Inhibition of Ileal Bile Acid Transport is Protective Against Cholestatic Liver Injury in Cyp2c70‐/‐Mice with Humanized Bile Acid Composition

Abstract

Cyp2c70 is the key murine liver enzyme responsible for the synthesis of 6‐hydroxylated muricholates. Cyp2c70 knockout (2c70‐/‐) mice present with a more human‐like bile acid (BA) composition devoid of protective, hydrophilic muricholic acids and with features of cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be protective in cholestatic models such as the Mdr2‐/‐ mouse. The aims of this study were to determine if IBAT inhibition is protective in 2c70‐/‐ mice and identify the underlying molecular mechanisms.Methods2c70‐/‐ mice (C57Bl/6J) were generated using CRISPR/Cas9 technology. Male and female wild type (WT) and 2c70‐/‐ mice were fed chow or chow containing 0.006% SC‐435 for 9 weeks following weaning at 3 weeks of age. Standard growth parameters were recorded; organs, gallbladder bile and blood were collected at necropsy for histologic, RNA and biochemical analyses.ResultsMale and female 2c70‐/‐ mice maintained on chow diet exhibited increased levels of serum liver injury markers ALT, AST, and BAs compared to WT mice. Evaluation of 2c70‐/‐ liver histology revealed increased hepatic inflammation, macrophage infiltration and bilary cell proliferation, which were markedly reduced with SC‐435 treatment. IBAT inhibition in 2c70‐/‐ mice improved liver histology, and reduced hepatic inflammation and bilary cell proliferation to levels indistinguishable from WT. The liver BA content was increased by 53% in 2c70‐/‐ mice, and was enriched in more hydrophobic BA species with an overall hydrophobicity similar that of humans (Figure 1). Treating 2c70‐/‐ mice with SC‐435 reduced the hepatic BA burden by 61%, but did not affect the overall hydrophobicity. RNA‐Seq and KEGG pathway analysis performed using liver RNA revealed genes involved in hepatic steroid and lipid metabolism (Sult2a1, Sult1e1, Hsd17b7), and detoxification (Cyp2c39, Cyp2c54, Cyp2c59, Cyp3a41a/b) were down‐regulated in 2c70‐/‐ mice and their expression were rescued with SC‐435 treatment. Conversely, genes in pathways related to immune cell activation and inflammation were significantly up‐regulated in 2c70‐/‐ mice and reduced to WT levels with IBAT inhibition (Figure 2).ConclusionsOur findings indicate that reducing the hepatic BA burden without altering the hydrophobic compostion is sufficient to protect 2c70‐/‐ mice from hepatic injury. The findings suggest that there may be a threshold in liver for pathological accretion of hydrophobic BAs and reducing the hepatic BA pool is sufficient to alleviate liver injury, independent of hydrophobicity. Moreover, these data support the inhibition IBAT activity, and other approaches to reduce hepatic return of BAs in the enterohepatic circulation, as therapeutic targets to treat cholestasis.