily and sham-operated mice were pair-fed so as to receive the same amount of food as BDL mice. Animals were sacrificed 7 d after surgery and livers and kidneys were harvested. The protocol was approved by the Yale Animal Care and Use Committee, and the animals received humane care as outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH publication 86-23, revised 1985).

Isolation of total RNA

Blood-free livers and kidneys were homogenized in GTC solution containing 4 mol/L guanidinium thiocyanate, 25 mmol/L Na-citrate, and 5 g/L N-lauroylsarcosine and subjected to CsCl gradient centrifugation. The recovered total RNA was further purified by phenol/chloroform extraction and ethanol precipitation. The RNA concentration was determined spectrophotometrically and the RNA quality was confirmed by formaldehyde-agarose gel electrophoresis. Equal amounts of liver and kidney, respectively, total RNA from each of four BDL and four sham-operated mice were pooled to minimize inter-animal variations and used for biotin-labeling.

DNA microarray hybridization and analysis

The biotin-labeled RNA from the different groups was hybridized with two replicates for each condition to individual high-density oligonucleotide microarray chips (GeneChip®MG-U74Av2) from Affymetrix (Santa Clara, CA) containing a total of 12 488 probe sets. Microarray expression data were generated with Affymetrix Microarray Suite 5.0 software and further analysis was carried out with GeneSpring®6.0 (Silicon Genetics, Redwood City, CA). Raw intensity values from each chip were normalized to the 50th percentile of the measurements taken from that chip to reduce chip-wide variations in intensity. Each gene was normalized to the average measurement of that gene in the respective paired controls to enable comparison of relative changes in gene expression levels between different conditions. Cross-gene error model was active based on the replicates. Comparisons of gene expression data were made between BDL and sham-operated mice. Signal and detection flag from Microarray Suite 5.0 were used as quality controls. Only genes with a minimum signal intensity of 600, a detection flag present in both replicates in at least one of the comparison conditions, and a two-fold and above change in gene expression were used for further analysis. For identification of differentially expressed genes in the different groups, a one-sample t-test with a P value cutoff of 0.05 was performed to determine if the average log of the ratio of the replicates was significantly different from 1.0, which was the value of the control samples after normalization. Finally, genes were categorized into GeneOntology (GO) and annotated using NetAffx®, an analysis web interface from Affymetrix.

Northern analysis

To validate alterations in gene expression on the microarray, changes in the expression of selected genes were confirmed in aliquots of the same RNA samples used for the microarray by Northern analysis as previously described[16]. The following primers were used for the generation of specific probes: cytochrome P450 7b1 (GenBank accession number U36993): 5’-GAATCTCAGCTTAGAGAGTAAGAG-3’ (sense), 5’-TTTGTACCTAAAGGAGACGGCAG-3’ (antisense); organic cation transporter 1 (Oct1) (GenBank accession number U38652): 5’-GCAGCCTGCCTCCTCATGATC-3’ (sense), 5’-GGTAAATCGTGTTTTCTTTGGCC-3’ (antisense); similar to putative integral membrane transport protein (GenBank accession number AI647632): 5’-TGATTACAAGAAATGTCAAGCAGG-3’ (sense), 5’-CCTCTTCCTGACTCCATCCATG-3’ (antisense).

Immunofluorescence

Indirect immunofluorescence with a polyclonal antibody against Oct1[17,18] (dilution 1:100; kindly provided by Prof. Dr. H. Koepsell, Würzburg, Germany) was conducted on liver specimens from sham-operated and BDL mice as previously described[19].

RESULTS
Gene expression profile in mouse liver in obstructive cholestasis

Of the total of 12 488 genes on the microarray chip, 44 and 50% were expressed in the livers of sham-operated and BDL mice, respectively. After 7 d of obstructive cholestasis 265 genes with GO annotation were up-regulated and 113 were down-regulated in livers of BDL mice by a factor of two or more in comparison with sham-operated pair-fed controls. Metabolism-related genes represented the largest functional group among the altered genes after BDL in liver (Table 1). It should be noted that the grouping of the altered genes was primarily done to achieve a clearer arrangement for the reader. Since a considerable number of the encoded proteins have multiple, little characterized or even unknown functions, we want to point out that the classification provided is subject to the personal opinions and emphasis of the authors (Table 1). Upon request, a complete list of the altered genes including genes without GO annotation that are not mentioned here can be obtained from the authors. Alternatively, the complete list of altered genes can be accessed via http://livercenter.yale.edu/datalist.html.

