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Am J Physiol Gastrointest Liver Physiol 294: G1411-G1420, 2008. First published April 24, 2008; doi:10.1152/ajpgi.00181.2007
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LIVER AND BILIARY TRACT

Pathophysiological basis of liver disease in cystic fibrosis employing a {Delta}F508 mouse model

Folke Freudenberg,1,2 Annemarie L. Broderick,1,2,3 Bian B. Yu,2 Monika R. Leonard,2 Jonathan N. Glickman,4 and Martin C. Carey1,2

1Department of Medicine, Harvard Medical School and Harvard Digestive Diseases Center, Boston; 2Department of Medicine, Gastroenterology Division, Brigham and Women's Hospital, Boston; 3Combined Program of Gastroenterology and Nutrition, Children's Hospital, Boston; and 4Pathology Department, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts

Submitted 25 April 2007 ; accepted in final form 20 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The molecular pathogenesis of cystic fibrosis (CF) liver disease is unknown. This study investigates its earliest pathophysiological manifestations employing a mouse model carrying {Delta}F508, the commonest human CF mutation. We hypothesized that, if increased bile salt spillage into the colon occurs as in the human disease, then this should lead to a hydrophobic bile salt profile and to "hyperbilirubinbilia" because of induced enterohepatic cycling of unconjugated bilirubin. Hyperbilirubinbilia may then lead to an increased bile salt-to-phospholipid ratio in bile and, following hydrolysis, precipitation of divalent metal salts of unconjugated bilirubin. We document in CF mice elevated fecal bile acid excretion and biliary secretion of more hydrophobic bile salts compared with control wild-type mice. Biliary secretion rates of bilirubin monoglucuronosides, bile salts, phospholipids, and cholesterol are increased significantly with an augmented bile salt-to-phospholipid ratio. Quantitative histopathology of CF livers displays mild early cholangiopathy in {approx}53% of mice and multifocal divalent metal salt deposition in cholangiocytes. We conclude that increased fecal bile acid loss leads to more hydrophobic bile salts in hepatic bile and to hyperbilirubinbilia, a major contributor in augmenting the bile salt-to-phospholipid ratio and endogenous β-glucuronidase hydrolysis of bilirubin glucuronosides. The confluence of these perturbations damages intrahepatic bile ducts and facilitates entrance of unconjugated bilirubin into cholangiocytes. This study of the earliest stages of CF liver disease provides a framework for investigating the molecular pathophysiology of more advanced disease in murine models and in humans with CF.

cystic fibrosis transmembrane conductance regulator; gallstones; enterohepatic cycling; bilirubin; metal salts

CYSTIC FIBROSIS (CF), the most common monogenic inherited disease in humans with northern European ancestry, is caused by more than 1,000 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (53, 57). CFTR is a chloride channel regulated by cAMP and is located on the apical plasma membranes of most absorptive and secretory cells including large cholangiocytes and cholecystocytes (19, 20, 34). Its dysfunction impairs Cl– secretion and therefore Cl–/HCO3– exchange. The most common mutation in the CFTR gene, the {Delta}F508 mutation, involves about 70% of CF patients (57). Hepatobiliary abnormalities occur in ~13–27% of pediatric patients with CF (29, 50) and 30–72% of adults with CF (40, 43, 69) and include cholangitis, cholestasis, hepatic steatosis, focal biliary cirrhosis (40), and gallstones (2, 43). The pathogenesis of any of these complications of CF is not understood. The aim of this study was to investigate the pathophysiological and histopathological changes in the hepatobiliary tree of a mouse model carrying the {Delta}F508 mutation in which liver disease is very mild or absent (76).

First, we assessed the histopathology of the liver, with particular reference to bile duct lesions. We also determined whether these CF mice demonstrate increased fecal bile acid loss and an altered bile salt profile, a notable feature of the human disease (49, 58, 74). We also characterized bilirubin molecular species and secretion rates, which can provide evidence for putative enterohepatic cycling of unconjugated bilirubin (UCB) since hyperbilirubinbilia (increased secretion of conjugated bilirubins into bile) has been demonstrated previously in ileectomized rats (9) and patients with ileal Crohn's disease (10). Any degree of enterohepatic cycling of bilirubin mimics chronic mild hemolytic states (9) and places an animal at risk for intraductal hydrolysis of conjugated bilirubins and insoluble metal salt formation as well as precipitation of "black" pigment stones in the gallbladder (72).

We studied histopathological changes in both sexes of mice as functions of age, and we demonstrate mild liver disease in ~53% of CF mice older than 100 days. Our work suggests that liver injury begins most likely at the level of the large cholangiocytes where the dysfunctional CFTR is located (1). We suggest that cholangiocytic injury is caused by a more hydrophobic bile salt pattern and an increased detergency from augmented bile salt-to-phospholipid ratio caused by hyperbilirubinbilia. This leads, in turn, to increased intrahepatic hydrolysis of bilirubin glucuronosides by endogenous β-glucuronidase and precipitation of unconjugated bilirubin in cholangiocytes that, in contrast to hepatocytes, lack a "disposal" mechanism for the bile pigment. Our findings in this murine model of CF suggest that the liver disease is exceedingly mild, allowing for observation of an unperturbed "window" on its causation by altered bile salt physiology arising in the distal gut. These subtle but overt alterations in bile chemistry may be translatable to more severe animal models of CF liver disease and to humans with CF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Heterozygous breeding pairs of {Delta}F508 mice (76) and wild-type (WT) mice (background: 75% C57BL/6, 25% 129SvEv) were kindly provided by Dr. Marie Egan, Yale University School of Medicine, New Haven, CT. They were housed on ground corncob bedding (The Andersons, Maumee, OH) in the animal facility of the Thorn Research Building at Brigham and Women's Hospital, Boston, MA. Mice were fed a diet containing 11% fat and replete with calories, vitamins, and minerals (Mouse Diet 5015; Labdiet, Richmond, IN) and were maintained on a regular 12-h:12-h light-dark cycle. Mice were given an oral isosmotic solution containing polyethylene glycol-3350 and electrolytes (GoLYTELY; Braintree Laboratories, Braintree, MA) ad libitum. Histopathological studies and fecal bile acid analyses were performed on age- and sex-matched homozygous CF and control (WT) mice. When we found little difference between sexes and because CF mice were scarce, we utilized age-matched mice of either sex for the remaining experiments. All experiments were performed at the same time of day on mice in the nonfasted state. At 3 wk of age, mice were genotyped after tail or ear clipping. DNA was isolated with the use of DNeasy kits (Qiagen, Valencia, CA), and the PCR product was amplified with Amplitaq Gold Master Mix (Applied Biosystems, Foster City, CA) with the primer sequences GAG TGT TTT CTT GAT GAT GTG and ACC TCA ACC AGA AAA ACC AG. The amplified DNA was restricted utilizing the enzyme RsaI (Applied Biosystems) and separated by agarose gel electrophoresis. Mice were weighed before surgery. All experiments were performed following protocols approved by the Harvard University Medical Area Standing Committee on Animals.

Liver histopathology. An age-matched study by a pathologist (J. Glickman) blinded as to genotype and mouse age was performed on 30 livers of CF mice and 48 livers of WT littermates aged from 1 day to ~400 days. After laparotomy and hepatectomy, livers were fixed at room temperature ({approx}22°C) for a minimum of 12 h in 10% formaldehyde, processed routinely, and embedded in paraffin. Five-micron-thick sections were stained with hematoxylin and eosin (H&E) and special stains (see below) and were examined microscopically. We employed a semiquantitative scoring system to assess inflammation as follows: 0, absent; 1, either mild (scattered portal or lobular inflammatory cells in single or small clusters) or a few foci in a minority of lobules; 2, moderate (numerous inflammatory cells in clusters, involving the majority of lobules); or 3, severe (sheets of inflammatory cells, invariably involving the majority of lobules). Fibrosis was quantified using Masson's trichrome staining protocol employing the following scoring system: 0, no increase; 1, portal, pericentral, or sinusoidal fibrosis; 2, septal fibrosis without bridging; 3, bridging fibrosis; and 4, cirrhosis (regenerative nodule formation). Hepatocyte regeneration was evaluated by H&E and reticulin stains and graded as follows: 0, absent; 1, one or two foci only; 2, multifocal, involving a minority of lobules; and 3, diffuse, involving the majority of lobules. Bile ductular proliferation was scored as absent, minimal (exemplified by prominent canals of Hering, increased cholangiocytes, but no extra ductules), 1+ (one to two foci and one extra bile ductule only), or 2+ (multifocal with multiple ductular profiles per focus). Scores were averaged for each group and reported as mean lesion scores. Deposition of Fe and Ca bilirubinates in the liver was assessed by Prussian blue and von Kossa staining, respectively, as well as Hall's bilirubin stain.

Fecal bile acid excretion. Mice were housed individually in metabolic cages and given free access to GoLYTELY and the high-fat (11% by weight) diet. At 72 h, 3-day stools were collected, and mice were weighed. Stools were dried under reduced pressure for 48 h and then ground to a powder using a mortar and pestle. Alkaline hydrolysis was performed on 100-mg aliquots of homogenized, dried stool. After acidification with HCl, fecal bile acids were extracted with diethyl ether and measured by an enzymatic assay (52).

Biliary bilirubin outputs and molecular species. Under general anesthesia [ketamine:xylazine:atropine, 90:10:0.13 mg/kg body weight (BW)], a midline abdominal incision was made and the cystic duct was identified and ligated, followed by ligation of the common bile duct near the Vaterian ampulla. A 0.28-mm (ID) polyethylene catheter (Intramedic; Becton Dickinson, Franklin Lakes, NJ) was inserted into the proximal common bile duct, and, after discarding the initial 5-min drainage, bile was collected for {approx}15 min into tared collection tubes. This procedure for bile collection minimizes interruption of the enterohepatic circulation. A 10-µl bile sample from each collection was used to quantify bilirubin concentrations and molecular species by HPLC (63). Total bilirubin secretion rates were normalized to 1 h of bile flow and 100 g BW.

Common biliary lipids in hepatic bile. Bile salt molecular species were determined by HPLC (56). Total bile salts were assayed by the 3{alpha}-hydroxysteroid dehydrogenase method (67). Biliary phospholipids were measured as inorganic phosphorus (4), and biliary cholesterol was hydrolyzed and extracted (38) before HPLC analysis (70). Bile salt hydrophobicity was quantified as a hydrophobicity index according to the method used by Heuman (35). Secretion rates of the major biliary lipids were calculated by normalizing concentrations per hour of bile flow and to 100 g BW. Cholesterol saturation indexes (CSIs) were calculated using critical tables (15) but without correcting for the muricholate content of mouse bile.

Bile flow, hepatic bile pH, and electrolytes. Following bile collection, biliary pH values were measured immediately by microelectrode (Thermo Electron, Beverly, MA), and bile volume was determined gravimetrically by numerically equating weight (g) with volume (ml). To obtain the 90-µl volumes needed for biliary Cl– measurements, hepatic bile was collected for ~1 h. Samples were frozen at –20°C until analyses could be performed at the clinical chemistry Core Laboratory, Children's Hospital, Boston, MA on a cobas c 501 system (Roche Diagnostics, Indianapolis, IN) utilizing an ion-specific electrode calibrated specifically for nonplasma, nonurine samples. Biliary Na+, K+, and PO43– concentrations were measured in hepatic bile samples ranging in volume from 20–90 µl on a cobas Integra 400 system (Roche Diagnostics). Samples were automatically diluted until values fell into the detectable range for the analyte. Both Na+ and K+ were measured by ion-selective electrodes, whereas measurement of phosphate was determined by a colorimetric reaction.

Ileal lumenal pH values. After ligating the ileum at both the ileocecal valve and 5 cm cephalad from the initial ligation, 1.0 ml of 0.15 M NaCl solution was injected into the ileal lumen, followed by gentle manipulation to ensure mixing. After 3 min of equilibration, which was shown in preliminary studies to lead to a steady-state value, pH was measured intralumenally by means of a microglass electrode.

Statistical treatments. Group values for each measurement are expressed as means ± SE. For comparisons between CF and WT mice, statistical significance was assessed using an unpaired two-tailed Student's t-test. P values less than 0.05 are considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General. Between 10 and 25 days, WT and CF mice were approximately the same weight. Over the course of this longitudinal study, all mice gained weight progressively (Fig. 1) with WT mice diverging at the earliest time points and mean weights leveling off at about 250 days. After the first 25-day period, both WT and CF mice displayed steady increases in weight, and differences between the groups remained similar. Of note is that mouse breeding (heterozygous male x heterozygous female or CF male x heterozygous female) yielded 40% fewer CF offspring than expected (data not shown). The mice studied in this work all appeared healthy up to the time of surgery and euthanasia.


Figure 1
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  		Fig. 1. Age-weight comparisons of individual cystic fibrosis (CF) ({circ}) and wild-type (WT) ({square}) mice obtained before surgery. At the earliest time points (circled), weights of CF and WT mice were similar, but by 50 days mouse weights had diverged. Weight gain for all mice followed a steady increase, thereafter leveling off at ~250 days. Second order, best-fit curves for CF (solid line) and WT (dotted line) mice demonstrate that weight differences between the genotypes remained similar for most of the study. It is clear that, on average, CF mice were substantially smaller than age-matched WT controls.

 
Liver histopathology. Sections of liver from representative CF (Fig. 2A) and WT (Fig. 2B) livers display variable, mild, patchy cholangiopathy characterized by reactive changes in the biliary epithelium, bile ductular proliferation, and mild portal fibrosis. These findings were present only rarely in WT mice (Fig. 2B). Intracellular fatty deposits were not observed in any of the CF livers. Curiously, some older WT mice developed steatohepatitis, most likely from efficient absorption of the 11% fat diet. So-called "inspissated" plugs were not detected in the hepatobiliary tree of any animal. None of the mice manifested any advanced degree of liver fibrosis or cirrhosis, and evidence for apoptosis was found in only a single sample out of 30 (data not shown). There were no sex differences in the liver histopathology in the case of either WT or CF mice.


Figure 2
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  		Fig. 2. Histopathology of the liver. Livers of CF (A) and WT (B) mice were fixed in formalin and embedded in paraffin. Five-micron-thick sections were stained with hematoxylin and eosin. Original magnification is x200. A representative CF liver (A) displays reactive changes in the biliary epithelium, bile ductular proliferation, and mild portal fibrosis (arrow) findings not present in WT (B) mice. To visualize Fe and Ca deposits, liver sections were prepared as described in MATERIALS AND METHODS and stained for Fe with Prussian blue stain or for Ca using the von Kossa protocol. Multifocal Fe deposition (C, arrow) was observed in 13% of CF and 6% of WT mice; multifocal Ca deposition (D, arrow) was found in 25% of CF and 3% of WT mice.

