Volume 118, Issue 1, Pages 163-172 (January 2000) Expression of the bile salt export pump is maintained after chronic cholestasis in the rat  John M. Lee, Michael Trauner, Carol J. Soroka, Bruno Stieger, Peter J. Meier, James L. Boyer  Gastroenterology  Volume 118, Issue 1, Pages 163-172 (January 2000) DOI: 10.1016/S0016-5085(00)70425-2 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of CBDL on the expression of Bsep, P-gp, and Mrp2 protein levels. Membrane fractions were isolated from total liver obtained from sham-operated and CBDL rats 1, 3, 5, 7, and 14 days after initial surgery and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). (A) Representative immunoblots. (B) Densitometric analysis on 4-6 independent control and CBDL samples at each time point. Data (mean ± SEM) are expressed as percentages of values in sham-operated controls (*P < 0.05 compared with controls). Bile duct ligation leads to a maximal decrease in Bsep expression 3 days after CBDL. Thereafter, a partial recovery of Bsep expression is evident. P-gp expression increases severalfold and remains elevated; Mrp2 expression remains significantly down-regulated at all time points after 3 days. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of CBDL on the expression of Bsep, P-gp, and Mrp2 protein levels. Membrane fractions were isolated from total liver obtained from sham-operated and CBDL rats 1, 3, 5, 7, and 14 days after initial surgery and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). (A) Representative immunoblots. (B) Densitometric analysis on 4-6 independent control and CBDL samples at each time point. Data (mean ± SEM) are expressed as percentages of values in sham-operated controls (*P < 0.05 compared with controls). Bile duct ligation leads to a maximal decrease in Bsep expression 3 days after CBDL. Thereafter, a partial recovery of Bsep expression is evident. P-gp expression increases severalfold and remains elevated; Mrp2 expression remains significantly down-regulated at all time points after 3 days. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of endotoxin (LPS, 16 hours) on the expression of Bsep and other canalicular transport proteins. Membrane fractions were isolated from total liver obtained from control (n = 4) and endotoxin-treated (n = 4) rats and subjected to SDS-PAGE. (A) Representative immunoblots. (B) Densitometric analysis on autoradiographs obtained after probing for Bsep, P-gp, and Mrp2. Data (mean ± SEM) are expressed as percentages of values in saline-injected controls (*P < 0.05 compared with controls). Endotoxin leads to a substantial decrease in Mrp2 expression; Bsep protein levels are also decreased, but not to the same degree. P-gp levels as assessed by C219 are unchanged. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of endotoxin (LPS, 16 hours) on the expression of Bsep and other canalicular transport proteins. Membrane fractions were isolated from total liver obtained from control (n = 4) and endotoxin-treated (n = 4) rats and subjected to SDS-PAGE. (A) Representative immunoblots. (B) Densitometric analysis on autoradiographs obtained after probing for Bsep, P-gp, and Mrp2. Data (mean ± SEM) are expressed as percentages of values in saline-injected controls (*P < 0.05 compared with controls). Endotoxin leads to a substantial decrease in Mrp2 expression; Bsep protein levels are also decreased, but not to the same degree. P-gp levels as assessed by C219 are unchanged. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of EE on the expression of Bsep and other canalicular transport proteins. Membrane fractions were isolated from total liver obtained from control and estrogen-treated rats after 5 and 10 days of treatment and subjected to SDS-PAGE. (A) Representative Western blots. (B) Densitometric analysis on autoradiographs obtained after probing for Bsep, P-gp, and Mrp2 on 4 (5-day group) and 8 (10-day group) independent liver samples from control and EE-treated rats. Data (mean ± SEM) are expressed as percentages of control values (*P < 0.05 compared with controls). Estrogen leads to a substantial decrease in Mrp2 expression; Bsep protein levels are also decreased, but not to the same degree. P-gp levels as assessed by C219 are unchanged. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of EE on the expression of Bsep and other canalicular transport proteins. Membrane fractions were isolated from total liver obtained from control and estrogen-treated rats after 5 and 10 days of treatment and subjected to SDS-PAGE. (A) Representative Western blots. (B) Densitometric analysis on autoradiographs obtained after probing for Bsep, P-gp, and Mrp2 on 4 (5-day group) and 8 (10-day group) independent liver samples from control and EE-treated rats. Data (mean ± SEM) are expressed as percentages of control values (*P < 0.05 compared with controls). Estrogen leads to a substantial decrease in Mrp2 expression; Bsep protein levels are also decreased, but not to the same degree. P-gp levels as assessed by C219 are unchanged. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of CBDL on Bsep and Ntcp steady-state mRNA levels. Total and poly(A)+ RNA was isolated from sham-operated and CBDL rats 1, 3, 5, 7, and 14 days after initial surgery, and mRNA levels were quantified using 32P-labeled cDNAs. (A) Representative Northern blots. (B) Phosphorimager analysis performed for both Bsep and Ntcp. Differences in loading were corrected by normalizing to GAPDH. Data (mean ± SEM) are expressed as percentages of values in sham-operated controls for 4-6 animals at each time point (*P < 0.05 compared with controls). Bile duct ligation leads to a maximal decrease in both Bsep and ntcp expression after 3 days CBDL. Thereafter, partial recovery of Bsep expression is evident. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of CBDL on Bsep and Ntcp steady-state mRNA levels. Total and poly(A)+ RNA was isolated from sham-operated and CBDL rats 1, 3, 5, 7, and 14 days after initial surgery, and mRNA levels were quantified using 32P-labeled cDNAs. (A) Representative Northern blots. (B) Phosphorimager analysis performed for both Bsep and Ntcp. Differences in loading were corrected by normalizing to GAPDH. Data (mean ± SEM) are expressed as percentages of values in sham-operated controls for 4-6 animals at each time point (*P < 0.05 compared with controls). Bile duct ligation leads to a maximal decrease in both Bsep and ntcp expression after 3 days CBDL. Thereafter, partial recovery of Bsep expression is evident. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of endotoxin (LPS, 16 hours) on Bsep, Mrp2, and Ntcp steady-state mRNA levels. Total and poly(A) RNA was isolated from control (n = 4) and endotoxin-treated (n = 4) rats, and mRNA levels were quantified using 32P-labeled cDNAs. (A) Representative autoradiographs. (B) Phosphorimager analysis performed for Bsep, Mrp2, and Ntcp after differences in loading were corrected by normalizing to GAPDH. Data (mean ± SEM) are expressed as percentages of control values (*P < 0.05 compared with controls). Endotoxin leads to substantial decreases in both Mrp2 and Ntcp expression; Bsep mRNA levels are also decreased, but not to the same degree. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of endotoxin (LPS, 16 hours) on Bsep, Mrp2, and Ntcp steady-state mRNA levels. Total and poly(A) RNA was isolated from control (n = 4) and endotoxin-treated (n = 4) rats, and mRNA levels were quantified using 32P-labeled cDNAs. (A) Representative autoradiographs. (B) Phosphorimager analysis performed for Bsep, Mrp2, and Ntcp after differences in loading were corrected by normalizing to GAPDH. Data (mean ± SEM) are expressed as percentages of control values (*P < 0.05 compared with controls). Endotoxin leads to substantial decreases in both Mrp2 and Ntcp expression; Bsep mRNA levels are also decreased, but not to the same degree. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of EE on Bsep, Mrp2, and Ntcp steady-state mRNA levels. Total and poly(A) RNA were isolated from control (n = 4) and estrogen-treated (n = 4) rats, and mRNA levels were quantified using 32P-labeled cDNAs. (A) Representative autoradiographs. (B) Phosphorimager analysis performed for Bsep, Mrp2, and Ntcp after differences in loading were corrected by normalizing to GAPDH. Data (mean ± SEM) are expressed as percentages of control values (*P < 0.05 compared with controls). EE leads to substantial decreases in Ntcp expression; Bsep and Mrp2 mRNA levels are unchanged. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of EE on Bsep, Mrp2, and Ntcp steady-state mRNA levels. Total and poly(A) RNA were isolated from control (n = 4) and estrogen-treated (n = 4) rats, and mRNA levels were quantified using 32P-labeled cDNAs. (A) Representative autoradiographs. (B) Phosphorimager analysis performed for Bsep, Mrp2, and Ntcp after differences in loading were corrected by normalizing to GAPDH. Data (mean ± SEM) are expressed as percentages of control values (*P < 0.05 compared with controls). EE leads to substantial decreases in Ntcp expression; Bsep and Mrp2 mRNA levels are unchanged. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Indirect immunofluorescence localization of Bsep in liver tissue from sham-operated and CBDL rat livers. Representative results are shown for selected time points. (A) Normal staining pattern for Bsep in liver from a representative control; fluorescent labeling delineates the canalicular membranes. (B-D) Staining pattern for Bsep in livers from animals 3, 5, and 14 days after CBDL, showing a less linear definition but a persisting immunofluorescence pattern at the canalicular membrane up to 14 days after bile duct ligation. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Indirect immunofluorescence localization of Bsep in rat livers from endotoxin (LPS)- and EE-treated animals with corresponding controls. (A) Normal staining pattern for Bsep in liver from a representative saline-injected control; fluorescent labeling delineates the canalicular membranes. (B) Staining pattern for Bsep in liver from animals treated with LPS (16 hours, 1 mg/kg body wt) showing a less defined but persisting immunofluorescence pattern at the canalicular membrane. (C) Normal staining pattern for Bsep in liver from a representative vehicle (1,2-propanediol)-injected control; fluorescent labeling delineates the canalicular membranes. (D) Staining pattern for Bsep in liver from animals treated with 17α-ethinylestradiol (10 days, 5 mg/kg body wt daily) showing a reduced immunofluorescence staining pattern at the canalicular membrane. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 The effects of obstructive cholestasis on in vivo bile salt output were determined after cannulation of the common bile duct 3 (●) and 14 (▴) days after CBDL or sham operation (■) (n = 5 in all groups). There was a nonsignificant reduction in bile salt output over the 60-minute collection time in the sham-operated animals, reflecting a reduction in the bile salt pool. An initial nonsignificant increase in bile salt output after 3 days of CBDL later subsided. Although the overall rate of bile salt output was significantly reduced after 14 days of CBDL (#P < 0.01 compared with controls), bile salt output continued throughout the 60-minute collection period. Gastroenterology 2000 118, 163-172DOI: (10.1016/S0016-5085(00)70425-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions