Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2-/- (Abcb4-/-) mouse cholangiopathy model by promoting biliary HCO⁻₃ output

Abstract

Chronic cholangiopathies have limited therapeutic options and represent an important indication for liver transplantation. The nuclear farnesoid X receptor (FXR) and the membrane G protein-coupled receptor, TGR5, regulate bile acid (BA) homeostasis and inflammation. Therefore, we hypothesized that activation of FXR and/or TGR5 could ameliorate liver injury in Mdr2(-/-) (Abcb4(-/-)) mice, a model of chronic cholangiopathy. Hepatic inflammation, fibrosis, as well as bile secretion and key genes of BA homeostasis were addressed in Mdr2(-/-) mice fed either a chow diet or a diet supplemented with the FXR agonist, INT-747, the TGR5 agonist, INT-777, or the dual FXR/TGR5 agonist, INT-767 (0.03% w/w). Only the dual FXR/TGR5 agonist, INT-767, significantly improved serum liver enzymes, hepatic inflammation, and biliary fibrosis in Mdr2(-/-) mice, whereas INT-747 and INT-777 had no hepatoprotective effects. In line with this, INT-767 significantly induced bile flow and biliary HCO 3- output, as well as gene expression of carbonic anhydrase 14, an important enzyme able to enhance HCO 3- transport, in an Fxr-dependent manner. In addition, INT-767 dramatically reduced bile acid synthesis via the induction of ileal Fgf15 and hepatic Shp gene expression, thus resulting in significantly reduced biliary bile acid output in Mdr2(-/-) mice.

Conclusion: This study shows that FXR activation improves liver injury in a mouse model of chronic cholangiopathy by reduction of biliary BA output and promotion of HCO 3--rich bile secretion.

Fig. 1

Dual FXR/TGR5 agonist INT-767 improves liver injury in Mdr2^−/− mice. Mdr2^−/− mice were either fed a chow diet or a diet supplemented with the FXR agonist, INT-747, the TGR5 agonist, INT-777, or the dual FXR/TGR5 agonist, INT-767 for 4 weeks. (A) Chemical structures of INT-747, INT-777, and INT-767 compounds with respective FXR and TGR5 EC₅₀. (B) INT-747 and INT-777 increased serum ALT and ALP, whereas INT-767 significantly reduced serum ALT, but not ALP, levels. Means of 6 mice/group ± SD. *P < 0.05 INT-747, INT-767, and INT-777 versus controls. (C) Representative histological pictures of H&E-stained livers. Bile duct proliferation and portal infiltration of inflammatory cells was reduced by INT-767. The INT-747-fed Mdr2^−/− mouse showed increased bile duct proliferation, expansion of biliary tract, and accumulation of inflammatory cells. No obvious alterations were detected in the INT-777-fed Mdr2^−/− mouse liver. Co, chow-fed littermates; pv, portal vein; bd, bile duct.

Fig. 2

INT-767 reduces hepatic inflammation and fibrosis in Mdr2^−/− mice. INT-767 inhibited the gene expression of macrophage marker F4/80 (A) and proinflammatory cytokines Tnf-α (B) and Il-1β (C) in Mdr2^−/− mice, whereas INT-747 induced Il-1β gene expression. INT-767 lowered hepatic HP levels (D) as well as Col1a1 gene expression (E) in Mdr2^−/− mice, whereas INT-747 increased and INT-777 did not modify HP or Col1a1 gene expression. (F) Spleen weight was normalized to body weight, and percent ratio is presented (SW/BW). Means of 5–6 mice/ group ± SD are presented. Gene-expression levels are normalized to the 36b4 housekeeping gene and the mean expression value of untreated Mdr2^−/− mice (Co) is accepted as 1. *P < 0.05 INT-747 and INT-767 versus Co.

Fig. 3

INT-747 and INT-767 inhibit BA synthesis in Mdr2^−/− mice. Both INT-747 and INT-767 dramatically inhibited Cyp7a1 (A) while inducing ileal Fgf15 (B) gene expression in Mdr2^−/− mice. (C) Only INT-767 significantly decreased, whereas INT-777 increased serum BA levels in Mdr2^−/− mice. Means of 6 animals/group ± SD are presented. Gene-expression levels are normalized to the 36b4 housekeeping gene, and the mean expression value of untreated Mdr2^−/− mice (Co) is accepted as 1. *P < 0.05 INT-747, INT-767, and INT-777 versus Co.

Fig. 4

INT-767 stimulates ${\text{HCO}}_3^{-}$-rich bile flow in Mdr2^−/− mice. Bile flow and composition were measured in Mdr2^−/− mice after 7 days of feeding either a chow or a diet supplemented with INT-747, INT-777, or INT-767. (A) INT-767 increased bile flow and (B) biliary ${\text{HCO}}_3^{-}$ output. (C) Biliary BA output was significantly decreased by INT-767. Neither INT-747 nor INT-777 modified bile flow, biliary ${\text{HCO}}_3^{-}$, or BA output in Mdr2^−/− mice. Means of 4–5 mice/ group ± SD are presented. *P < 0.05 INT-767 versus chow-fed Mdr2^−/− mice (Co).

Fig. 5

Induction of ${\text{HCO}}_3^{-}$-rich choleresis by INT-767 depends on Fxr, but not Tgr5. Bile flow and biliary ${\text{HCO}}_3^{-}$ output were measured in Fxr^+/+ and Fxr^−/− mice after 7 days of feeding on a chow or INT-747-, INT-777-, or INT-767-supplemented diet. (A) INT-767 induced bile flow only in Fxr^+/+ mice, whereas INT-747 and INT-777 did not change bile flow in both Fxr^+/+ and Fxr^−/− mice. (B) Only INT-767 induced biliary ${\text{HCO}}_3^{-}$ output in Fxr^+/+, but not in Fxr^−/−, mice. Means of 4–5 mice/group ± SD are presented. *P < 0.05 INT-767-fed versus chow-fed Fxr^+/+ mice (Co). ^#P < 0.05 Fxr^−/− versus Fxr^+/+ mice. Bile flow (C) and biliary ${\text{HCO}}_3^{-}$ (D) output were measured in age-matched Tgr5-Tg and Tgr5^+/+ female mice. Bile flow, as well as biliary ${\text{HCO}}_3^{-}$ output, was undistinguishable between Tgr5-Tg and Tgr5^+/+ mice. Means of 4–5 mice/group ± SD. are presented.

Fig. 6

INT-767 reduces biliary output of endogenous BAs in an Fxr-dependent manner. Output of biliary BAs (A), cholesterol (B), and PLs (C) was reduced in Fxr^+/+ mice by INT-767 while remaining unchanged in Fxr^−/− mice. INT-747 modestly reduced biliary BA output (A), whereas INT-777 reduced PL (B) and Cholesterol (C) output in Fxr^+/+ mice. (D) Biliary concentrations of respective INT compounds in bile samples of Fxr^+/+ and Fxr^−/− mice. Means of 4–5 animals/group ± SD are presented. *P < 0.05 INT-747, INT-767 and INT-777-fed versus chow-fed Fxr^+/+ mice (Co). ^#P < 0.05 Fxr^−/− versus Fxr^+/+ mice.

Fig. 7

INT-767 significantly induces carbonic anhydrase 14 (Ca14) gene expression via Fxr. INT-767 significantly stimulated hepatic Ca14 gene expression in Mdr2^−/− (A) and Fxr^+/+ mice (B), whereas no alterations were detected in Fxr^−/− mice. Means of 5–6 animals/group ± SD are presented. Gene-expression levels are normalized to the 36b4 housekeeping gene, and the mean expression value of chow-fed Mdr2^−/−, Fxr^+/+, and Fxr^−/− mice is accepted as 1 (Co). *P < 0.05 INT-767-fed Fxr^+/+ and Mdr2^−/− mice versus respective controls. ^#P < 0.05 Fxr^−/− versus Fxr^+/+ mice. (C) INT-767 (10 µM) significantly induced CA14 gene expression in HepG2 cells. Values are presented as means of 3 samples per group ± SD. Expression levels are normalized to 36B4. *P < 0.05 INT-767- versus control medium-incubated (Co) cells. RU, relative units.

Fig. 8

Proposed model of INT-767-mediated beneficial effects in Mdr2^−/− mice. INT-767 activates Fxr in the ileum and liver. By the induction of ileal Fgf15 and hepatic Shp, INT-767 profoundly inhibits endogenous BA synthesis, resulting in a significant reduction of biliary output of hydrophobic BAs and cholesterol. In addition, INT-767 via Fxr activation induces the expression of hepatocellular membrane-bound Ca14, which, in turn, promotes ${\text{HCO}}_3^{-}$ output and ${\text{HCO}}_3^{-}$-rich choleresis because of (1) complex formation with ${\text{HCO}}_3^{-}$ transporter Ae2 and (2) hydration of ${\text{HCO}}_3^{-}$ with H⁺ into CO₂ and H₂O, thus increasing the local transmembrane ${\text{HCO}}_3^{-}$ gradient to further facilitate ${\text{HCO}}_3^{-}$ export. CO₂, being easily permeable across the cell membrane, can enter cells where it is rehydrated to H⁺ and ${\text{HCO}}_3^{-}$, thus contributing to ${\text{HCO}}_3^{-}$ recycling. Collectively, INT-767, by reducing the concentration of detergent BAs and increasing ${\text{HCO}}_3^{-}$-rich choleresis, contributes to less reactive alterations of bile duct epithelium and results in decreased portal inflammation and fibrosis.