 
Table 1 quantifies histopathological changes (i.e., lobular inflammation, fibrosis, and bile duct alterations) in WT and CF mice as functions of age. At euthanasia, signs of lobular inflammation were found in approximately half of CF mice older than 100 days compared with nearly a third of age-matched WT mice, with both the percentage of affected CF mice and their mean lesion scores reaching a plateau at 100 days; however, none of the differences between age-matched WT and CF mice were statistically significant. The percentage of both WT and CF mice affected with fibrosis and their corresponding mean lesion scores demonstrated no age-related trend; however, the differences were significant for mice aged 101–200 days. Whereas the incidence and mean lesion scores for bile duct alterations remained generally constant in WT mice, the proportion of CF mice affected and their mean lesion scores rose steadily with passage of time. Differences for percentages affected and mean lesion scores of bile ductular histopathology were statistically significant for comparisons of older age-matched CF and WT mice. Mean lesion scores for all histopathological changes as functions of age indicate that liver disease was very mild in this CF model and remained so for the duration of the study. Nonetheless, 47% of CF mice did not exhibit any bile duct lesions even though some animals were more than 12 mo old.


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  		Table 1. Chronology of liver histopathology in WT and CF mice

 
Prussian blue and von Kossa staining for Fe (Fig. 2C) and Ca (Fig. 2D) salts revealed multifocal hepatic deposition of both metals in cholangiocytes and adjacent portal tract connective tissue. Multifocal Fe deposition was found in 13% of CF and 6% of WT mice and multifocal Ca deposition in 25% of CF and 3% of WT mice. CF mice displayed a trend toward greater Fe deposition with increasing age (data not displayed). Hall's bilirubin stain was positive but insufficiently sensitive by light microscopy to verify an appreciable difference between CF and WT mice.

Fecal bile acid excretion. Figure 3 displays 24-h fecal bile acid outputs for male and female WT and CF mice normalized per 100 g BW. Fecal excretion levels are significantly higher in all CF compared with WT mice, with values of 22.8 ± 3.1 and 12.7 ± 1.8 µmol/d per 100 g BW in CF and WT males, respectively (n = 6 per group), and 30.3 ± 4.8 and 15.8 ± 2.7 µmol/d per 100 g BW in CF and WT females (n = 8 per group); (P = 0.02 for both comparisons). Figure 3 also shows that mean fecal bile acid excretions of female mice were somewhat higher than those of age-matched males; however, sex differences for CF and WT mice were not statistically significant.


Figure 3
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  		Fig. 3. Fecal bile acid excretion. Fecal bile acid excretion expressed in µmol/day and per 100 g body weight (BW) was measured by enzymatic assay of 3-day stool collections. Bile acid excretion is increased approximately twofold in both female and male {Delta}F508 CF compared with WT mice. *P = 0.02 for both comparisons, n = 6 per group (males) and 8 per group (females).

 
Bile salt molecular species in hepatic bile. Table 2 displays percentages of the common bile salt species present in hepatic bile of CF and WT mice. Concentrations of tauro {alpha}-muricholate and tauro β-muricholate, both primary bile salts in the mouse (26), were combined because the peaks of these hydrophilic bile salts could not be baseline-separated by the HPLC method used (56). In CF mice, we found the percentage of tauromuricholates significantly decreased compared with WT mice, with a reciprocal increase that was also significant for taurocholate. The secondary bile salt taurodeoxycholate was increased in CF compared with WT mice, whereas the percentage of the primary bile salt taurochenodeoxycholate is similar in both CF and WT mice. Bile salt hydrophobicity indexes were significantly higher in CF mice compared with WT mice.


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  		Table 2. Distribution of bile salt molecular species and bile salt hydrophobicity indexes in hepatic bile of WT and {Delta}F508 CF mice

 
Bilirubin outputs and molecular species. Secretion rates of total conjugated bilirubins (Fig. 4A) are increased significantly in CF mice (35.6 ± 4.0 nmol/h per 100 g BW, n = 8, compared with 22.4 ± 2.8 nmol/h per 100 g BW in WT mice, n = 7; P = 0.02). This was due principally to the secretion rate of bilirubin monoglucuronoside (BMG) (Fig. 4B), the principal bilirubin conjugate in mice, which was increased significantly in CF compared with WT mice (24.4 ± 2.7 and 16.0 ± 1.8 nmol/h per 100 g BW, respectively; n = 9 and 7; P = 0.03). Secretion rates of total bilirubin diconjugates, i.e., bilirubin diglucuronoside (BDG) plus all other diconjugates (BDX) (Fig. 4C), were appreciably increased (10.4 ± 1.7 in CF compared with 6.4 ± 1.1 nmol/h per 100 g BW in WT; n = 8 and 7; P = 0.08) without reaching significance. Although the secretion rate of UCB was nearly doubled in CF compared with WT mice (Fig. 4D) (0.7 ± 0.2 and 0.4 ± 0.1 nmol/h per 100 g BW, respectively; n = 9 and 7; P = 0.3), that difference also failed to reach statistical significance. To determine if a correlation between liver histopathology and bilirubin secretion rates existed, we separated CF mice with histological evidence of liver disease (CF + LD) from those with normal histology (CF – LD). Figure 4E displays BMG secretion rates for both CF groups compared with WT mice. This reveals that BMG secretion rates are significantly elevated in CF mice with histopathological liver lesions (29.0 ± 2.6 nmol/h per 100 g BW, n = 6; P = 0.02), whereas BMG secretion rates for CF mice without histological changes (13.5 ± 1.5 nmol/h per 100 g BW, n = 2) are similar to those of WT mice (16.0 ± 1.8 nmol/h per 100 g BW).


Figure 4
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  		Fig. 4. Bilirubin molecular species and secretion rates. Hepatic bile from 15-min collections was immediately injected onto an HPLC column to separate and quantify bilirubin species. Bilirubin secretion rates were calculated for the various bilirubin molecular species and are displayed in nmol/h and per 100 g BW for WT and CF mice. *Denotes statistically significant difference between groups. A: when secretion rates for all conjugated bilirubin (BR) species were combined, secretion rates of conjugated bilirubins were increased by {approx}60% in CF compared with WT mice (n = 8 and 7, respectively; *P = 0.02). B: secretion rates of bilirubin monoglucuronoside (BMG), the bilirubin species most prevalent in mice, were elevated by {approx}50% in CF (n = 9) compared with WT (n = 7) mice (*P = 0.03). C: because of non-baseline separation of bilirubin diglucuronoside (BDG) and other bilirubin diconjugate peaks, we combined the concentrations of all bilirubin diconjugates (BDG + BDX) before calculating secretion rates. Although BDG + BDX secretion rates were also increased by {approx}60%, the means of CF and WT groups (n = 8 and 7 per group, respectively) were not significantly different (P = 0.08). D: mean secretion rates of unconjugated bilirubin (UCB) were substantially increased in CF compared with WT mice, but this difference was not statistically significant (n = 9 and 7, respectively; P = 0.3). E: correlations between histopathological changes ("liver disease," LD) and bilirubin secretion rates. By dividing CF mice into 2 groups based on the presence (n = 6) or absence (n = 2) of LD, we show that BMG secretion rates in bile are elevated only for CF mice with early LD (CF + LD), as inferred from histopathological changes (*P = 0.02), whereas in CF mice without liver disease, i.e., normal histology (CF – LD), BMG secretion rates are statistically the same as in WT mice.

 
Biliary pH and electrolytes. Figure 5A demonstrates that pH values of hepatic bile, although slightly lower in CF mice, are not significantly different from those of WT mice (8.6 ± 0.06 and 8.7 ± 0.06, respectively; n = 11 per group; P = 0.3). Hepatic bile Cl– concentrations (Fig. 5B) show opposite trends, being higher (nonsignificantly) in CF than in WT mice (88.4 ± 3.7 and 81.5 ± 2.5 mM, respectively; n = 8 and 6; P = 0.18). No appreciable differences between CF and WT mice were found for PO43– concentrations or concentrations of the monovalent counterions (Na+ and K+) (data not displayed).