Table 1 Fold increase/decrease in liver and kidney, GenBank accession number, and classification of altered genes in mice 7 d after bile duct ligation in comparison with pair-fed sham-operated controls.
LiverKidneyAccession numberDescription
Cell death
3.6AF011428CD5 antigen-like
3.2AW046181Serum/glucocorticoid regulated kinase
2.6AV373612Bcl2-associated athanogene 3
-2.6X65128Growth arrest specific 1
-2.7AA770736Induced in fatty liver dystrophy 2
2.8M61737Fat-specific gene 27
2.7AV003873Clusterin
2.3D14077Clusterin
-7AJ000062Deoxyribonuclease I
Stress response
1036.1X03505Serum amyloid A 3
5M13521Serum amyloid A 2
2.8M12566Orosomucoid 2
2.5J04633Heat shock protein 1, alpha
2.5X60676Serine (or cysteine) proteinase inhibitor, clade H, member 1
7.4M96827Haptoglobin
-2.2Z36774Serine (or cysteine) proteinase inhibitor, clade F, member 2
Immune and inflammatory response
31.95M94584Chitinase 3-like 3
17.3M19681Chemokine (C-C motif) ligand 2
10.7X53798Chemokine (C-X-C motif) ligand 2
9.64.2J04596Chemokine (C-X-C motif) ligand 1
9.4AW120786Chemokine (C-X-C motif) ligand 14
9.2U18424Macrophage receptor with collagenous structure
8.4AV370035Chemokine (C-C motif) receptor 5
7.1U56819Chemokine (C-C) receptor 2
6.95.5AF002719Secretory leukocyte protease inhibitor
6.1M18237Immunoglobulin kappa chain variable 8 (V8)
5.9U34277Phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma)
5.4M83218S100 calcium binding protein A8 (calgranulin A)
3.63.7X04673Adipsin
3.6AI844520Interferon gamma inducible protein 30
3.6AF081789Complement component 1, q subcomponent, receptor 1
3.5X12905Properdin factor, complement
3.3L32838Interleukin 1 receptor antagonist
3.2U96752Histocompatibility 2, Q region locus 1
3.23.4M22531Complement component 1, q subcomponent, beta polypeptide
3.2X15591Cytotoxic T lymphocyte-associated protein 2 alpha
3.1X63782Lymphocyte antigen 6 complex, locus D
2.9M58004Chemokine (C-C motif) ligand 6
2.9M21932Histocompatibility 2, class II antigen A, beta 1
2.9U16985Lymphotoxin B
2.9M14639Interleukin 1 alpha
2.94.2X58861Complement component 1, q subcomponent, alpha polypeptide
2.8U77461Complement component 3a receptor 1
2.8M31314Fc receptor, IgG, high affinity I
2.8X52643Histocompatibility 2, class II antigen A, alpha
2.7AB007599Lymphocyte antigen 86
2.6X15592Cytotoxic T lymphocyte-associated protein 2 beta
2.6AF013715Periplakin
2.62.1X66295Complement component 1, q subcomponent, gamma polypeptide
2.53.1L38444T-cell specific GTPase
2.5AA596710Leukotriene B4 12-hydroxydehydrogenase
2.5AB019505Interleukin 18 binding protein
2.4M34815Chemokine (C-X-C motif) ligand 9
2.42X00496Ia-associated invariant chain
2.42.8AJ007970Guanylate nucleotide binding protein 2
2.3D86382Allograft inflammatory factor 1
2.3L22181Formyl peptide receptor 1
2.2AF038149Paired-Ig-like receptor B
2.1AW060457Immunoglobulin superfamily, member 7
2.1U03003Defensin related cryptdin 6
2M29855Colony stimulating factor 2 receptor, beta 2, low-affinity (granulocyte-macrophage)
2AF003525Defensin beta 1
-2.8M29007Complement component factor h
-4.6L22977X-linked lymphocyte-regulated 3b
13.6U47810Complement component factor i
6.5K02782Complement component 3
6X06454Complement component 4 (within H-2S)
4.2AI563854Tumor-associated calcium signal transducer 2
3.8AA986114T-cell immunoglobulin and mucin domain containing 2
3.5U49513Chemokine (C-C motif) ligand 9
3Y08830Tumor-associated calcium signal transducer 2
2.4AA270365Cytokine receptor-like factor 1
2.2AI152789Sema domain, immunoglobulin domain (Ig), and GPI membrane anchor, (semaphorin) 7A
Signal transduction
12.9U88328Suppressor of cytokine signaling 3
5.4Z48043Coagulation factor II (thrombin) receptor-like 1
52.7M14044Annexin A2
42.2AJ001633Annexin A3
3.6AI641895Shroom
3.6U90715Coxsackievirus and adenovirus receptor
3.6AI317205Mitogen activated protein kinase kinase kinase 1
3.4J03023Hemopoietic cell kinase
3.1AW209098IQ motif containing GTPase activating protein 1
3AW049806RIKEN cDNA 1700093E07 gene
3X84797Hematopoietic cell specific Lyn substrate 1
2.93AB015978Oncostatin M receptor
2.6X93328EGF-like module containing, mucin-like, hormone receptor-like sequence 1
2.3D63423Annexin A5
2.32.1M69260Annexin A1
2.2M68902Hemopoietic cell phosphatase
2.2AF020313Amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein
2.13.6AV374868Suppressor of cytokine signaling 3
2.1AA608387Interleukin 13 receptor, alpha 1
-2AC002397Gene rich cluster, C9 gene
-2AW125649Guanine nucleotide binding protein, alpha 12
-2.4AI839138Thioredoxin interacting protein
-2.6AV321519Sorting nexin 17
-2.7AA691492RIKEN cDNA D530020C15 gene
-5.6D17444Leukemia inhibitory factor receptor
-11.7AV349152Regulator of G-protein signaling 16
-15.3U94828Regulator of G-protein signaling 16
2.3AF084466Ras-related associated with diabetes
2.1AF009246RAS, dexamethasone-induced 1
-2.1AF054623Frizzled homolog 1 (Drosophila)
-2.2D85605Cholecystokinin A receptor
-2.2AI834895Membrane progestin receptor alpha
-2.3AW046638PDZ domain containing 1
Cell growth and maintenance
8.7M33960Serine (or cysteine) proteinase inhibitor, clade E, member 1
6.9X98471Epithelial membrane protein 1
5.84.9X66449S100 calcium binding protein A6 (calcyclin)
5.4AF055638Growth arrest and DNA-damage-inducible 45 gamma
5.1M17298Nerve growth factor, beta
3.6AI849928Cyclin D1
3.5X59846Growth arrest specific 6
3.2M64292B-cell translocation gene 2, anti-proliferative
3.2AW048937Cyclin-dependent kinase inhibitor 1A (P21)
3.1AF009366Neural precursor cell expressed, developmentally down-regulated gene 9
2.7M21019Harvey rat sarcoma oncogene, subgroup R
2.7X06368Colony-stimulating factor 1 receptor
2.2X81579Insulin-like growth factor binding protein 1
2.1AI851454Cysteine rich protein 2
2AA529583Mortality factor 4 like 2
-2.1X95280G0/G1 switch gene 2
-2.2M31680Growth hormone receptor
-2.5U15012Growth hormone receptor
3.4AI852641Nuclear protein 1
2.8M34094Midkine
2.8AF058798Stratifin
2.1X81580Insulin-like growth factor binding protein 2
Protein biosynthesis
2.3Y11460Integrin beta 4 binding protein
2.1NM_011690Valyl-tRNA synthetase 2
-2AV055186Ribosomal protein, large, P1
Proteolysis and protein degradation
7.62.4X61232Carboxypeptidase E
6.1AW060527Ubiquitin-conjugating enzyme E2 variant 2
4AJ000990Legumain
45AJ223208Cathepsin S
3.7AL078630Ubiquitin D
2U35833Ubiquitin-like 1 (sentrin) activating enzyme E1B
-2.2AI844932F-box only protein 8
-2.4L21221Proprotein convertase subtilisin/kexin type 4
-2.6AV359471Ubiquitin specific protease 15
-2.2J04946Angiotensin converting enzyme
-2.5L15193Meprin 1 beta
Protein amino acid phosphorylation and dephosphorylation
3.2D89728Serine/threonine kinase 10
3.2M97590Protein tyrosine phosphatase, non-receptor type 1
2.6D37801Protein tyrosine phosphatase, non-receptor type 21
2X61940Dual specificity phosphatase 1
-2.1L31783Uridine monophosphate kinase
Cell adhesion and extracellular matrix
24.7L36244Matrix metalloproteinase 7
20.6U43525Proteinase 3
10.7M82831Matrix metalloproteinase 12
10.42.3D00613Matrix gamma-carboxyglutamate (gla) protein
93.1X16834Lectin, galactose binding, soluble 3
8.9L02918Procollagen, type V, alpha 2
8.1M31039Integrin beta 2
7.2M62470Thrombospondin 1
6.2X13986Secreted phosphoprotein 1
5.92.4U03419Procollagen, type I, alpha 1
5.8D14010Regenerating islet-derived 1
4.92.1X52046Procollagen, type III, alpha 1
4.72.5M90551Intercellular adhesion molecule
4.2X58251Procollagen, type I, alpha 2
4L57509Discoidin domain receptor family, member 1
3.44.3U12884Vascular cell adhesion molecule 1
3.23.3M84487Vascular cell adhesion molecule 1
3.2L29454Fibrillin 1
3Z22532Syndecan 1
2.9M23552Serum amyloid P-component
2.8X04017Secreted acidic cysteine rich glycoprotein
2.7M38337Milk fat globule-EGF factor 8 protein
2.7AA763466Procollagen, type I, alpha 1
2.5AA919594Elastin
2.5M70642Connective tissue growth factor
2.5D88577C-type (calcium dependent, carbohydrate recognition domain) lectin, superfamily member 13
2.3M15832Procollagen, type IV, alpha 1
2.2X59990Catenin alpha 1
2.2U82624Amyloid beta (A4) precursor protein
2.1X53928Biglycan
2.1U89915F11 receptor
2X04647Procollagen, type IV, alpha 2
22.2V00755Tissue inhibitor of metalloproteinase 1
2X91144Selectin, platelet (p-selectin) ligand
-2.1AF101164CEA-related cell adhesion molecule 2
-2.2AI840501Camello-like 1
2.1L19932Transforming growth factor, beta induced
Cytoskeleton and structural elements
57.3M36120Keratin complex 1, acidic, gene 19
4.8V00830Keratin complex 1, acidic, gene 10
4.5U38967Thymosin, beta 4, X chromosome
3.62.3AI852553Thymosin, beta 10
3.6U42471Wiskott-Aldrich syndrome homolog (human)
3.4U29539Lysosomal-associated protein transmembrane 5
3.4M22479Tropomyosin 1, alpha
3.22.2M28739Tubulin, beta 2
3.2AW215736RIKEN cDNA 2310057H16 gene
3.24.5M22832Keratin complex 1, acidic, gene 18
3.1AI505453Myosin heavy chain IX
3X15662Keratin complex 2, basic, gene 8
2.8X60671Villin 2
2.7D49733Lamin A
2.7AW125446Golgi phosphoprotein 2
2.6AW050256Tubulin, beta 3
2.6AI839417Moesin
2.4AW125698Myosin heavy chain IX
2.4AW212775Actin-related protein 2/3 complex, subunit 1B
2.4AV356071Lysosomal-associated protein transmembrane 5
2.2M28727Tubulin, alpha 2
2.2AI835858Tropomyosin 4
2.2M12347Actin, alpha 1, skeletal muscle
2.1D88793Cysteine and glycine-rich protein 1
2.1AF020185Dynein, cytoplasmic, light chain 1
2.1AI837625Cysteine and glycine-rich protein 1
2.13.2X54511Capping protein (actin filament), gelsolin-like
22.1X04663Tubulin, beta 5
2AI841606Actin-binding LIM protein 1
2M21495Actin, gamma, cytoplasmic
2AI849152Clathrin, light polypeptide (Lcb)
2M60474Myristoylated alanine rich protein kinase C substrate
-2.2AW123904Gamma-aminobutyric acid (GABA(A)) receptor-associated protein-like 1
3.3AB000713Caudin 4
2.6AA755126Keratin complex 2, basic, gene 7
2.6AF087825Claudin 7
2.3AI195392Actinin, alpha 1
Transport
13.166.5X81627zLipocalin 2
12.12L48687Sodium channel, voltage-gated, type I, beta polypeptide
7.2U04827Fatty acid binding protein 7, brain
3.7M24417ATP-binding cassette, sub-family B (MDR/TAP), member 1A
3.4AI842825Glycolipid transfer protein
3.2NM_033444Chloride intracellular channel 1
3.2X99347Lipopolysaccharide binding protein
2.9L13732Solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1
2.8U72680FXYD domain-containing ion transport regulator 5
2.8X60367Retinol binding protein 1, cellular
2.5U27315Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4
2.4AI842065Expressed sequence AW538430
2.3AI849583RIKEN cDNA 6330416G13 gene
2.3AI852578Solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2
2.1D87661Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide
2.1U28960Phospholipid transfer protein
-2-2.2AA670737RIKEN cDNA 1700013L23 gene
-2.1M16360Major urinary protein 5
-2.1AF072757Solute carrier family 27 (fatty acid transporter), member 2
-2.1M16358Major urinary protein 4
-2.1L28836ATP-binding cassette, sub-family D (ALD), member 3
-2.3U38652Solute carrier family 22 (organic cation transporter), member 1
-2.3M16357Major urinary protein 3
-2.3U95131Solute carrier family 10 (sodium/bile acid cotransporter family), member 1
-2.3-2.3AV355798Major urinary protein 2
-2.3AV104178Serine (or cysteine) proteinase inhibitor, clade A, member 6
-2.4U95132Solute carrier family 10 (sodium/bile acid cotransporter family), member 1
-2.4M16359Major urinary protein 1
-2.4AB028737ATP-binding cassette, sub-family C (CFTR/MRP), member 6
-2.6-2D00073Transthyretin
-2.9AJ011080Afamin
-3.8-3.8AI647632Similar to putative integral membrane transport protein
-4.4AI255271Major urinary protein 2
-6.4Y14660Fatty acid binding protein 1, liver
-6.6X70533Serine (or cysteine) proteinase inhibitor, clade A, member 6
4M55413Group-specific component
2.7AF047838Chloride channel calcium activated 1
2.7AI849587Protein distantly related to the gamma subunit family
2.6D00466Apolipoprotein E
2.4AI661431Aquaporin 2
2.3AI197481Amiloride binding protein 1 (amine oxidase, copper-containing)
-2.1AI606956Solute carrier family 2 (facilitated glucose transporter), member 5
-2.1AW122706Solute carrier family 7 (cationic amino acid transporter, y+ system), member 8
-2.2AI120514Solute carrier family 26 (sulfate transporter), member 1
-2.3AI837530Solute carrier family 9 (sodium/hydrogen exchanger), member 8
Cell surface markers and membrane proteins
12.9X13333CD14 antigen
6.7X97227CD53 antigen
6.4M65027Glycoprotein 49 A
5.9D16432CD63 antigen
5.32.3AW209486Prostate stem cell antigen
53.4AF024637TYRO protein tyrosine kinase binding protein
3.6M58661CD24a antigen
3.3U37438Deleted in malignant brain tumors 1
3.3M55561CD52 antigen
3.3AI854863RIKEN cDNA 1200015A22 gene
3AF039663Prominin 1
2.7AI849180Integral membrane protein 2C
2.6AI787183RIKEN cDNA 0610011I04 gene
2.52.5X68273CD68 antigen
2.2AB031386RIKEN cDNA 1810009M01 gene
2L11332CD38 antigen
-2.5AI843959RIKEN cDNA 5730403B10 gene
3.3AW261569RIKEN cDNA D630035O19 gene
2AI847784CD34 antigen
-2.5L23108CD36 antigen
Transcription factors and nucleic acid binding proteins
8.17.2V00727FBJ osteosarcoma oncogene
6.23.6AW124113Brain abundant, membrane attached signal protein 1
4.92.2AW049031Core promoter element binding protein
4M90397B-cell leukemia/lymphoma 3
3.8M31885Inhibitor of DNA binding 1
3.83AA614971Molecule possessing ankyrin-repeats induced by lipopolysaccharide
3.23.6X61800CCAAT/enhancer binding protein (C/EBP), delta
3.2AF017258Ribonuclease, RNase A family, 2
2.7AB016424RNA binding motif protein 3
2.62.4U19118Activating transcription factor 3
2.5AF016294E74-like factor 3
2.4L03215SFFV proviral integration 1
2.3AI642098RIKEN cDNA 4921515A04 gene
2.3U20735Jun-B oncogene
2.2M60523Inhibitor of DNA binding 3
2.2D26089Minichromosome maintenance deficient 4 homolog (S. cerevisiae)
2.12.2U20344Kruppel-like factor 4 (gut)
-2U36799Retinoblastoma-like 2
-2AF038995DEAD (Asp-Glu-Ala-Asp) box polypeptide 6
-2.1L20450Zinc finger protein 97
-2.1X77602Upstream transcription factor 2
-2.2AF064088TGFB inducible early growth response 1
-2.2U95945One cut domain, family member 1
-2.4U62674Histone 2, H2aa1
-2.4AA002843Nuclear factor I/X
-2.7AI834950Nuclear receptor subfamily 1, group D, member 1
-2.8AW047343D site albumin promoter binding protein
-3.4X57638Peroxisome proliferator activated receptor alpha
4.7AI840339Ribonuclease, RNase A family 4
2.7M28845Early growth response 1
2.3X16995Nuclear receptor subfamily 4, group A, member 1
Metabolism
8.5M13018Cysteine-rich protein 1 (intestinal)
6.1AV327760Stearoyl-Coenzyme A desaturase 2
637.5X51547P lysozyme structural
5.9AW046124Cytochrome b-245, alpha polypeptide
5.14.6M21050Lysozyme
4.9X97047Pyruvate kinase, muscle
4.2AV368209Pyruvate kinase, muscle
4.1U43384Cytochrome b-245, beta polypeptide
4.1AA726364Lipoprotein lipase
4AI846517Cytochrome b-561
4AI854821RIKEN cDNA 0610041P13 gene
3.8U13705Glutathione peroxidase 3
3.8U12961NAD(P)H dehydrogenase, quinone 1
3.62.1M26270Stearoyl-Coenzyme A desaturase 2
3.6M34141Prostaglandin-endoperoxide synthase 1
3.6X078883-hydroxy-3-methylglutaryl-Coenzyme A reductase
3.5M31775Cytochrome b-245, alpha polypeptide
3.5AI847162RIKEN cDNA 1300017C10 gene
3.4U87147Flavin containing monooxygenase 3
3.4AA690863ATPase, class VI, type 11A
3.4J04696Glutathione S-transferase, mu 2
3.3X56824Heme oxygenase (decycling) 1
3AJ238894Acyl-Coenzyme A thioesterase 3, mitochondrial
3D42048Squalene epoxidase
2.9AW060927Lanosterol synthase
2.8J03953Glutathione S-transferase, mu 3
2.4U49350Cytidine 5'-triphosphate synthase
2.4AI594518Chitinase, acidic
2.2J02980Alkaline phosphatase 2, liver
2.2U27455Serine palmitoyltransferase, long chain base subunit 2
2.2AI327450Phospholipase A2, group IB, pancreas
2.2AF077527Syndecan binding protein
2.2AA710635Colipase, pancreatic
2.1M627663-hydroxy-3-methylglutaryl-Coenzyme A reductase
2.1AW049778Mevalonate (diphospho) decarboxylase
2.1AF0573687-dehydrocholesterol reductase
2U49385Cytidine 5'-triphosphate synthase 2
-2AW123316Methylcrotonoyl-Coenzyme A carboxylase 1 (alpha)
-2AA824102Hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 7
-2AF098009Fatty acid amide hydrolase
-2.1AV216468Expressed in non-metastatic cells 1, protein
-2.1L42996Dihydrolipoamide branched chain transacylase E2
-2.1AI846934Lipin 1
-2.1AV071102Cytochrome c oxidase, subunit VIc
-2.1AI839995Sarcosine dehydrogenase
-2.1X61397Carbonic anhydrase 8
-2.1AF022894Sulfotransferase family 1B, member 1
-2.1U24493Tryptophan 2,3-dioxygenase
-2.2AA675075Proline dehydrogenase (oxidase) 2
-2.2AV276715Aldehyde dehydrogenase family 3, subfamily A2
-2.3L11333Esterase 31
-2.3L11163Acyl-Coenzyme A dehydrogenase, short chain
-2.3AI840013Peroxisomal delta3, delta2-enoyl-Coenzyme A isomerase
-2.4M27347Elastase 1, pancreatic
-2.4U32684Paraoxonase 1
-2.4M77015Hydroxysteroid dehydrogenase-3, delta<5>-3-beta
-2.4AF030343Enoyl coenzyme A hydratase 1, peroxisomal
-2.4AF047542Cytochrome P450, family 2, subfamily c, polypeptide 37
-2.4AF047727Cytochrome P450, family 2, subfamily c, polypeptide 40
-2.4Z14050Dodecenoyl-Coenzyme A delta isomerase (3,2 trans-enoyl-Coenyme A isomerase)
-2.5D17674Cytochrome P450, family 2, subfamily c, polypeptide 29
-2.5AI8448462,4-dienoyl CoA reductase 1, mitochondrial
-2.6X83202Hydroxysteroid 11-beta dehydrogenase 1
-2.6U14390Aldehyde dehydrogenase family 3, subfamily A2
-2.6AW0125883-ketoacyl-CoA thiolase B
-2.7AI530403Acetyl-Coenzyme A acyltransferase 1
-2.7X51971Carbonic anhydrase 5a, mitochondrial
-2.7AF031170Hydroxysteroid dehydrogenase-6, delta<5>-3-beta
-2.8AI266885RIKEN cDNA 1700124F02 gene
-2.9AF030513Retinol dehydrogenase 6
-3U15977Fatty acid Coenzyme A ligase, long chain 2
-3X04283Cytochrome P450, family 1, subfamily a, polypeptide 2
-3.4X63349Dopachrome tautomerase
-3.6M15268Aminolevulinic acid synthase 2, erythroid
-4D63764Pyruvate kinase liver and red blood cell
-4.1AF026074Sulfotransferase related gene X1
-4.1Y14004Cytosolic acyl-CoA thioesterase 1
-4.3-3.8AV141027Cytochrome P450, family 7, subfamily b, polypeptide 1
-4.3AJ132098Vanin 1
-4.6AW226939Carboxylesterase 3
-5.1U49861Deiodinase, iodothyronine, type I
-6.1-3.4U36993Cytochrome P450, family 7, subfamily b, polypeptide 1
-6.4U127913-hydroxy-3-methylglutaryl-Coenzyme A synthase 2
-6.6-2.8M88694Thioether S-methyltransferase
-6.9AF090317Cytochrome P450, family 8, subfamily b, polypeptide 1
-14AB018421Cytochrome P450, family 4, subfamily a, polypeptide 10
-17.9Y11638Cytochrome P450, family 4, subfamily a, polypeptide 14
-282.5AJ006474Carbonic anhydrase 3
-37.8M21855Cytochrome P450, family 2, subfamily b, polypeptide 9
-93.8L41519Hydroxysteroid dehydrogenase-5, delta<5>-3-beta
6.4AB006034Cytochrome P450, family 27, subfamily b, polypeptide 1
4.8U49430Ceruloplasmin
3AF032466Arginase type II
2.9J05277Hexokinase 1
2.6Z19521Low density lipoprotein receptor
2.6U04204Aldo-keto reductase family 1, member B8
AI848668Sterol-C4-methyl oxidase-like
2.6U31966Carbonyl reductase 1
2.5U49915Adipocyte complement related protein
2.4AW124337Microsomal glutathione S-transferase 1
2.3U18975UDP-N-acetyl-alpha-D-galactosamine:(N-acetylneuraminyl)-galactosylglucosylceramide-beta-1,4-N-acetylgalactosaminyltransferase
2.2L06047Glutathione S-transferase, alpha 4
2.1AA718169Resistin
2.1D88994AMP deaminase 3
2AA710564N-acetylneuraminate pyruvate lyase
-2U19265Glucosaminyl (N-acetyl) transferase 1, core 2
-2AB005450Carbonic anhydrase 14
-2.1M75886Hydroxysteroid dehydrogenase-2, delta<5>-3-beta
-2.1AB020239Adenylate kinase 4
-2.2U48896UDP-glucuronosyltransferase 8
-2.2U89352Lysophospholipase 1
-2.2M12330Ornithine decarboxylase, structural
-2.3U90535Flavin containing monooxygenase 5
-2.3AF009605Phosphoenolpyruvate carboxykinase 1, cytosolic
-2.3AB015426Fucosyltransferase 9
-2.4U89906Alpha-methylacyl-CoA racemase
-2.5AA840463Lysophospholipase 1
-3.5X06358UDP-glucuronosyltransferase 2 family, member 5
Other
18.68.2U69488G7e protein
10.92.4X67644Immediate early response 3
7.6U78770Trefoil factor 2 (spasmolytic protein 1)
2.9AI117936Mus musculus 11 days embryo head cDNA, RIKEN full-length enriched library, clone: 6230409N14 product:unknown EST, full insert sequence
2.7AI852545Transgelin 2
2.62AW121336RIKEN cDNA 1600023A02 gene
2.6X58196H19 fetal liver mRNA
2.52.2U25844Serine (or cysteine) proteinase inhibitor, clade B, member 6a
2.4AA980164SPARC related modular calcium binding 2
2.4D38410Trefoil factor 3, intestinal
2.2U44426Tumor protein D52
2.1U22262Apolipoprotein B editing complex 1
2.14.6AW230891Leucine-rich alpha-2-glycoprotein
-2.1U32170Regucalcin
-2.3AI854813Mus musculus 3 days neonate thymus cDNA, RIKEN full-length enriched library, clone: A630086H07 product:RAS GTPASE-ACTIVATING-LIKE PROTEIN IQGAP2 homolog [Homo sapiens], full insert sequence
-2.3AW049373RIKEN cDNA 2310016A09 gene
-2.8AI326963Angiopoietin-like 4
-3AA797604Angiopoietin-like 4
-3.410.7AB011030Protein related to DAN and cerberus
9.8AA986050Fibrinogen, gamma polypeptide
6.8M64086Serine (or cysteine) proteinase inhibitor, clade A, member 3N
5AA880891Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 10
2.6Fibrinogen, alpha polypeptide
2.1X61597Serine (or cysteine) proteinase inhibitor, clade A, member 3C
2X59520Cholecystokinin
2D13003Reticulocalbin
-2.1AI314227RIKEN cDNA 0610006H10 gene
-2.2AW122036Mus musculus transcribed sequence with strong similarity to protein ref:NP_005351.2(H.sapiens) v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian); v-maf musculoaponeurotic fibrosarcoma (avian) oncogene homolog; Avian musculoaponeurotic fibrosarcoma (MAF) protooncogene [Homo sapiens]
-3.4M93264Pregnancy zone protein
Gene expression profile in mouse kidney in obstructive cholestasis

