Impaired uptake of conjugated bile acids and hepatitis b virus pres1-binding in na(+) -taurocholate cotransporting polypeptide knockout mice

Abstract

The Na(+) -taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. It has recently been recognized as the receptor mediating hepatocyte-specific entry of hepatitis B virus and hepatitis delta virus. Myrcludex B, a peptide inhibitor of hepatitis B virus entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1-knockout mouse model (Slc10a1 encodes NTCP). Primary Slc10a1(-/-) hepatocytes showed absence of sodium-dependent taurocholic acid uptake, whereas sodium-independent taurocholic acid uptake was unchanged. In vivo, this was manifested as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with ursodeoxycholic acid. Biliary BA output remained intact, while fecal BA excretion was reduced in hypercholanemic Slc10a1(-/-) mice, explained by increased Asbt and Ostα/β expression. These mice further showed reduced Asbt expression in the kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by down-regulation of OATP1A1 and up-regulation of OATP1A4. Furthermore, sodium-dependent taurocholic acid uptake was inhibited by Myrcludex B in wild-type hepatocytes, while Slc10a1(-/-) hepatocytes were insensitive to Myrcludex B. Finally, positron emission tomography showed a complete abrogation of hepatic binding of labeled Myrcludex B in Slc10a1(-/-) mice.

Conclusion: The Slc10a1-knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and hepatitis B virus preS1/Myrcludex B binding in vivo; the NTCP-independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally insufficient to clear BAs from the circulation.

Figure 1

(A) Conventional knockout of Slc10a1 in mouse embryonic stem cells by homologous recombination. A schematic representation of the mouse Slc10a1 gene. Exon 1 was replaced by the β-galactosidase/puromycin (βgeo/puro) selection cassette located in the targeting vector (pKOS 73). Primers of the wild-type allele (wt-fw and wt-rv) and primers of the targeted allele (mut-fw and mut-rv) are shown. (B) Genomic DNA was isolated for genotyping of the offspring mice. Analysis by PCR detected fragments of the Slc10a1 WT (412-bp) and targeted (624-bp) alleles. Homozygous WT (+/+) mice as well as heterozygous (+/−) or homozygous KO (−/−) male and female mice are shown. All PCR products were separated by 1.0% agarose gel electrophoresis. (C) Slc10a1 mRNA expression in female and male WT, heterozygous (HET), and Slc10a1^−/− (KO) mouse livers, analyzed by qRT-PCR. Slc10a1 expression values were calculated relative to the geometric mean of Rplp0 and Tbp and normalized to male WT mice (set at 1.0). Data represent mean ± SEM from six or seven male (light gray) or female mice (dark gray). *P < 0.05. (D) Levels of NTCP protein (∼45-50 kDa) in liver plasma membranes of WT and KO male mice (n – 3), analyzed by western blot. Equal volumes of protein were loaded, as confirmed by the sodium/potassium adenosine triphosphatase (∼90 kDa). (E) Immunofluorescent NTCP staining in WT and KO fresh frozen liver tissue. Scale bar – 40 μm.

Figure 2

(A) Results of a TCA uptake assay in primary hepatocytes of WT and Slc10a1^−/− (KO) mice. Uptake of TCA was determined after preincubation for 30 minutes in sodium-containing buffer (Na, black bar), sodium-free buffer (NMDG, light gray bar), sodium-containing buffer plus 1 μM Myrcludex B (Na + Myr B, dark gray bar), and sodium-free buffer plus 1 μM Myrcludex B (NMDG + Myr B, white bar). Bars represent the mean ± SEM for two independent experiments, each performed in triplicate. *P < 0.05. (B) Total serum BA concentrations in male and female WT and Slc10a1^−/− (KO) mice. Data are given as mean ± SEM on a ¹⁰log scale for seven to 16 mice in each group, quantified using the total BA assay. Serum composition of (C) conjugated and (D) unconjugated BA species as quantified using high-pressure liquid chromatography. Results for WT (white bars), Slc10a1^−/− mice with normal serum BA concentrations (light gray bars), and Slc10a1^−/− mice with high serum BA concentrations (dark gray bars) are shown. Conjugated BA (C-BA) concentrations are the sum of tauro-α-muricholic acid, tauro-β-muricholic acid, tauroursodeoxycholic acid, TCA, taurochenodeoxycholic acid, and taurodeoxycholic acid. Unconjugated BA (U-BA) concentrations are the sum of ω-muricholic acid, α-muricholic acid, β-muricholic acid, cholic acid, UDCA, chenodeoxycholic acid, and DCA. Data are given as mean ± SEM on a ¹⁰log scale for three to six mice in each group. *P < 0.05 (compared to WT levels). (E) Serum BA composition of WT mice, Slc10a1^−/− mice with normal serum BA concentrations, and KO mice high serum BA concentrations. Conjugated BA (dark gray) and unconjugated BA (light gray) species are shown as a percentage of total, as quantified using high-pressure liquid chromatography. (F) Total serum BA levels in relation to body weights of WT (open dots) and KO (black dots) 8-week-old mice (n – 11–14).

Figure 3

Effects of BA supplementation on serum BA concentrations. After 4 days of 0.1% UDCA supplementation, serum composition of (A) conjugated and (B) unconjugated BA species was measured in 5-week-old WT (open dots) and Slc10a1^−/− (black dots) mice. Data are given as a scatter plot on a ¹⁰log scale from five mice in each group. *P < 0.05. (C) Total serum BA levels were measured before starting 0.1% UDCA supplementation (pre-UDCA) and after 1 week (post-UDCA) in 8- to 10-week-old Slc10a1^−/− mice (black dots). As a control total serum BA levels of WT mice (open dots) were measured after 0.1% UDCA supplementation. Data are given on a ¹⁰log scale from four WT and eight Slc10a1^−/− mice.

Figure 4

(A) In vivo serum TCA clearance and (B) cumulative biliary excretion in WT (open dots) and normocholanemic (black dots) Slc10a1^−/− mice. The TCA kinetics after an intravenous TCA bolus (150 μmol/kg body weight) are given as a percentage of the whole bolus (–100%). Serum and bile kinetics are given as the mean ± SEM (2-40 minutes postinjection, n – 5). Mice were considered normocholanemic if the total BA concentration was <20 μM before the start of the experiment. (C) Total serum BA (TBA) clearance during hypercholanemia. Concentrations from four hypercholanemic Slc10a1^−/− mice are given relative to the concentration at the start of gallbladder cannulation (set at 100%).

Figure 5

Effects of NTCP deficiency on the enterohepatic circulation. Bile was collected for 30 minutes after gallbladder cannulation and ligation of the common bile duct. (A) Bile flow (microliters per minute per 100 g body weight) is shown during this period for WT (open dots), normocholanemic Slc10a1^−/− (black dots), and hypercholanemic Slc10a1^−/− (black triangles) mice. (B) Bile acid output (nanomoles per minute per 100 g body weight) during the 10- to 20-minute collection period for WT (white bar), normocholanemic Slc10a1^−/− (light gray bar), and hypercholanemic Slc10a1^−/− (dark gray bar) mice. Data are given as mean ± SEM (n – 8-12). (C) Total fecal BA excretion (micromoles per 24 hours per kilogram of body weight) and (D) relative mRNA expression of BA transporters in the distal ileum for WT mice (white bars) and for Slc10a1^−/− mice with low (light gray bars) and high (dark gray bars) serum BA concentrations, using the geometric mean of control genes Hprt and Ppib. (E) Urinary BA concentrations (μmol/L) and (F) relative mRNA expression of renal BA transporters for WT (white bars) and for Slc10a1^−/− mice with low (light gray bars) and high (dark gray bars) serum BA concentrations, using the geometric mean of control genes Rplp0 and Hprt. Data in (C-F) are given as mean ± SEM (n – 4-6) from 6- to 8-week-old mice. *P < 0.05 (compared to WT levels).

Figure 6

(A) Oatp mRNA expression as determined by qRT-PCR. Relative mRNA expression levels were calculated for WT mice (white bars) and for Slc10a1^−/− mice with low (light gray bars) and high (dark gray bars) serum BA concentrations using the geometric mean of control genes Rplp0 and Tbp. The two sexes were pooled equally. Values are given as mean ± SEM for four to six mice per group. (B) Western blot analysis of the OATP1B2 (∼80 kDa), OATP1A1, and OATP1A4 (both ∼85 kDa) proteins. Protein expression levels were determined relative to the sodium/potassium adenosine triphosphatase (∼90 kDa), and values are given as mean ± SEM for four or five mice per group. *P < 0.05 (compared to WT levels).

Figure 7

Myrcludex B-derived peptide binding assessed by microPET. (A) Female WT (+/+, first panel), heterozygous (+/-, second panel), and homozygous Slc10a1^−/− (-/-, third panel) mice were imaged postinjection of ⁶⁸Ga-WT peptide or ⁶⁸Ga-control peptide (fourth panel). (Top) Uncorrected planar projection, (bottom) coronal PET section. (B) Time activity curves of ⁶⁸Ga-WT peptide in liver and kidney of WT (black line with dots), heterozygous (blue line with squares), and Slc10a1^−/− (red line with triangles) male mice during 60 minutes postinjection. Abbreviation: SUV BW, standardized uptake value body weight.