Figure 5
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  		Fig. 5. Biliary pH and Cl– secretion rates in CF and WT mice. Hepatic bile pH was measured by microelectrode immediately following each 15-min bile collection. A: although slightly lower in CF mice, hepatic bile pH is not significantly different between the CF and WT models (n = 11 per group; P = 0.3). B: Cl– concentrations ([Cl–]) of hepatic bile samples are increased nonsignificantly in CF mice (n = 8) compared with WT controls (n = 6); P = 0.18.

 
Hepatic bile flow and biliary lipid secretion rates. Figure 6A plots normalized hepatic bile flow and demonstrates that the values are significantly higher in CF than in WT mice (593.3 ± 73.2 and 335.2 ± 36.4 µl/h per 100 g BW, respectively; n = 9 and 15; P = 0.002). The assayed absolute concentrations of bile salts, phospholipids, and cholesterol in hepatic bile are similar in CF and WT mice with mean CSIs (uncorrected for presence of muricholates) of 0.65 and 0.67, respectively. However, there was a 19% increase in bile salt-to-phospholipid ratio in bile of CF compared with WT mice (n = 6 and 11, respectively). Figure 6B plots normalized bile salt secretion rates and shows a significant threefold increase in CF compared with WT mice (44.1 ± 12.3 compared with 15.6 ± 3.4 µmol/h per 100 g BW; P = 0.02). Similarly, phospholipid secretion rates (Fig. 6C) are doubled in CF compared with WT mice (4.1 ± 0.9 compared with 1.9 ± 0.3 µmol/h per 100 g BW; P < 0.01), and cholesterol secretion rates (Fig. 6D) are nearly tripled (0.91 ± 0.28 compared with 0.34 ± 0.05 µmol/h per 100 g BW; P = 0.02). To obtain an estimate of the contributions of bile salt-dependent and bile salt-independent bile flows to the increased bile flow in CF mice, we plotted (Fig. 6E) the volume of bile flow (Fig. 6A) for both WT and CF mice against their respective bile salt outputs (Fig. 6B). The slope of each regression line (volume of hepatic bile water in µl/µmol bile salts) is a quantitative measure of the bile salt-dependent bile flow. The extrapolated y-intercept of each line (i.e., when bile salt output is theoretically zero) gives the bile salt-independent bile flow. Although the difference between slopes is not quite significant, Fig. 6E suggests that bile salt-dependent bile flow in CF mice is less than 50% of that of WT mice (slopes = 7.8 and 18.3; n = 5 and 10, respectively; P = 0.096), whereas the bile salt-independent bile flow in CF mice is nearly three times that of WT mice (y-intercept = 351 for CF and 125 for WT mice). These findings are consistent with the higher Cl– values in the hepatic biles of CF mice that we documented in Fig. 5B.


Figure 6
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  		Fig. 6. Hepatic bile flow and biliary lipid secretion rates in hepatic bile. After cannulation of the common bile duct and collection of bile over a 15-min period into a tared collection tube, hepatic bile flow was calculated by equating 1 mg with 1 µl. *Indicates statistically significant difference between CF and WT mice. A: bile flow was normalized to volume/h and per 100 g BW. Normalized hepatic bile flow is {approx}75% greater in CF (n = 9) compared with WT (n = 15) mice (*P = 0.002). We assayed bile salts, phospholipids, and cholesterol in hepatic bile of {Delta}F508 CF (n = 6) and WT (n = 11) mice by standard validated methods. All secretion rates are expressed as µmol/h and per 100 g BW. Secretion rates of bile salts (B), phospholipids (C), and cholesterol (D) are all significantly increased in CF compared with WT mice (*P < 0.05 for bile salt and cholesterol secretion rates; *P < 0.01 for phospholipid secretion rates). E: to assess the contribution of bile salt-dependent and -independent bile flow to the increased bile flow observed in {Delta}F508 mice (A), we plotted bile flow of CF ({circ}) and WT ({square}) mice against their respective bile salt secretion rates. The slope of each line (µl/µmol) is the bile salt-dependent flow, whereas the y-intercept (biliary water when bile salt output is extrapolated to a theoretical zero) is the bile salt-independent flow. Our data suggest (P = 0.096 for the difference in slopes) that bile salt-dependent bile flow is halved, whereas bile salt-independent bile flow is nearly tripled in {Delta}F508 compared with WT mice (see DISCUSSION).

 
Ileal lumenal pH. As determined from preliminary experiments, before measuring the pH by glass electrode, we allowed the injected saline solution to mix with endogenous secretions for 3 min in the doubly ligated, closed ileal loops. The final recorded pH values in the distal ileum are significantly lower (P = 0.003) in CF (6.7 ± 0.1) than in WT mice (7.2 ± 0.1); n = 8 per group.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study provides a systematic description of the earliest and, in several cases, subtle alterations in the biochemistry, biophysics, and pathophysiology of the small and large intestines and hepatobiliary system in a CF mouse model carrying the most common human CF mutation, {Delta}F508. Although others have demonstrated advanced liver histopathology in a mouse model with more severe systemic CF disease (25), we chose for our experiments the mouse model described by Zeiher et al. (76) in which liver disease is mild, if it occurs at all. This model allowed us to obtain valid indexes for pH, electrolytes, and biliary outputs of flow, common biliary lipids, and especially bilirubins in hepatic bile without the values being perturbed by severe dysfunctional liver disease. The {Delta}F508 dysfunction is constructed on a 75% C57BL/6 and 25% 129SvEv background and resembles the human disease with the same genetic defect but with the difference that residual CFTR activity is present. As in the human disease, ~53% of these animals develop some histopathological evidence of liver disease, but many mice never acquire microscopic alterations in their livers. Interestingly, there is a high incidence of meconium ileus (in the absence of corncob bedding; see MATERIALS AND METHODS) in this model, and the {Delta}F508 mice exhibit significant growth retardation compared with WT mice (Fig. 1). On the other hand, this {Delta}F508 murine model displays minimal pulmonary phenotype, most likely because of ancillary Cl– channels and, in this regard, is markedly different from humans with the same CFTR mutation.

We documented the development of histopathological changes in ~53% of CF animals, with morphological features of cholangiopathy including bile ductular proliferation and mild portal fibrosis (Fig. 2A), characteristics of very early and mild liver disease. As anticipated, no evidence was found histologically for bile ductular obstruction from so-called inspissated bile. As inferred histopathologically, this work suggests that the earliest evidence of hepatic disease appears to be damage to cholangiocytes, not hepatocytes. We proposed that the damage resulted from intralumenal alterations in the biochemistry and physical chemistry of biliary lipids and lipopigments, i.e., bilirubins, and focused on the possibility that there might be hyperbilirubinbilia and deposition of salts of unconjugated bilirubin and that hydrophobic bile acids and an altered bile salt/phospholipid ratio might be involved. This led us to focus on distal (i.e., ileal and colonic) causes for these alterations as the possible source and origin of the liver insult.

We first examined steady-state fecal bile acid outputs and biliary lipid compositions in CF and WT mice. As in the human disease where {approx}30% of CF patients exhibit bile salt malabsorption (49, 74), we found that fecal bile acid excretion is increased approximately twofold in our CF mouse model (Fig. 3). Two principal hypotheses have been proposed for this observation in humans. 1) Zentler-Munro et al. (78) showed that increased acidity of the upper small intestine in CF leads to protonation and precipitation of glycine-conjugated bile salts as crystals (16, 60). However, acid (i.e., proton)-induced bile salt insolubility cannot explain our findings since the bile salt pool in mice consists mostly (>95%) of taurine-conjugated bile salts. The sulfonate group of the side chain of taurine-conjugated bile salts cannot be protonated at gut lumenal pH values found in CF mice since their pKa values are less than 2 (60). 2) Others have shown that increased fecal bile acid loss may be due to the binding of bile salts to undigested protein, starch, or lipids, thus preventing bile salt resorption in the ileum (49, 73, 77). This mechanism cannot apply either to our mouse model since the {Delta}F508 mutation and several other CF mouse models exhibit no major pathological changes in the pancreas, in contrast to humans (21, 23, 32, 33, 68, 76). Although not a formal part of this study, we confirmed high levels of pancreatic amylase and lipase in the proximal small intestines of our CF mice (F. Freudenberg and M. C. Carey, unpublished observations). In addition, we found no evidence by Western blot for altered expression of the SLC10A2/apical sodium-dependent bile acid transporter (ASBT) receptor in the ileum (F. Freudenberg and M. C. Carey, unpublished observations). Nonetheless, ex vivo studies by Lack and Weiner (41) of the ileal mucosa's affinity for bile salts have shown that its function is highly sensitive to lumenal pH, with small decreases resulting in a pronounced lower uptake. We interpolate from their published work (41) that the significant difference of ~0.5 pH units in ileal lumenal pH (see RESULTS) would decrease the ileal transport of taurocholate by ~9% per enterohepatic cycle. This could explain most of the fecal bile acid loss in the CF mouse since the bile salt pool circulates frequently with nocturnal eating (36). Another plausible explanation for fecal bile acid loss could be the increased thickness of viscous intestinal mucin gel in the CF mouse model, but whether mucin gel binds bile salts appreciably is controversial (61, 75). However, mucin gel may act as a diffusion barrier (6, 42) to retard bile acid resorption by ileal SLC10A2/ASBT in both mice and humans. Nonetheless, we propose that the electrochemical reason noted above (41) is the most likely cause for the approximate doubling of fecal bile acid loss that we observed in our {Delta}F508 mice (Fig. 3). The increased fecal bile acid excretion also reflects more secondary bile acid formation by bacteria in the colon and is consistent with the altered bile acid molecular species observed in hepatic bile (Table 2) since the murine liver is not 100% efficient in 7{alpha}-rehydroxylation of the bile acid nucleus. We propose that the excess bile acids in the colon are also responsible for induced enterohepatic cycling of bilirubin (9) found in these mice.

With further respect to the higher proportion of more hydrophobic bile salts (Table 2) in bile of CF mice, we note that the molecular bile salt profile found in a mouse model with severe fecal bile acid loss due to disruption of SLC10A2 is similar (22); besides, more hydrophobic bile salts are better conserved by passive resorption in the colon (17). In addition, the higher bile salt-to-phospholipid ratio (30) may be another source of detergent damage to cholangiocytes and may further aggravate cholangiocyte injury by facilitating the entry of locally formed UCB into cells. Most likely the increased bile salt-to-phospholipid ratio in hepatic bile is secondary to uncoupling of phospholipid from bile salt secretion at the canalicular level as a result of hypersecretion of conjugated bilirubins (3, 71). These explanations validate, in part, the "classic" theoretical postulate that so-called "toxic" components of bile (believed to be mainly bile acids) might impair cholangiocyte integrity and cause bile duct damage leading to CF liver disease (29, 62).

In addition to the secretion rates of the lipopigment bilirubin molecules being significantly increased in {Delta}F508 mice (Fig. 4, A–C), we also found (Fig. 4E) a strong correlation between mice with elevated BMG secretion rates and those animals developing histopathological changes in the liver. Furthermore, in mice with mild CF liver disease, we observed histological staining consistent with multifocal hepatic deposition, presumably as bilirubinates of Fe and Ca metal salts, in cholangiocytes and in portal tracts (Fig. 2, C and D, respectively). It is unlikely that Ca or Fe precipitated with carbonate or phosphate since the pH of hepatic bile is not consistent with supersaturation with Ca salts of these anions (47, 48). Moreover, conjugated bile salts, even glycine-conjugated ones, are resistant to Ca precipitation in the typical concentrations found in hepatic bile (24, 39). In contrast, the exquisite sensitivity of UCB to forming insoluble salts with Ca2+ is demonstrated by the ion product of Ca(HUCB)2 (i.e., the monoacidic Ca salt of UCB) in model bile, which is of the order of 10–15 M2 (12). Hyperbilirubinbilia of any cause is an accepted risk factor for increased intrahepatic hydrolysis by endogenous β-glucuronidase and deposition of UCB and possibly BMG (66) as metal salts, not only in the gallbladder as black pigment stones but also, as shown by us here, in cholangiocytes. Cholangiocytes lack a bilirubin conjugation/transport system and export pump (65) and therefore cannot dispose of UCB as effectively as do hepatocytes. Moreover, UCB is cytotoxic as well as being disruptive to plasma membranes (11, 13, 18, 51). We speculate that cytotoxicity of elevated UCB levels in cholangiocytes, either as the free dianion or as metal salts, might be a major contributor to CF liver disease (14), augmenting injury by the increased detergency of hepatic bile (7).

The calculated relative lipid compositions expressed as CSIs of hepatic bile are similar in CF and WT mice (see RESULTS). Published studies in humans with severe CF disease indicate that significantly higher relative concentrations of bile cholesterol, secondary to bile salt malabsorption, are not always found (2, 5, 58, 64). Nonetheless, because of increased bile flow, possibly secondary to alternate Cl– channel compensation, the normalized (but not absolute) secretion rates of all three major biliary lipids (Fig. 6, B–D), as well as bilirubins (Fig. 4), were significantly higher in CF compared with WT mice. Therefore, taking all data together, it is likely that the cholangiocyte damage in CF mice is related to increased detergent exposure over time, primarily to more hydrophobic bile salts (Table 2), and to hyperbilirubinbilia, which, although innocuous per se, increases bile salt-to-phospholipid ratios and augments UCB formation.

It is evident (Fig. 6E) that bile salt-dependent bile flow (i.e., slope of the regression line plotting bile flow and bile salt output) is lower in CF than in WT mice. At the phospholipid/bile salt ratios in both CF and WT mice (values <0.15; data not shown), bile salt-dependent bile flow is predicated by the number of simple micelles (i.e., those not containing phospholipids) coexisting with mixed micelles (16, 44). Because the number of these osmotically active particles increases with hydrophilicity (46), the decreased bile salt-dependent bile flow observed in the CF mice is consistent with a more hydrophobic bile salt profile (Table 2). The increased bile salt-independent bile flow in the CF mice is consistent with Cl– levels being higher in CF than in WT mice. This suggests either that CFTR is stimulated by cholehepatic shunting of bile salts (37) or that alternate Cl– channels are upregulated. It is interesting to note in this regard that, in two different {Delta}F508 mouse models (21, 68), Bijvelds et al. (8) showed that ileal bile salt absorption activates CFTR-mediated salt and water secretion. These authors suggested that an analogous ASBT/CFTR interaction might take place at the level of the intrahepatic bile ductules in CF.