In the kidneys of sham-operated and BDL mice, 49 and 51% of the 12 488 genes on the microarray chip were expressed, respectively. Seven days after surgery, 112 genes with GO annotation were up-regulated and 36 were down-regulated in the kidneys of BDL mice at least two-fold when compared with the sham-operated pair-fed controls. Thus the number of altered genes in kidney seven days after BDL was considerably smaller than that in liver (148 vs 378). Of the 112 GO genes up-regulated in kidney after BDL, 53 were also up-regulated in cholestatic liver. In contrast, of the 36 genes down-regulated in kidney, 7 were also down-regulated in liver (Table 1). What was particularly striking is that many of the most highly up-regulated genes in liver were also the same genes that were most highly up-regulated in kidney, irrespective of their functional class (Table 1). This suggests that both the liver and the kidney may be responding to similar transcriptional signaling molecules in this cholestatic model. For example, the acute phase gene, serum amyloid A3, was up-regulated 10.0-fold in liver and 36.1-fold in kidney, the gene encoding chemokine (C-X-C motif) ligand 1 was increased 9.6-fold in liver and 4.2-fold in kidney, and the gene encoding the transport molecule lipocalin 2 was up-regulated 13.1-fold in liver and 66.5-fold in kidney. In addition, a number of cell adhesion and extracellular matrix genes were similarly up-regulated in both liver and kidney. However, only one membrane transporter gene was up-regulated in both tissues, the gene encoding the β1 subunit of the voltage-gated sodium channel (Table 1). Interestingly, several genes for nucleic acid binding proteins were also highly up-regulated in both liver and kidney including the genes encoding the transcription factors FBJ osteosarcoma oncogene (alias c-Fos), CCAAT/enhancer binding protein (C/EBP), delta, and activating transcription factor 3.

