Vitamin D modulates biliary fibrosis in ABCB4 deficient mice

Background: Vitamin D deficiency is a hallmark of many chronic liver diseases. Impaired vitamin D-VDR signalling might represent an aggravating factor during liver injury (Zuniga et al. 2011) and recent studies suggest that vitamin D exerts a protective role in biliary-type liver diseases (Chignard et al. 2012). To assess the effect of vitamin D on biliary fibrosis, we now treated ATP binding cassette transporter knockout (Abcb4-/-) mice as established model of chronic cholangitis and biliary fibrosis with 25-hydroxyvitamin D.

Methods: Abcb4-/- (n=60) and wild-type mice (n=54) were fed a vitamin D-supplemented diet (2400 IE 25-hydroxvitamin D/kg food), a vitamin D-deficient diet (100 IE/kg) or a control diet (600 IE/kg) for 12 weeks. Serum 25-hydroxyvitamin D concentrations were measured by chemiluminescence immunoassays. Liver injury was determined by liver enzyme activities (ALT, AP), histopathological stages of fibrosis and hepatic collagen contents. Steady-state mRNA expression levels of Col1a2, Timp1, Tgf-ß, Vdr and Camp were analyzed by quantitative RT-PCR.

Results: Serum vitamin D levels depend on genotype and diet. Of note, Abcb4-/- mice on controls diet display lower vitamin D concentrations as compared to wild-type mice (38.4±1.9 vs. 46.8±1.7ng/ml; p<0.01), whereas after vitamin D supplementation Abcb4-/- animals demonstrate higher vitamin D levels in comparison to wild-type mice (67.2±2.4 vs. 42.0±2.3ng/ml; p<0.001). Fibrosis stages differ significantly between Abcb4-/- mice fed different diets, while hepatic collagen contents in wild-type mice are not affected by diet. Abcb4-/- mice receiving vitamin D-deficient diet develop significantly (p<0.01) higher fibrosis stages (F-score 2.8±0.1) as compared to knockout mice that were fed control or vitamin D-sufficient diets (F-scores 1.6±0.2 and 2.1±0.1, respectively). In line with liver histology, we detected significantly (p<0.05) higher collagen contents in Abcb4-/- mice fed the vitamin D-deficient diet (351±22µg hydroxyproline/g liver) as compared to mice on control diet (294±15µg/g); the lowest collagen contents were observed after vitamin D supplementation (275±14µg/g). Additionally, vitamin D deficient Abcb4-/- mice reveal significantly (p<0.05) reduced expression of Timp1, Tgf-ß and Vdr in comparison to Abcb4-/- animals on regular diet.

Conclusions: Liver fibrosis severity in Abcb4-/- mice depends on vitamin D status. Our findings indicate that vitamin D modulate biliary injury and fibrogenesis in vivo. We speculate that adequate vitamin D intake might have antifibrotic effects in patients with chronic liver diseases.