We found only a slightly less alkaline hepatic bile pH in our mouse model (Fig. 5A), possibly due, as evidenced by our Cl– data, from a compensation by other ductular Cl– channels (27, 28, 31, 45, 54, 55, 59). In addition to increasing bile salt-independent bile flow, this most likely normalized hepatic bile pH by providing ample Cl– for Cl–/HCO3– exchange. However, the dysfunctional CFTR is also expressed on cholecystocytes, and we found that the pH of gallbladder bile in these CF mice was significantly more acidic than that of WT mice (F. Freudenberg and M. C. Carey, unpublished observations). If CFTR were the only Cl– channel on cholangiocytes in humans, this would explain why CF liver disease is much more severe than in our mouse model. Contrariwise, if, in humans, there are individualized genetic mechanisms that maintain hepatic bile pH within the normal range, then this could explain why some patients acquire liver disease and others, despite possessing the same mutation in the CFTR gene, do not.

In summary, this longitudinal study on a large cohort of {Delta}F508 CF mice of both sexes suggests that several pathophysiological alterations in biliary lipid and lipopigment metabolism are likely responsible for early and mild histopathological changes observed in 53% of CF mice. Increased fecal bile acid loss in the CF mouse apparently results from decreased ileal pH that, in turn, leads to a more hydrophobic bile acid profile in hepatic bile from anaerobic bacterial catabolism of primary bile salts in the colon. Moreover, excess bile salt spillage into the colon also induces enterohepatic cycling of bilirubin, and the resulting hyperbilirubinbilia is most likely responsible for the decreased biliary secretion rate of phospholipids compared with bile salts in {Delta}F508 mice. Following β-glucuronidase hydrolysis, hyperbilirubinbilia is also ultimately responsible for depositing insoluble metal salts of UCB, and possibly BMG, in cholangiocytes. This not only augments bile detergency but also impairs cholangiocyte integrity by a number of interrelated mechanisms. To further unravel the complexities of more severe liver disease in CF, systematic studies in mouse models with more advanced liver disease, as well as studies in humans with CF, are required.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-036588, DK-073687, and DK-052911 (M. C. Carey), by Deutsche Forschungsgemeinschaft Grant FR 1733/ 1-1 (F. Freudenberg), and by Training Grant 5T32 DK07477, as well as a Foundation for Digestive Health and Nutrition/AstraZeneca Fellowship/Faculty transition award (A. Broderick).