In contrast, only seven genes were commonly down-regulated in both liver and kidney. These included the RIKEN cDNA 1700013L23 gene and the genes encoding similar to putative integral membrane transport protein, major urinary protein 2, transthyretin, cytochrome P450 7b1 (GenBank accession numbers AV141027 and U36993), and thioether S-methyltransferase.

Northern analysis of selected genes

Gene expression results from the microarray were confirmed by Northern analysis for selected genes that included cytochrome P450 7b1, organic cation transporter 1 (Oct1; solute carrier family 22, member 1) and similar to putative integral membrane transport protein from aliquots of the RNA samples utilized for the microarray (Figure 1).

Figure 1
Figure 1 Confirmation of microarray data by Northern analysis of selected genes. Northern blots were performed with aliquots of pooled RNA from livers and kidneys from each 4 pair-fed sham-operated and 4 bile duct ligated mice, 7 d after surgery. Cyp7b1: Cytochrome P450 7b1; Oct1: Organic cation transporter 1; Spmt: Similar to putative integral membrane transport protein; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; Sham: Sham-surgery; BDL: Bile duct ligation.
Tissue immunofluorescence of Oct1 in liver

Indirect immunofluorescence was performed to illustrate the decreased expression of the organic cation transporter Oct1 in BDL mouse liver. Figures 2A and B demonstrate that the findings are consistent with the microarray and the Northern blot results and corroborate that obstructive cholestasis leads to a down-regulation of Oct1 in mouse liver similarly as demonstrated previously in rat liver following BDL[19,20].

Figure 2
Figure 2 Indirect immunofluorescence of organic cation transporter 1 in murine liver sections. A: A low magnification view (x 20) shows antibody labeling at the basolateral membranes of hepatocytes of the pericentral zone of the liver lobule in the liver section of a sham-operated mouse, 7 d after surgery; B: In contrast, there is only a weak signal for organic cation transporter 1 after bile duct ligation.
DISCUSSION

Ligation of the common bile duct in rodents is a well-established model of obstructive cholestasis. While most previous studies have been limited to investigations of small numbers of genes and their encoded proteins, we have been able to simultaneously monitor the responses of large numbers of genes in this cholestatic model by using high-density oligonucleotide microarray technology. In contrast to a recent study which investigated gene expression in obstructive cholestasis only in the livers of BDL mice[21], we additionally monitored alterations of gene expression in the kidneys because the kidney is functionally closely linked to the liver and provides an alternative excretory route for cholephilic substances in cholestasis[5]. One of the interesting conclusions from this analysis is the finding that many of the most highly up-regulated genes were shared in both liver and kidney, possibly due to a common response to similar transcriptional signaling molecules in both tissues. The interpretation and discussion of our data is based on the assumption that changes in gene expression lead to changes in protein expression although it is known that changes at the mRNA level do not always result in changes in protein expression in certain time periods[22]. As others have done, we first evaluated the observed changes in gene expression in terms of what is already known about the effects of cholestasis. We then attempted to identify novel regulatory processes that have not yet been investigated[22].

