Pharmacological inhibition of apical sodium-dependent bile acid transporter changes bile composition and blocks progression of sclerosing cholangitis in multidrug resistance 2 knockout mice

Abstract

Deficiency of multidrug resistance 2 (mdr2), a canalicular phospholipid floppase, leads to excretion of low-phospholipid "toxic" bile causing progressive cholestasis. We hypothesize that pharmacological inhibition of the ileal, apical sodium-dependent bile acid transporter (ASBT), blocks progression of sclerosing cholangitis in mdr2(-/-) mice. Thirty-day-old, female mdr2(-/-) mice were fed high-fat chow containing 0.006% SC-435, a minimally absorbed, potent inhibitor of ASBT, providing, on average, 11 mg/kg/day of compound. Bile acids (BAs) and phospholipids were measured by mass spectrometry. Compared with untreated mdr2(-/-) mice, SC-435 treatment for 14 days increased fecal BA excretion by 8-fold, lowered total BA concentration in liver by 65%, reduced total BA and individual hydrophobic BA concentrations in serum by >98%, and decreased plasma alanine aminotransferase, total bilirubin, and serum alkaline phosphatase levels by 86%, 93%, and 55%, respectively. Liver histology of sclerosing cholangitis improved, and extent of fibrosis decreased concomitant with reduction of hepatic profibrogenic gene expression. Biliary BA concentrations significantly decreased and phospholipids remained low and unchanged with treatment. The phosphatidylcholine (PC)/BA ratio in treated mice corrected toward a ratio of 0.28 found in wild-type mice, indicating decreased bile toxicity. Hepatic RNA sequencing studies revealed up-regulation of putative anti-inflammatory and antifibrogenic genes, including Ppara and Igf1, and down-regulation of several proinflammatory genes, including Ccl2 and Lcn2, implicated in leukocyte recruitment. Flow cytometric analysis revealed significant reduction of frequencies of hepatic CD11b(+) F4/80(+) Kupffer cells and CD11b(+) Gr1(+) neutrophils, accompanied by expansion of anti-inflammatory Ly6C(-) monocytes in treated mdr2(-/-) mice.

Conclusion: Inhibition of ASBT reduces BA pool size and retention of hydrophobic BA, favorably alters the biliary PC/BA ratio, profoundly changes the hepatic transcriptome, attenuates recruitment of leukocytes, and abrogates progression of murine sclerosing cholangitis.

Figure 1. SC-435 increases fecal BA excretion and lowers liver and serum BA levels in mdr2−/− mice

30-day-old female mdr2−/− and age- and sex-matched mdr2+/+ received chow (Lab 5020) containing 0.006% SC-435, or the same chow without the compound (controls). Feces were collected for 48 hours prior to day of treatment (DOT) 14; BA were extracted and subjected to enzymatic quantification (A). Concentrations of individual BA in liver and serum samples, collected on DOT 14, were determined by tandem mass spectrometry, and plotted as total liver and serum BA concentration in B and C, respectively. Individual species were grouped as hydrophilic and hydrophobic BAs in D. Statistical analysis: one-way ANOVA with Tukey’s multiple comparison test was applied to the data, with *, **, *** denoting p-values of <0.05, <0.01, <0.001, respectively.

Figure 2. SC-435 prevents wasting and reduces biomarkers of hepatocellular injury and cholestasis in mdr2−/− mice

Body weights were recorded for mice of the four groups during the 14 days of intervention (A). ALT, total bilirubin, and alkaline phosphatase levels were determined on blood sampled by cardiocentesis on DOT 14. Statistical analysis: unpaired t test was applied to the data from treated and control mdr2−/− mice in A, one-way ANOVA with Tukey’s multiple comparison test in B.

Figure 3. SC-435 treatment is associated with reduced periportal inflammation and fibrosis in mdr2−/− mice

Sections from paraffin embedded liver samples obtained from mdr2−/− mice on DOT 14 were subjected to H&E and Trichrome staining. Representative photomicrographs of sections are shown in A, with arrowheads denoting bile duct profiles. The components of sclerosing cholangitis score (inflammation, ductal proliferation, necrosis, and fibrosis) were analyzed on a 1 to 4+ scale (B). Trichrome stained slides from livers of SC-435-treated and control mdr2−/− mice were subjected to automated Aperio-based tissue image analysis to quantify the percentage of liver fibrosis (C). Total hepatic RNA from 44-day old mice of the four groups was subjected to qPCR and relative expression of the pro-inflammatory gene TNFα and of the profibrogenic genes SMA and TIMP1 was computed by ΔΔCT analysis using HPRT as the house keeping gene (D). Statistical analysis: unpaired t test was applied to the data of the two groups (SC-435 vs control) in mdr2−/− mice in B, with *, **, *** denoting p-values of <0.05, <0.01, <0.001, respectively, and one-way ANOVA in C and D.

Figure 4. SC-435 treatment is associated with hepatic upregulation of genes controlling BA pool size and putative hepatoprotective genes and downregulation of pro-inflammatory genes

RNAseq studies were performed on total hepatic RNA from mice of the four treatment groups. Distribution of up- and downregulated genes (>2-fold difference) compared to untreated mice of the respective genotype (knockout [KO] and wild type [WT]) across the different treatment groups is depicted in a Venn diagram (A). Based on pathway enrichment analysis, 12 pathways comprising significantly up- or downregulated genes in SC-435 treated mdr2−/− compared to mdr2−/− control mice are depicted with p values for these pathways plotted as −log₁₀ (B). 23 genes from the up- and downregulated pathways, respectively, are displayed in the heatmaps with expression levels normalized to control mdr2+/+ mice (C).

Figure 5. Despite increase in hepatic BA synthesis rate, SC-435 treatment is associated with reduced biliary BA concentration

Concentrations for the biological precursor 7α-hydroxy-4-cholesten-3-one (C4), which correlates with BA synthesis, were determined by tandem mass spectrometry in plasma samples obtained on DOT 14 (A). Bile was aspirated from the gallbladders on DOT14 and subjected to MS-based quantification of PC, cholesterol, and BA concentrations. The biliary molar ratios of PC/BA (linkage coefficient) were calculated based on these measurements, and mean values of the bile constituents were plotted in a ternary phase diagram. Control mdr2−/− mice plot in region A, which is a 2 phase zone of the Wang and Carey diagram, characterized by very low PC/BA ratio, cholesterol supersaturation and formation of cholesterol crystals. SC-435 treated mdr2−/− and both groups of wild type mice plot in the micellar 1 phase zone of the diagram (B). Statistical analysis: unpaired t test was applied to compare the data between the two groups (SC-435 vs control) in mdr2−/− and mdr2+/+ mice.

Figure 6. SC-435 treatment is associated with decreased bile duct epithelial injury and partial restoration of ultrastructural integrity

Paraffin-embedded liver sections from SC-435 treated and control mice (n=2-3/group) were subjected to Ki67 immunohistochemistry. Ki67 positive cells were enumerated in large interlobular bile ducts (10 bile duct profiles/ liver). Statistical analysis: one-way ANOVA was applied to compare the data between the groups (A). In B, glutaraldehyde-fixed liver sections were analyzed by electron microscopy. Left panel depicts a bile duct profile in control mdr2+/+ mice in low magnification, with * denoting the lumen. The other images show higher magnification of the basement membrane (denoted by arrowheads) in mdr2+/+ mice, which is disrupted in control mdr2−/− mice; integrity is restored in SC-435-treated mdr2−/− mice. 10 bile duct profiles were examined per group (n=2 mice/group), of which 4 were surrounded by intact basement membrane in control and 7 in SC-435 treated mice.

Figure 7. SC-435 treatment is associated with decreased population of the liver with macrophages, neutrophils and a shift towards anti-inflammatory monocytes

Single cell suspensions were prepared from livers of mice from the four treatment groups and subjected to flow cytometry. The two top panels delineate the gating strategies: Following out-gating of cell duplets, debris and lineage positive CD3⁺ or CD19⁺ cells, macrophages (Kupffer cells) were identified as F4/80⁺CD11b⁺ and neutrophils as Gr1^highCD11b⁺ cells. F4/80⁻ and Gr1⁻ CD11b⁺ myeloid were further distinguished into inflammatory (Ly6C⁺) and anti-inflammatory (Ly6C⁻) monocytes. Frequencies of these cell populations were enumerated as denoted in the graphs, and differences in means between the groups were tested for statistical significance with one-way ANOVA.