   ACKNOWLEDGMENTS
 
We thank Dr. Marie Egan and Ms. Elisa Ferreira (Yale University Medical Center, New Haven, CT) for providing heterozygous breeding pairs of CF mice together with helpful advice regarding their breeding and husbandry. We are grateful to Dr. Paul A. Dawson (Wake Forest University School of Medicine, Winston-Salem, NC) for providing antibodies to SLC10A2/ASBT; to Ms. Shou-An Liu (Brigham and Women's Hospital, Gastroenterology Division) for invaluable help with mouse surgery and husbandry; to Mr. Christopher MacDonald (Clinical Chemistry Core Laboratory, Children's Hospital, Boston, MA) for skilled assistance in measuring Cl– concentrations; and to Dr. Vincent Ricchiuti (Director, General Clinical Research Center Core Laboratory, Brigham and Women's Hospital) and his laboratory for their expert measurement of Na+, K+, and PO43– concentrations in hepatic bile samples.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. Carey, Brigham and Women's Hosp., Gastroenterology Div., 75 Francis St., Boston, MA 02115 (e-mail: mccarey{at}rics.bwh.harvard.edu)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alpini G, Roberts S, Kuntz SM, Ueno Y, Gubba S, Podila PV, LeSage G, LaRusso NF. Morphological, molecular, and functional heterogeneity of cholangiocytes from normal rat liver. Gastroenterology 110: 1636–1643, 1996.[CrossRef][Web of Science][Medline]
  2. Angelico M, Gandin C, Canuzzi P, Bertasi S, Cantafora A, De Santis A, Quattrucci S, Antonelli M. Gallstones in cystic fibrosis: a critical reappraisal. Hepatology 14: 768–775, 1991.[CrossRef][Web of Science][Medline]
  3. Apstein MD. Inhibition of biliary phospholipid and cholesterol secretion by bilirubin in the Sprague-Dawley and Gunn rat. Gastroenterology 87: 637–638, 1984.
  4. Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem 234: 466–468, 1959.[Free Full Text]
  5. Becker M, Staab D, Leiss O, von Bergmann K. Biliary lipid composition in patients with cystic fibrosis. J Pediatr Gastroenterol Nutr 8: 308–312, 1989.[Web of Science][Medline]
  6. Behrens I, Stenberg P, Artursson P, Kissel T. Transport of lipophilic drug molecules in a new mucus-secreting cell culture model based on HT29-MTX cells. Pharm Res 18: 1138–1145, 2001.[CrossRef][Web of Science][Medline]
  7. Benedetti A, Alvaro D, Bassotti C, Gigliozzi A, Ferretti G, La Rosa T, Di Sario A, Baiocchi L, Jezequel AM. Cytotoxicity of bile salts against biliary epithelium: a study in isolated bile ductule fragments and isolated perfused rat liver. Hepatology 26: 9–21, 1997.[Web of Science][Medline]
  8. Bijvelds MJ, Jorna H, Verkade HJ, Bot AG, Hofmann F, Agellon LB, Sinaasappel M, de Jonge HR. Activation of CFTR by ASBT-mediated bile salt absorption. Am J Physiol Gastrointest Liver Physiol 289: G870–G879, 2005.[Abstract/Free Full Text]
  9. Brink MA, Méndez-Sánchez N, Carey MC. Bilirubin cycles enterohepatically after ileal resection in the rat. Gastroenterology 110: 1945–1957, 1996.[Medline]
  10. Brink MA, Slors JF, Keulemans YC, Mok KS, De Waart DR, Carey MC, Groen AK, Tytgat GN. Enterohepatic cycling of bilirubin: a putative mechanism for pigment gallstone formation in ileal Crohn's disease. Gastroenterology 116: 1420–1427, 1999.[CrossRef][Medline]
  11. Brito MA, Silva RF, Brites D. Bilirubin induces loss of membrane lipids and exposure of phosphatidylserine in human erythrocytes. Cell Biol Toxicol 18: 181–192, 2002.[CrossRef][Web of Science][Medline]
  12. Cahalane MJ, Neubrand MW, Carey MC. Physical-chemical pathogenesis of pigment gallstones. Semin Liver Dis 8: 317–328, 1988.[Web of Science][Medline]
  13. Calligaris S, Cekic D, Roca-Burgos L, Gerin F, Mazzone G, Ostrow JD, Tiribelli C. Multidrug resistance associated protein 1 protects against bilirubin-induced cytotoxicity. FEBS Lett 580: 1355–1359, 2006.[CrossRef][Web of Science][Medline]
  14. Calligaris SD, Bellarosa C, Giraudi P, Wennberg RP, Ostrow JD, Tiribelli C. Cytotoxicity is predicted by unbound and not total bilirubin concentration. Pediatr Res 62: 576–580, 2007.[Web of Science][Medline]
  15. Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 19: 945–955, 1978.[Abstract]
  16. Carey MC. Physical-chemical properties of bile acids and their salts. In: Sterols and Bile Acids. New Comprehensive Biochemistry, edited by Danielsson H and Sjövall J. Amsterdam: Elsevier, 1985, p. 345–403.
  17. Carey MC, Duane WC. Enterohepatic circulation. In: The Liver: Biology and Pathobiology (3rd ed.), edited by Arias IM, Boyer JL, Fausto N, Jakoby WB, Schacter DA and Shafritz DA. New York: Raven, 1994, p. 719–767.
  18. Cesaratto L, Calligaris SD, Vascotto C, Deganuto M, Bellarosa C, Quadrifoglio F, Ostrow JD, Tiribelli C, Tell G. Bilirubin-induced cell toxicity involves PTEN activation through an APE1/Ref-1-dependent pathway. J Mol Med 85: 1099–1112, 2007.[CrossRef][Web of Science][Medline]
  19. Chinet T, Fouassier L, Dray-Charier N, Imam-Ghali M, Morel H, Mergey M, Dousset B, Parc R, Paul A, Housset C. Regulation of electrogenic anion secretion in normal and cystic fibrosis gallbladder mucosa. Hepatology 29: 5–13, 1999.[CrossRef][Web of Science][Medline]
  20. Cohn JA, Strong TV, Picciotto MR, Nairn AC, Collins FS, Fitz JG. Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells. Gastroenterology 105: 1857–1864, 1993.[Web of Science][Medline]
  21. Colledge WH, Abella BS, Southern KW, Ratcliff R, Jiang C, Cheng SH, MacVinish LJ, Anderson JR, Cuthbert AW, Evans MJ. Generation and characterization of a delta F508 cystic fibrosis mouse model. Nat Genet 10: 445–452, 1995.[CrossRef][Web of Science][Medline]
  22. Dawson PA, Haywood J, Craddock AL, Wilson M, Tietjen M, Kluckman K, Maeda N, Parks JS. Targeted deletion of the ileal bile acid transporter eliminates enterohepatic cycling of bile acids in mice. J Biol Chem 278: 33920–33927, 2003.[Abstract/Free Full Text]
  23. Delaney SJ, Alton EW, Smith SN, Lunn DP, Farley R, Lovelock PK, Thomson SA, Hume DA, Lamb D, Porteous DJ, Dorin JR, Wainwright BJ. Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations. EMBO J 15: 955–963, 1996.[Web of Science][Medline]
  24. Donovan JM, Leonard MR, Batta AK, Carey MC. Calcium affinity for biliary lipid aggregates in model biles: complementary importance of bile salts and lecithin. Gastroenterology 107: 831–846, 1994.[Medline]
  25. Durie PR, Kent G, Phillips MJ, Ackerley CA. Characteristic multiorgan pathology of cystic fibrosis in a long-living cystic fibrosis transmembrane regulator knockout murine model. Am J Pathol 164: 1481–1493, 2004.[Abstract/Free Full Text]
  26. Elliott WH. Metabolism of bile acids in liver and extrahepatic tissues. In: Sterols and Bile Acids. New Comprehensive Biochemistry, edited by Danielsson H and Sjövall J. Amsterdam: Elsevier, 1985, p. 303–329.
  27. Feranchak AP. Hepatobiliary complications of cystic fibrosis. Curr Gastroenterol Rep 6: 231–239, 2004.[CrossRef][Medline]
  28. Feranchak AP, Roman RM, Doctor RB, Salter KD, Toker A, Fitz JG. The lipid products of phosphoinositide 3-kinase contribute to regulation of cholangiocyte ATP and chloride transport. J Biol Chem 274: 30979–30986, 1999.[Abstract/Free Full Text]
  29. Feranchak AP, Sokol RJ. Cholangiocyte biology and cystic fibrosis liver disease. Semin Liver Dis 21: 471–488, 2001.[CrossRef][Web of Science][Medline]
  30. Fickert P, Fuchsbichler A, Wagner M, Zollner G, Kaser A, Tilg H, Krause R, Lammert F, Langner C, Zatloukal K, Marschall HU, Denk H, Trauner M. Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice. Gastroenterology 127: 261–274, 2004.[CrossRef][Web of Science][Medline]
  31. Fitz JG, Basavappa S, McGill J, Melhus O, Cohn JA. Regulation of membrane chloride currents in rat bile duct epithelial cells. J Clin Invest 91: 319–328, 1993.[Web of Science][Medline]
  32. Grubb BR, Boucher RC. Pathophysiology of gene-targeted mouse models for cystic fibrosis. Physiol Rev 79: S193–S214, 1999.[Medline]
  33. Guilbault C, Saeed Z, Downey GP, Radzioch D. Cystic fibrosis mouse models. Am J Respir Cell Mol Biol 36: 1–7, 2007.[Abstract/Free Full Text]
  34. Hayden UL, Carey HV. Cellular localization of cystic fibrosis transmembrane regulator protein in piglet and mouse intestine. Cell Tissue Res 283: 209–213, 1996.[CrossRef][Web of Science][Medline]
  35. Heuman DM. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions. J Lipid Res 30: 719–730, 1989.[Abstract]
  36. Hofmann AF. Enterohepatic circulation of bile acids. In: Handbook of Physiology. The Gastrointestinal System. Salivary, Gastric, Pancreatic, and Hepatobiliary Secretion. Bethesda, MD: Am. Physiol. Soc., 1989, sect. 6, vol. III, pt. 1, chapt. 29, p. 567–596.
  37. Hulzebos CV, Voshol PJ, Wolters H, Kruit JK, Ottenhof R, Groen AK, Stellaard F, Verkade HJ, Kuipers F. Bile duct proliferation associated with bile salt-induced hypercholeresis in Mdr2 P-glycoprotein-deficient mice. Liver Int 25: 604–612, 2005.[CrossRef][Web of Science][Medline]
  38. Jeske DJ, Dietschy JM. Regulation of rates of cholesterol synthesis in vivo in the liver and carcass of the rat measured using [3H]water. J Lipid Res 21: 364–376, 1980.[Abstract]
  39. Jones CA, Hofmann AF, Mysels KJ, Roda A. The effect of calcium and sodium ion concentration on the properties of dilute aqueous solutions of glycine conjugated bile salts: phase behavior and solubility products of the calcium salts of the common glycine conjugated bile acids. J Colloid Interface Sci 114: 452–470, 1986.[CrossRef][Web of Science]
  40. Kopelman H. Cystic fibrosis. 6. Gastrointestinal and nutritional aspects. Thorax 46: 261–267, 1991.[Free Full Text]
  41. Lack L, Weiner IM. The ileal bile salt transport system: effect of the charged state of the substrate on activity. Biochim Biophys Acta 135: 1065–1068, 1967.[Medline]
  42. Larhed AW, Artursson P, Björk E. The influence of intestinal mucus components on the diffusion of drugs. Pharm Res 15: 66–71, 1998.[CrossRef][Web of Science][Medline]
  43. Lindblad A, Glaumann H, Strandvik B. Natural history of liver disease in cystic fibrosis. Hepatology 30: 1151–1158, 1999.[CrossRef][Web of Science][Medline]
  44. Mazer NA, Benedek GB, Carey MC. Quasielastic light-scattering studies of aqueous biliary lipid systems. Mixed micelle formation in bile salt-lecithin solutions. Biochemistry 19: 601–615, 1980.[CrossRef][Web of Science][Medline]
  45. McGill JM, Gettys TW, Basavappa S, Fitz JG. GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells. J Membr Biol 133: 253–261, 1993.[Web of Science][Medline]
  46. Montet JC, Parquet M, Sacquet E, Montet AM, Infante R, Amic J. β-Muricholic acid; potentiometric and cholesterol-dissolving properties. Biochim Biophys Acta 918: 1–10, 1987.[Medline]
  47. Moore EW, Vérine HJ. Pancreatic calcification: formation constants of CaHCO3+ and CaCO30 complexes determined with Ca2+ electrode. Am J Physiol Gastrointest Liver Physiol 241: G182–G190, 1981.[Abstract/Free Full Text]
  48. Moore EW, Vérine HJ. Pathogenesis of pancreatic and biliary CaCO3 lithiasis: the solubility product (K'sp) of calcite determined with the Ca++ electrode. J Lab Clin Med 106: 611–618, 1985.[Web of Science][Medline]
  49. O'Brien S, Mulcahy H, Fenlon H, O'Broin A, Casey M, Burke A, FitzGerald MX, Hegarty JE. Intestinal bile acid malabsorption in cystic fibrosis. Gut 34: 1137–1141, 1993.[Abstract/Free Full Text]
  50. Oppenheimer EH, Esterly JR. Hepatic changes in young infants with cystic fibrosis: possible relation to focal biliary cirrhosis. J Pediatr 86: 683–689, 1975.[CrossRef][Web of Science][Medline]
  51. Ostrow JD, Pascolo L, Brites D, Tiribelli C. Molecular basis of bilirubin-induced neurotoxicity. Trends Mol Med 10: 65–70, 2004.[CrossRef][Web of Science][Medline]
  52. Porter JL, Fordtran JS, Santa Ana CA, Emmett M, Hagey LR, Macdonald EA, Hofmann AF. Accurate enzymatic measurement of fecal bile acids in patients with malabsorption. J Lab Clin Med 141: 411–418, 2003.[CrossRef][Web of Science][Medline]
  53. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, Drumm ML, Iannuzzi MC, Collins FS, Tsui LC. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066–1073, 1989.[Abstract/Free Full Text]
  54. Roman RM, Feranchak AP, Salter KD, Wang Y, Fitz JG. Endogenous ATP release regulates Cl– secretion in cultured human and rat biliary epithelial cells. Am J Physiol Gastrointest Liver Physiol 276: G1391–G1400, 1999.[Abstract/Free Full Text]
  55. Roman RM, Wang Y, Fitz JG. Regulation of cell volume in a human biliary cell line: activation of K+ and Cl– currents. Am J Physiol Gastrointest Liver Physiol 271: G239–G248, 1996.[Abstract/Free Full Text]
  56. Rossi SS, Converse JL, Hofmann AF. High pressure liquid chromatographic analysis of conjugated bile acids in human bile: simultaneous resolution of sulfated and unsulfated lithocholyl amidates and the common conjugated bile acids. J Lipid Res 28: 589–595, 1987.[Abstract]
  57. Rowe SM, Miller S, Sorscher EJ. Cystic fibrosis. N Engl J Med 352: 1992–2001, 2005.[Free Full Text]
  58. Roy CC, Weber AM, Morin CL, Combes JC, Nussle D, Megevand A, Lasalle R. Abnormal biliary lipid composition in cystic fibrosis. Effect of pancreatic enzymes. N Engl J Med 297: 1301–1305, 1977.[Abstract]
  59. Schlenker T, Fitz JG. Ca2+-activated C1– channels in a human biliary cell line: regulation by Ca2+/calmodulin-dependent protein kinase. Am J Physiol Gastrointest Liver Physiol 271: G304–G310, 1996.[Abstract/Free Full Text]
  60. Small DM. The physical chemistry of cholanic acids. In: The Bile Acids: Chemistry, Physiology, and Metabolism, edited by Kritchevsky D and Nair PP. New York: Plenum, 1971, p. 249–353.
  61. Smith BF, LaMont JT. Hydrophobic binding properties of bovine gallbladder mucin. J Biol Chem 259: 12170–12177, 1984.[Abstract/Free Full Text]
  62. Sokol RJ, Straka MS, Dahl R, Devereaux MW, Yerushalmi B, Gumpricht E, Elkins N, Everson G. Role of oxidant stress in the permeability transition induced in rat hepatic mitochondria by hydrophobic bile acids. Pediatr Res 49: 519–531, 2001.[Web of Science][Medline]
  63. Spivak W, Yuey W. Application of a rapid and efficient h.p.l.c. method to measure bilirubin and its conjugates from native bile and in model bile systems. Potential use as a tool for kinetic reactions and as an aid in diagnosis of hepatobiliary disease. Biochem J 234: 101–109, 1986.[Web of Science][Medline]
  64. Strandvik B, Einarsson K, Lindblad A, Angelin B. Bile acid kinetics and biliary lipid composition in cystic fibrosis. J Hepatol 25: 43–48, 1996.[CrossRef][Web of Science][Medline]
  65. Trauner M, Wagner M, Fickert P, Zollner G. Molecular regulation of hepatobiliary transport systems: clinical implications for understanding and treating cholestasis. J Clin Gastroenterol 39: S111–S124, 2005.[CrossRef][Web of Science][Medline]
  66. Trotman BW, Bernstein SE, Bove KE, Wirt GD. Studies on the pathogenesis of pigment gallstones in hemolytic anemia: description and characteristics of a mouse model. J Clin Invest 65: 1301–1308, 1980.[Web of Science][Medline]
  67. Turley SD, Dietschy JM. Re-evaluation of the 3 alpha-hydroxysteroid dehydrogenase assay for total bile acids in bile. J Lipid Res 19: 924–928, 1978.[Abstract]
  68. van Doorninck JH, French PJ, Verbeek E, Peters RH, Morreau H, Bijman J, Scholte BJ. A mouse model for the cystic fibrosis delta F508 mutation. EMBO J 14: 4403–4411, 1995.[Web of Science][Medline]
  69. Vawter GF, Shwachman H. Cystic fibrosis in adults: an autopsy study. Pathol Annu 14: 357–382, 1979.[Medline]
  70. Vercaemst R, Union A, Rosseneu M. Separation and quantitation of free cholesterol and cholesteryl esters in a macrophage cell line by high-performance liquid chromatography. J Chromatogr 494: 43–52, 1989.[Web of Science][Medline]
  71. Verkade HJ, Wolbers MJ, Havinga R, Uges DR, Vonk RJ, Kuipers F. The uncoupling of biliary lipid from bile acid secretion by organic anions in the rat. Gastroenterology 99: 1485–1492, 1990.[Web of Science][Medline]
  72. Vítek L, Carey MC. Enterohepatic cycling of bilirubin as a cause of ‘black’ pigment gallstones in adult life. Eur J Clin Invest 33: 799–810, 2003.[CrossRef][Web of Science][Medline]
  73. Watkins JB, Tercyak AM, Szczepanik P, Klein PD. Bile salt kinetics in cystic fibrosis: influence of pancreatic enzyme replacement. Gastroenterology 73: 1023–1028, 1977.[Medline]
  74. Weber AM, Roy CC, Morin CL, Lasalle R. Malabsorption of bile acids in children with cystic fibrosis. N Engl J Med 289: 1001–1005, 1973.[Web of Science][Medline]
  75. Wiedmann TS, Liang W, Herrington H. Interaction of bile salts with gastrointestinal mucins. Lipids 39: 51–58, 2004.[CrossRef][Web of Science][Medline]
  76. Zeiher BG, Eichwald E, Zabner J, Smith JJ, Puga AP, McCray PB Jr, Capecchi MR, Welsh MJ, Thomas KR. A mouse model for the delta F508 allele of cystic fibrosis. J Clin Invest 96: 2051–2064, 1995.[Web of Science][Medline]
  77. Zentler-Munro PL. Cystic fibrosis—a gastroenterological cornucopia. Gut 28: 1531–1547, 1987.[Free Full Text]
  78. Zentler-Munro PL, Fitzpatrick WJ, Batten JC, Northfield TC. Effect of intrajejunal acidity on aqueous phase bile acid and lipid concentrations in pancreatic steatorrhoea due to cystic fibrosis. Gut 25: 500–507, 1984.[Abstract/Free Full Text]




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