Volume 44, Issue 4, Pages 768-775 (April 2006) Mice lacking Mrp3 (Abcc3) have normal bile salt transport, but altered hepatic transport of endogenous glucuronides  Noam Zelcer, Koen van de Wetering, Rudi de Waart, George L. Scheffer, Hanns-Ulrich Marschall, Peter R. Wielinga, Annemieke Kuil, Cindy Kunne, Alexander Smith, Martin van der Valk, Jan Wijnholds, Ronald Oude Elferink, Piet Borst  Journal of Hepatology  Volume 44, Issue 4, Pages 768-775 (April 2006) DOI: 10.1016/j.jhep.2005.07.022 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Tissue distribution of Mrp3 in mice. (A) Detection of Mrp3 in total tissue lysates (30μg protein per lane) by immunoblot analysis. α-Tubulin was used as loading control. (B) Immunolocalization of Mrp3 in tissue sections of WT and Mrp3(−/−) mice. Journal of Hepatology 2006 44, 768-775DOI: (10.1016/j.jhep.2005.07.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Detection of Mrp3 and Mrp2 in BDL livers. (A) Mrp3 was analyzed in liver sections from untreated or 3-day BDL WT mice as indicated in the figure. Control stainings in which the αMrp3 antiserum was replaced by normal rabbit serum are shown in the lower panels. (B) Total liver lysates from untreated WT, 3-day BDL WT, and 3-day BDL Mrp3(−/−) (n=3 mice per group) were prepared and 40μg protein loaded per lane. Mrp3 and Mrp2 were detected as described in methods. L.c, Gel loading was controlled by ponceau staining of blotted membranes (not shown) and with a non-specific cross-reacting band. Journal of Hepatology 2006 44, 768-775DOI: (10.1016/j.jhep.2005.07.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Serum levels of total bile salts and bilirubin glucuronide after 3 days of BDL in WT and Mrp3(−/−) mice. Serum from BDL mice was collected after 3 days. (A) Total serum bile salts (n=10 mice per group). The serum bile salt levels of untreated WT and Mrp3(−/−) were 1.6±0.9 and 1.9±1.2μM, respectively (n=5, P=0.65). (B) Serum concentrations of bilirubin glucuronide. Each bar and error represent the mean±SD of the total serum bilirubin glucuronide concentration (n=15 mice per group). The serum bilirubin levels of untreated WT and Mrp3(−/−) are not different (see Supplementary Table 1). Journal of Hepatology 2006 44, 768-775DOI: (10.1016/j.jhep.2005.07.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Time-, concentration- and ATP-dependent transport of HDC-GlcA and HC-GlcA by MRP3. Membrane vesicles containing MRP3 (circles) or control membranes (squares) from Sf9 insect cells were incubated at 37°C in the presence of 4mM ATP (closed symbols) or 4mM AMP (open symbols). (A, B) Time-dependent transport of HDC-GlcA and HC-GlcA, respectively. (C, D) Concentration-dependent transport of HDC-GlcA and HDC-GlcA, respectively. Note that lines representing transport in WT vesicles and in MRP3-containing vesicles in the presence of AMP overlap and are therefore not visible separately. Insets represent Lineweaver–Burk transformations of the presented data. Values are presented as means±SD. Journal of Hepatology 2006 44, 768-775DOI: (10.1016/j.jhep.2005.07.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Cumulative excretion of HDC-GlcA and THDC in perfusate and bile after perfusion of mouse liver with HDC. Livers of WT and Mrp3(−/−) mice (three mice per group) were perfused with Krebs-bicarbonate buffer supplemented with 225nmol HDC per min and the cumulative excretion of THDC (A) and HDC-GlcA (B) in perfusate and bile was determined as described in Section 2. Journal of Hepatology 2006 44, 768-775DOI: (10.1016/j.jhep.2005.07.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions