Volume 130, Issue 2, Pages 465-481 (February 2006) 24-norUrsodeoxycholic Acid Is Superior to Ursodeoxycholic Acid in the Treatment of Sclerosing Cholangitis in Mdr2 (Abcb4) Knockout Mice  Peter Fickert, Martin Wagner, Hanns–Ulrich Marschall, Andrea Fuchsbichler, Gernot Zollner, Oleksiy Tsybrovskyy, Kurt Zatloukal, Jie Liu, Michael P. Waalkes, Cathleen Cover, Helmut Denk, Alan F. Hofmann, Hartmut Jaeschke, Michael Trauner  Gastroenterology  Volume 130, Issue 2, Pages 465-481 (February 2006) DOI: 10.1053/j.gastro.2005.10.018 Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 1 norUDCA reduces serum alanine transaminase (ALT) and alkaline phosphatase (AP) levels, whereas UDCA significantly increases both enzymes in Mdr2−/− mice. Control diet–fed wild-type animals (WT), control diet–fed Mdr2−/− mice (KO), UDCA-fed Mdr2−/− mice (KO + UDCA), and norUDCA-fed Mdr2−/− mice (KO + norUDCA) are shown. Values are mean ± SD from 5 animals per group. P < .05: *WT vs KO, †KO vs KO + UDCA, ‡KO vs KO + norUDCA. Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 2 norUDCA ameliorates sclerosing cholangitis in Mdr2−/− mice. (A) Liver histology (H&E staining) in control diet–fed wild-type mouse (WT), control diet–fed Mdr2−/− mouse (KO), UDCA-fed Mdr2−/− mouse (KO + UDCA), and norUDCA-fed Mdr2−/− mouse (KO + norUDCA) is shown. The pronounced large bile duct disease in KO was significantly reduced in KO + UDCA and virtually absent in KO + norUDCA. (B) Sirius red staining showing significant fibrosis with periductal collagen fibers (red) in KO. There was a moderate reduction of fibrosis in KO + UDCA and a pronounced reduction in KO + norUDCA (original magnification, 10×). bd, bile duct; pv, portal vein. Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 3 norUDCA reduces ductular proliferation in Mdr2−/− mice. (A) Immunohistochemical staining of bile duct epithelial cells by using an anti-CK19 antibody in control diet–fed wild-type mouse (WT), control diet–fed Mdr2−/− mouse (KO), UDCA-fed Mdr2−/− mouse (KO + UDCA), and norUDCA-fed Mdr2−/− mouse (KO + norUDCA) is shown (original magnification, 20×). Pronounced ductular proliferation in KO was slightly reduced in KO + UDCA but virtually absent in KO + norUDCA. (B) Hepatic CK19 protein levels were determined in homogenates from all experimental groups by Western blotting. Densitometry data are expressed as the n-fold change relative to control diet–fed wild-type mice. Values are the average obtained from 3 animals per group. Note the significant increase in CK19 protein levels in KO and KO + UDCA compared to WT, whereas CK19 protein levels were significantly reduced in KO + norUDCA. The specificity of these findings is confirmed by unchanged β-actin expression levels. P < .05: *WT vs KO and KO + UDCA; †KO vs KO + norUDCA. Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 4 norUDCA significantly reduces hepatic hydroxyproline content in Mdr2−/− mice. Hepatic hydroxyproline content was determined in liver homogenates from wild-type mice (WT), control diet–fed Mdr2−/− mice (KO), UDCA-fed Mdr2−/− mice (KO + UDCA), and norUDCA-fed Mdr2−/− mice (KO + norUDCA). Hepatic hydroxyproline content was significantly increased in KO compared with WT and was nearly reduced to baseline levels in KO + norUDCA animals. Values are mean ± SD from 5 animals per group. P < .05: *WT vs KO, †KO vs KO + norUDCA. Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 5 norUDCA and UDCA reduce α-SMA expression of periductal myofibroblasts. (A) Immunohistochemical staining of periductal myofibroblasts by using an anti–α-SMA antibody in control diet–fed wild-type mouse (WT), control diet–fed Mdr2−/− mouse (KO), UDCA-fed Mdr2−/− mouse (KO + UDCA), and norUDCA-fed Mdr2−/− mouse (KO + norUDCA) is shown (original magnification, 40×). Pronounced periductal fibrosis in KO was shown with an increased number of periductal α-SMA–positive periductal myofibroblasts, which were reduced in KO + UDCA and in KO + norUDCA. (B) α-SMA levels were assessed in liver homogenates from all experimental groups by Western blotting. Densitometry data are expressed as the n-fold change relative to WT. Values are the average obtained from 3 animals per group. Note the significant decrease in α-SMA protein levels in KO + UDCA and KO + norUDCA . P < .05: *WT vs KO, †KO vs KO + UDCA and KO + norUDCA. bd, bile duct; ha, hepatic arteric; pv, portal vein. Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 6 norUDCA reduces portal vascular cell adhesion molecule 1 (VCAM-1) expression in Mdr2−/− mice. Immunohistochemical staining for VCAM-1 (red) in small (A) and large (B) bile ducts of a control diet–fed wild-type mouse (WT), control diet–fed Mdr2−/− mouse (KO), UDCA-fed Mdr2−/− mouse (KO + UDCA), and a norUDCA-fed Mdr2−/− mouse (KO + norUDCA) is shown. (A) WT liver stains negative for VCAM. No obvious difference exists in portal VCAM-1 staining between KO and KO + UDCA, whereas in KO + norUDCA, VCAM-1 staining is significantly reduced. (B) At the level of the large bile ducts, both UDCA and norUDCA reduce cholangiocellular VCAM-1 staining, but the reduction is more pronounced in KO + norUDCA. (C) Morphometric analysis of VCAM-1 staining showing a significant increase in VCAM expression in KO and KO + UDCA but a reduction in KO + norUDCA. (D) VCAM-1 protein levels were assessed in liver homogenates from all experimental groups by Western blotting. Densitometry data are expressed as the n-fold change relative to WT. Values are the average obtained from 3 animals per group. Note the significant decrease in VCAM protein levels in KO + norUDCA. P < .05: *WT vs KO, †KO vs KO + UDCA, ‡KO vs KO + norUDCA (original magnification: A, 63×; B, 40×). bd, bile duct. Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 7 norUDCA reduces portal neutrophils in Mdr2−/− mice. Immunohistochemistry for CD11b (red, staining neutrophils) in a control diet–fed wild-type mouse (WT), control diet–fed Mdr2−/− mouse (KO), UDCA-fed Mdr2−/− mouse (KO + UDCA), and a norUDCA-fed Mdr2−/− mouse (KO + norUDCA) of (A) small and (B) large bile ducts is shown. (C) Quantification of CD11b-positive cells per 20 portal fields. norUDCA significantly reduces the number of CD11b-positive cells in Mdr2−/− mice. Values are mean ± SD from 3 animals per group. P < .05: *WT vs KO, †WT vs KO + norUDCA, ‡KO vs KO + norUDCA. bd, bile duct (original magnification: A, 63×; B, 40×). Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 8 norUDCA reduces oxidative stress in Mdr2−/− mice. Immunohistochemical staining for 5-hydroxynonenal (HNE) protein adducts (red) in small (A) and large (B) bile ducts of a control diet–fed wild-type mouse (WT), control diet–fed Mdr2−/− mouse (KO), UDCA-fed Mdr2−/− mouse (KO + UDCA), and norUDCA-fed Mdr2−/− mouse (KO + norUDCA) is shown. (A) WT liver stains negative for HNE. No obvious difference in portal HNE staining between KO and KO + UDCA was observed. In contrast, KO + norUDCA shows significantly reduced HNE staining compared with KO (original magnification: A, 40×; B, 63×). bd, bile duct. Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 9 norUDCA inhibits proliferation of hepatocytes and cholangiocytes of large bile ducts in Mdr2−/− mice. Immunohistochemical staining for Ki-67 (red) in hepatocytes (A) and cholangiocytes (B) in a control diet–fed wild-type mouse (WT), control diet–fed Mdr2−/− mouse (KO), UDCA-fed Mdr2−/− mouse (KO + UDCA), and norUDCA-fed Mdr2−/− mouse (KO + norUDCA) is shown. (A) Numerous Ki-67–positive hepatocytes in KO and KO + UDCA and scattered positive nuclei in KO + norUDCA are shown. (B) Numerous Ki-67–positive cholangiocytes in KO and few positive cholangiocytes (arrowheads) in KO + UDCA and KO + norUDCA are shown. (C) The number of Ki-67–positive hepatocytes per 30 high-power fields and the number of Ki-67–positive cholangiocytes per 20 portal fields are shown. Only norUDCA significantly reduced the number of both proliferating hepatocytes and cholangiocytes of large ducts. Values are mean ± SD from 3 animals per group. P < .05: *WT vs KO, †WT vs KO + UDCA, ‡KO vs KO + norUDCA. bd, bile duct. Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 10 norUDCA induces basolateral Mrp3 and Mrp4 protein levels in Mdr2−/− mice. Liver membranes were isolated from control diet–fed Mdr2−/− mice (KO), UDCA-fed (KO + UDCA) mice, and norUDCA-fed (KO + norUDCA) Mdr2−/− mice and analyzed by Western blotting. Densitometry data are expressed as the n-fold change relative to KO. Values are the average obtained from 5 animals per group. Note the significant increase in Mrp3 and Mrp4 protein levels in KO + UDCA and KO + norUDCA, which is more pronounced in KO + norUDCA. *P < .05, KO vs KO + UDCA and KO + norUDCA. Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 11 Electrospray mass spectrometry of bile from norUDCA-fed Mdr2−/− mice. Quasi-molecular negative ions at m/z 377, 393, and 409 semiquantitatively represent the abundance of unconjugated dihydroxylated, trihydroxylated, and tetrahydroxylated nor-bile acids, at m/z 484, 500, 553, and 569 represent dihydroxylated and trihydroxylated nor-bile acids conjugated with taurine or glucuronic acid. Ions at m/z 484 and m/z 514 represent sulfated norUDCA and taurine-conjugated C-24 bile acids. Gastroenterology 2006 130, 465-481DOI: (10.1053/j.gastro.2005.10.018) Copyright © 2006 American Gastroenterological Association Terms and Conditions