For example, our microarray data largely confirm previous results obtained by conventional determination of transcription in obstructive cholestasis, such as the up-regulation of the canalicular cation transporter multidrug resistance P-glycoprotein 1a (Mdr1a, Abcb1a)[23] or the down-regulation of the basolateral sodium-taurocholate cotransporting polypeptide (Ntcp, Slc10a1)[24]. In addition, our gene expression profile obtained from cholestatic liver also closely matched the gene expression profile recently generated by Campbell et al[21], although there are a substantial number of additional gene alterations in our data set. This difference can be explained since Campbell et al[21] excluded genes with expression levels of less than 1 000, whereas we included genes with a mininum signal intensity of 600 and above. This approach led to the identification of a number of novel gene alterations of functional significance for the cholestatic phenotype. For instance, the decrease in expression of the gene encoding Oct1 in BDL liver in the present microarray, an alteration not reported by Campbell et al[21] but previously reported by Ogawa et al[20] in the rat, led us to study this important basolateral cationic drug transporter in more detail. We were subsequently able to demonstrate that Oct1 is indeed down-regulated in rat liver, but not in kidney, in obstructive cholestasis at the mRNA as well as the protein levels and that this decrease results in reduced hepatic uptake of the Oct1 substrate tetraethylammonium[19]. Northern analysis and immunofluorescence microscopy of hepatic Oct1 performed in the present study indicated a similar pattern in mouse and confirmed the results of our microarray.

A number of other observations emerge from this analysis that deserve further study. For example, among the cell growth-related genes, the number of genes up-regulated in liver after BDL surpassed by far the number of down-regulated genes, a pattern which might reflect the extensive fibroproliferative process and tissue remodeling that takes place in this model of obstructive cholestasis. Similarly, a large number of genes related to cell adhesion, the extracellular matrix, and the cytoskeleton were found to be altered that have not been identified yet. We presume that many of these genes may play an important but as yet to be identified role in the fibrogenic response of the liver to bile duct obstruction. Alterations in the composition of the extracellular matrix are typical features of hepatic fibrosis[13], including substantial increases of collagens and non-collagenous components[25,26]. Accordingly, we observed a uniform up-regulation of genes encoding the procollagen types Iα1, Iα2, IIIα1, IVα1, IVα2, and Vα2 in this mouse model of obstructive cholestasis. In addition, two members of the matrix metalloproteinase family, the matrix metalloproteinases 7 and 12, were up-regulated more than ten-fold following BDL when compared with the sham-operated controls. Matrix metalloproteinases represent a group of calcium-dependent enzymes involved in physiological and pathological degradation of extracellular matrix and tissue-remodeling[27]. Matrix metalloproteinase 7 (matrilysin), an enzyme which is associated with poor prognosis in hepatocellular[28] and cholangiocellular carcinomas[29], has been closely related to the fibro-proliverative process in chronic hepatitis C[30] but not in cholestatic liver diseases. In contrast, matrix metalloproteinase 12, to our knowledge, has not been associated with liver fibrosis before and deserves future attention. Interestingly, the genes encoding tissue inhibitor of metalloproteinase 1, vascular cell adhesion molecule 1 and intercellular adhesion molecule were up-regulated both in liver and kidney of BDL mice. Genes encoding the procollagen types Iα1 and IIIα1 were also increased in the kidney of BDL mice although at lower levels than in the liver. The up-regulation of fibrosis-associated factors in kidney following BDL might be due to a paracrine action of fibrogenic mediators such as connective tissue growth factor whose hepatic expression is increased in cholestasis as previously described[31,32] and confirmed in our microarray. However, the functional relevance of the increased expression of these fibrotic genes in the kidney remains to be determined. Alternatively, the simultaneous up-regulation of important regulators of transcription following BDL such as FBJ osteosarcoma oncogene, core promoter element binding protein, and activating transcription factor 3 in both liver and kidney supports the idea of coordinated gene regulation in different tissues as response to a specific stimulus. Another non-collagenous component of the extracellular matrix which was up-regulated in BDL liver is the gene for the matricellular protein secreted acidic cysteine rich glycoprotein. Matricellular proteins are a group of matrix-associated factors that mediate cell-matrix interactions but do not serve primarily as structural elements[13]. In particular, the expression of secreted acidic cysteine rich glycoprotein has been associated with cell proliferation, migration, and extracellular matrix remodeling in tissues, and secreted acidic cysteine rich glycoprotein has been found to be increased in different models of hepatic fibrosis[33].

The expression of a number of genes encoding membrane proteins and transporters that were not previously known to be affected by cholestasis was also of interest. For example, the gene encoding the β1 subunit of the voltage-gated sodium channel which is important for the maturation and function of this channel[34] was up-regulated in liver as well as in kidney of BDL mice. In contrast, the expression of the gene encoding the ATP-binding cassette transporter multidrug resistance-associated protein 6 (Mrp6, Abcc6) was reduced in cholestatic mouse liver as previously described for the rat[20]. Since mutations of human MRP6 are associated with pseudoxanthoma elasticum, a disorder characterized by calcification of the elastic fibres and abnormalities of the collagen fibrils[35], it is tempting to speculate that reduced hepatic Mrp6 expression in cholestasis might have functional implications for the development of liver fibrosis. Other genes up-regulated in cholestatic liver were the genes encoding the macrophage receptor markers CD14 antigen and CD68 antigen. Hepatic expression of both markers is increased in patients with biliary atresia[36], and expression of CD68 antigen may be an indicator of prognosis[37]. The functional significance of the concomitant CD68 antigen elevation in BDL kidney is unclear at the moment but illustrates again the close linkage between liver and kidney in this model of cholestasis and supports again a concept of coordinated gene regulation in different tissues.

In accordance with previous studies[38], obstructive cholestasis decreased the expression of a number of genes encoding cytochrome P450 isoenzymes in liver. Since BDL results in an increase in liver concentrations of bile acids[15], the down-regulation of the cytochrome P450 7b1 (oxysterol 7α-hydroxylase) and cytochrome P450 8B1 (sterol 12α-hydroxylase) genes, that encode key enzymes in the conversion of cholesterol to bile acids[39], may represent adaptive responses to minimize the liver levels of cytotoxic bile salts. The increase of the gene encoding cytochrome P450 27b1 (25-hydroxyvitamin D3 1α-hydroxylase) in BDL kidney is another interesting observation. Cytochrome P450 27b1 catalyzes the conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3, the last step in vitamin D activation, which takes place in kidney[40]. Thus the increase in renal cytochrome P450 27b1 expression may reflect an adaptive response to compensate for 25-hydroxyvitamin D deficiency in cholestasis. This may be a pathophysiologically important mechanism since patients with primary biliary cirrhosis often present with deficiencies of 25-hydroxyvitamin D but normal or even elevated levels of 1, 25-dihydroxyvitamin D[41].

In summary, the present study provides a comprehensive gene expression profile from mouse liver and kidney in obstructive cholestasis. Changes in gene expression were validated by Northern analysis, immunofluorescence, or comparison with the literature. The findings in this study provide new insights for generating novel hypotheses concerning the adaptive responses of gene expression in this mouse model of cholestasis.

ACKNOWLEDGMENTS

We thank Albert Mennone and Kathy Harry (both Liver Center, Yale University School of Medicine, New Haven, CT, USA) for excellent technical assistance and Prof. Dr. Hermann Koepsell (Institut für Anatomie und Zellbiologie, Bayerische Julius-Maximilians-Universität, Würzburg, Germany) for providing the Oct1 antibody.

Footnotes

S- Editor Pan BR L- Editor Kumar M E- Editor Bai SH

References
1.  Trauner M, Boyer JL. Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev. 2003;83:633-671.  [PubMed]  [DOI]  [Cited in This Article: ]
2.  Gartung C, Ananthanarayanan M, Rahman MA, Schuele S, Nundy S, Soroka CJ, Stolz A, Suchy FJ, Boyer JL. Down-regulation of expression and function of the rat liver Na+/bile acid cotransporter in extrahepatic cholestasis. Gastroenterology. 1996;110:199-209.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 179]  [Cited by in F6Publishing: 143]  [Article Influence: 7.2]  [Reference Citation Analysis (0)]
3.  Dumont M, Jacquemin E, D'Hont C, Descout C, Cresteil D, Haouzi D, Desrochers M, Stieger B, Hadchouel M, Erlinger S. Expression of the liver Na+-independent organic anion transporting polypeptide (oatp-1) in rats with bile duct ligation. J Hepatol. 1997;27:1051-1056.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 73]  [Cited by in F6Publishing: 9]  [Article Influence: 3.2]  [Reference Citation Analysis (0)]
4.  Vos TA, Hooiveld GJ, Koning H, Childs S, Meijer DK, Moshage H, Jansen PL, Müller M. Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, Spgp, in endotoxemic rat liver. Hepatology. 1998;28:1637-1644.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 273]  [Cited by in F6Publishing: 191]  [Article Influence: 12.4]  [Reference Citation Analysis (0)]
5.  Lee J, Azzaroli F, Wang L, Soroka CJ, Gigliozzi A, Setchell KD, Kramer W, Boyer JL. Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat. Gastroenterology. 2001;121:1473-1484.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 119]  [Cited by in F6Publishing: 91]  [Article Influence: 6.0]  [Reference Citation Analysis (0)]
6.  Soroka CJ, Cai SY, Boyer JL. Effects of cholestasis on the regulation of membrane transporter expression in intestine and kidney. Hepatology. 2002;36:462A.  [PubMed]  [DOI]  [Cited in This Article: ]
7.  Beuers U, Denk GU, Soroka CJ, Wimmer R, Rust C, Paumgartner G, Boyer JL. Taurolithocholic acid exerts cholestatic effects via phosphatidylinositol 3-kinase-dependent mechanisms in perfused rat livers and rat hepatocyte couplets. J Biol Chem. 2003;278:17810-17818.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 58]  [Cited by in F6Publishing: 20]  [Article Influence: 3.2]  [Reference Citation Analysis (0)]
8.  Beuers U, Probst I, Soroka C, Boyer JL, Kullak-Ublick GA, Paumgartner G. Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes. Hepatology. 1999;29:477-482.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 59]  [Cited by in F6Publishing: 48]  [Article Influence: 2.7]  [Reference Citation Analysis (0)]
9.  Rust C, Gores GJ. Apoptosis and liver disease. Am J Med. 2000;108:567-574.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 137]  [Cited by in F6Publishing: 26]  [Article Influence: 6.5]  [Reference Citation Analysis (0)]
10.  Patel T, Gores GJ. Apoptosis and hepatobiliary disease. Hepatology. 1995;21:1725-1741.  [PubMed]  [DOI]  [Cited in This Article: ]
11.  Oude Elferink RP, Groen AK. Mechanisms of biliary lipid secretion and their role in lipid homeostasis. Semin Liver Dis. 2000;20:293-305.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 48]  [Cited by in F6Publishing: 26]  [Article Influence: 2.4]  [Reference Citation Analysis (0)]
12.  Bedossa P, Paradis V. Liver extracellular matrix in health and disease. J Pathol. 2003;200:504-515.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 167]  [Cited by in F6Publishing: 103]  [Article Influence: 9.3]  [Reference Citation Analysis (0)]
13.  Schuppan D, Ruehl M, Somasundaram R, Hahn EG. Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis. 2001;21:351-372.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 356]  [Cited by in F6Publishing: 257]  [Article Influence: 17.8]  [Reference Citation Analysis (0)]
14.  Holloway AJ, van Laar RK, Tothill RW, Bowtell DD. Options available--from start to finish--for obtaining data from DNA microarrays II. Nat Genet. 2002;32 Suppl:481-489.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 165]  [Cited by in F6Publishing: 64]  [Article Influence: 9.2]  [Reference Citation Analysis (0)]
15.  Bohan A, Chen WS, Denson LA, Held MA, Boyer JL. Tumor necrosis factor alpha-dependent up-regulation of Lrh-1 and Mrp3(Abcc3) reduces liver injury in obstructive cholestasis. J Biol Chem. 2003;278:36688-36698.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 114]  [Cited by in F6Publishing: 35]  [Article Influence: 6.3]  [Reference Citation Analysis (0)]
16.  Lee JM, Trauner M, Soroka CJ, Stieger B, Meier PJ, Boyer JL. Expression of the bile salt export pump is maintained after chronic cholestasis in the rat. Gastroenterology. 2000;118:163-172.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 175]  [Cited by in F6Publishing: 24]  [Article Influence: 8.3]  [Reference Citation Analysis (0)]
17.  Meyer-Wentrup F, Karbach U, Gorboulev V, Arndt P, Koepsell H. Membrane localization of the electrogenic cation transporter rOCT1 in rat liver. Biochem Biophys Res Commun. 1998;248:673-678.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 85]  [Cited by in F6Publishing: 59]  [Article Influence: 3.7]  [Reference Citation Analysis (0)]
18.  Karbach U, Kricke J, Meyer-Wentrup F, Gorboulev V, Volk C, Loffing-Cueni D, Kaissling B, Bachmann S, Koepsell H. Localization of organic cation transporters OCT1 and OCT2 in rat kidney. Am J Physiol Renal Physiol. 2000;279:F679-F687.  [PubMed]  [DOI]  [Cited in This Article: ]
19.  Denk GU, Soroka CJ, Mennone A, Koepsell H, Beuers U, Boyer JL. Down-regulation of the organic cation transporter 1 of rat liver in obstructive cholestasis. Hepatology. 2004;39:1382-1389.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 52]  [Cited by in F6Publishing: 30]  [Article Influence: 3.1]  [Reference Citation Analysis (0)]
20.  Ogawa K, Suzuki H, Hirohashi T, Ishikawa T, Meier PJ, Hirose K, Akizawa T, Yoshioka M, Sugiyama Y. Characterization of inducible nature of MRP3 in rat liver. Am J Physiol Gastrointest Liver Physiol. 2000;278:G438-G446.  [PubMed]  [DOI]  [Cited in This Article: ]
21.  Campbell KM, Sabla GE, Bezerra JA. Transcriptional reprogramming in murine liver defines the physiologic consequences of biliary obstruction. J Hepatol. 2004;40:14-23.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 13]  [Cited by in F6Publishing: 8]  [Article Influence: 0.8]  [Reference Citation Analysis (0)]
22.  Deaciuc IV, Doherty DE, Burikhanov R, Lee EY, Stromberg AJ, Peng X, de Villiers WJ. Large-scale gene profiling of the liver in a mouse model of chronic, intragastric ethanol infusion. J Hepatol. 2004;40:219-227.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 40]  [Cited by in F6Publishing: 27]  [Article Influence: 2.4]  [Reference Citation Analysis (0)]
23.  Schrenk D, Gant TW, Preisegger KH, Silverman JA, Marino PA, Thorgeirsson SS. Induction of multidrug resistance gene expression during cholestasis in rats and nonhuman primates. Hepatology. 1993;17:854-860.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 66]  [Cited by in F6Publishing: 39]  [Article Influence: 2.4]  [Reference Citation Analysis (0)]
24.  Zollner G, Fickert P, Silbert D, Fuchsbichler A, Stumptner C, Zatloukal K, Denk H, Trauner M. Induction of short heterodimer partner 1 precedes downregulation of Ntcp in bile duct-ligated mice. Am J Physiol Gastrointest Liver Physiol. 2002;282:G184-G191.  [PubMed]  [DOI]  [Cited in This Article: ]
25.  Rojkind M, Giambrone MA, Biempica L. Collagen types in normal and cirrhotic liver. Gastroenterology. 1979;76:710-719.  [PubMed]  [DOI]  [Cited in This Article: ]
26.  Schuppan D. Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins. Semin Liver Dis. 1990;10:1-10.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 246]  [Cited by in F6Publishing: 179]  [Article Influence: 7.9]  [Reference Citation Analysis (0)]
27.  Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver Dis. 2001;21:373-384.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 336]  [Cited by in F6Publishing: 265]  [Article Influence: 16.8]  [Reference Citation Analysis (0)]
28.  Yamamoto H, Itoh F, Adachi Y, Sakamoto H, Adachi M, Hinoda Y, Imai K. Relation of enhanced secretion of active matrix metalloproteinases with tumor spread in human hepatocellular carcinoma. Gastroenterology. 1997;112:1290-1296.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 76]  [Cited by in F6Publishing: 28]  [Article Influence: 3.2]  [Reference Citation Analysis (0)]
29.  Miwa S, Miyagawa S, Soeda J, Kawasaki S. Matrix metalloproteinase-7 expression and biologic aggressiveness of cholangiocellular carcinoma. Cancer. 2002;94:428-434.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 36]  [Cited by in F6Publishing: 27]  [Article Influence: 1.9]  [Reference Citation Analysis (0)]
30.  Lichtinghagen R, Michels D, Haberkorn CI, Arndt B, Bahr M, Flemming P, Manns MP, Boeker KH. Matrix metalloproteinase (MMP)-2, MMP-7, and tissue inhibitor of metalloproteinase-1 are closely related to the fibroproliferative process in the liver during chronic hepatitis C. J Hepatol. 2001;34:239-247.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 92]  [Cited by in F6Publishing: 30]  [Article Influence: 4.6]  [Reference Citation Analysis (0)]
31.  Paradis V, Dargere D, Vidaud M, De Gouville AC, Huet S, Martinez V, Gauthier JM, Ba N, Sobesky R, Ratziu V. Expression of connective tissue growth factor in experimental rat and human liver fibrosis. Hepatology. 1999;30:968-976.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 233]  [Cited by in F6Publishing: 198]  [Article Influence: 10.6]  [Reference Citation Analysis (0)]
32.  Sedlaczek N, Jia JD, Bauer M, Herbst H, Ruehl M, Hahn EG, Schuppan D. Proliferating bile duct epithelial cells are a major source of connective tissue growth factor in rat biliary fibrosis. Am J Pathol. 2001;158:1239-1244.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 116]  [Cited by in F6Publishing: 36]  [Article Influence: 5.8]  [Reference Citation Analysis (0)]
33.  Frizell E, Liu SL, Abraham A, Ozaki I, Eghbali M, Sage EH, Zern MA. Expression of SPARC in normal and fibrotic livers. Hepatology. 1995;21:847-854.  [PubMed]  [DOI]  [Cited in This Article: ]
34.  Kupershmidt S, Yang T, Roden DM. Modulation of cardiac Na+ current phenotype by beta1-subunit expression. Circ Res. 1998;83:441-447.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 20]  [Cited by in F6Publishing: 1]  [Article Influence: 0.9]  [Reference Citation Analysis (0)]
35.  Bergen AA, Plomp AS, Schuurman EJ, Terry S, Breuning M, Dauwerse H, Swart J, Kool M, van Soest S, Baas F. Mutations in ABCC6 cause pseudoxanthoma elasticum. Nat Genet. 2000;25:228-231.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 377]  [Cited by in F6Publishing: 191]  [Article Influence: 18.0]  [Reference Citation Analysis (0)]
36.  Tracy TF Jr, Dillon P, Fox ES, Minnick K, Vogler C. The inflammatory response in pediatric biliary disease: macrophage phenotype and distribution. J Pediatr Surg. 1996;31:121-125; discussion 125-126;.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 62]  [Cited by in F6Publishing: 16]  [Article Influence: 2.5]  [Reference Citation Analysis (0)]
37.  Kobayashi H, Puri P, O'Briain DS, Surana R, Miyano T. Hepatic overexpression of MHC class II antigens and macrophage-associated antigens (CD68) in patients with biliary atresia of poor prognosis. J Pediatr Surg. 1997;32:590-593.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 67]  [Cited by in F6Publishing: 14]  [Article Influence: 2.8]  [Reference Citation Analysis (0)]
38.  Tateishi T, Watanabe M, Nakura H, Tanaka M, Kumai T, Kobayashi S. Liver damage induced by bile duct ligation affects CYP isoenzymes differently in rats. Pharmacol Toxicol. 1998;82:89-92.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 12]  [Cited by in F6Publishing: 5]  [Article Influence: 0.5]  [Reference Citation Analysis (0)]
39.  Chiang JY. Bile acid regulation of gene expression: roles of nuclear hormone receptors. Endocr Rev. 2002;23:443-463.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 319]  [Cited by in F6Publishing: 226]  [Article Influence: 17.7]  [Reference Citation Analysis (0)]
40.  Omdahl JL, Bobrovnikova EV, Annalora A, Chen P, Serda R. Expression, structure-function, and molecular modeling of vitamin D P450s. J Cell Biochem. 2003;88:356-362.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 20]  [Cited by in F6Publishing: 8]  [Article Influence: 1.1]  [Reference Citation Analysis (0)]
41.  Kaplan MM, Elta GH, Furie B, Sadowski JA, Russell RM. Fat-soluble vitamin nutriture in primary biliary cirrhosis. Gastroenterology. 1988;95:787-792.  [PubMed]  [DOI]  [Cited in This Article: ]