Side chain structure determines unique physiologic and therapeutic properties of norursodeoxycholic acid in Mdr2-/- mice

Abstract

24-norursodeoxycholic acid (norUDCA), a side chain-modified ursodeoxycholic acid derivative, has dramatic therapeutic effects in experimental cholestasis and may be a promising agent for the treatment of cholestatic liver diseases. We aimed to better understand the physiologic and therapeutic properties of norUDCA and to test if they are related to its side chain length and/or relative resistance to amidation. For this purpose, Mdr2(-/-) mice, a model for sclerosing cholangitis, received either a standard diet or a norUDCA-, tauro norursodeoxycholic acid (tauro- norUDCA)-, or di norursodeoxycholic acid (di norUDCA)-enriched diet. Bile composition, serum biochemistry, liver histology, fibrosis, and expression of key detoxification and transport systems were investigated. Direct choleretic effects were addressed in isolated bile duct units. The role of Cftr for norUDCA-induced choleresis was explored in Cftr(-/-) mice. norUDCA had pharmacologic features that were not shared by its derivatives, including the increase in hepatic and serum bile acid levels and a strong stimulation of biliary HCO(3)(-)-output. norUDCA directly stimulated fluid secretion in isolated bile duct units in a HCO(3)(-)-dependent fashion to a higher extent than the other bile acids. Notably, the norUDCA significantly stimulated HCO(3)(-)-output also in Cftr(-/-) mice. In Mdr2(-/-) mice, cholangitis and fibrosis strongly improved with norUDCA, remained unchanged with tauro- norUDCA, and worsened with di norUDCA. Expression of Mrp4, Cyp2b10, and Sult2a1 was increased by norUDCA and di norUDCA, but was unaffected by tauro- norUDCA.

Conclusion: The relative resistance of norUDCA to amidation may explain its unique physiologic and pharmacologic properties. These include the ability to undergo cholehepatic shunting and to directly stimulate cholangiocyte secretion, both resulting in a HCO(3)(-)-rich hypercholeresis that protects the liver from cholestatic injury.

Fig. 1

norUDCA is more effective than tauro-norUDCA and dinorUDCA in reducing biliary fibrosis in Mdr2^−/− mice. (A) Sirius red staining of liver in standard chow-fed (control), norUDCA-fed, tauro-norUDCA-fed (T-norUDCA), and dinorUDCA-fed Mdr2^−/− mice. Compared with striking reduction of fibrosis with periductal collagen fibers (red) in norUDCA-treated Mdr2^−/− mice, the effects of tauro-norUDCA and dinorUDCA were much weaker (magnification ×20). bd, bile duct; pv, portal vein. (B) Western blot analysis of hepatic α-SMA protein levels (marker for activated myofibroblasts) in liver homogenates of standard chow-fed (control), norUDCA-fed, tauro-norUDCA-fed (T-norUDCA), and dinorUDCA-fed Mdr2^−/− mice. Densitometry data are expressed as n-fold change relative to standard chow-fed Mdr2^−/− mice. Values are expressed as the mean ± standard deviation. Note a significant decrease in α-SMA protein levels by norUDCA treatment. *P < 0.05 versus control.

Fig. 2

norUDCA, but not tauro-norUDCA and dinorUDCA, reduce ductular proliferation in Mdr2^−/− mice. (A) Immunohistochemistry of cholangiocytes using an anti-K19 antibody in standard chow-fed (control), norUDCA-fed, tauro-norUDCA-fed (T-norUDCA), and dinorUDCA-fed Mdr2^−/− mice (magnification ×20). Ductular proliferation in standard chow-fed Mdr2^−/− mice was reduced by norUDCA and remained unchanged after tauro-norUDCA. dinorUDCA even increased ductular proliferation in Mdr2^−/− mice. (B) Western blot analysis of hepatic K19 protein levels in liver homogenates of standard chow-fed (control), norUDCA-fed, tauro-norUDCA-fed (T-norUDCA), and dinorUDCA-fed Mdr2^−/− mice. Densitometry data are expressed as n-fold change relative to standard chow-fed Mdr2^−/− mice. Values are expressed as the mean ± standard deviation. Note the significant decrease in K19 protein levels by norUDCA and significant increase by dinorUDCA. *P < 0.05 versus control. #P < 0.05 versus norUDCA.

Fig. 3

Effects of nor-bile acids on hepatic messenger RNA expression of phase I and II detoxification enzymes and alternative bile acid transporter. Relative messenger RNA expression of Cyp2b10, Sult2a1, and Mrp4 of standard chow-fed (control), norUDCA-fed, tauro-norUDCA-fed (T-norUDCA), and dinorUDCA-fed Mdr2^−/− mice. Values are expressed as n-fold change compared with the control group (mean ± standard deviation of five animals). norUDCA and dinorUDCA induced both detoxification enzymes Cyp2b10 and Sult2a1 as well as alternative export pump Mrp4. The effects of T-norUDCA were less pronounced. *P < 0.05 versus control. #P < 0.05 versus norUDCA.