1. Research on bile acids transporters
（Kagawa T. Hepatobiliary transport of bile acids. In “Bile Acids in Gastroenterology: Basic and Clinical”, 2017, pp. 9-25）
Bile acids are the major driving force of bile excretion from hepatocytes; they are synthesized from cholesterol via at least 17 enzymatic reactions. They play a critical role in cholesterol disposal and the absorption of fat as well as fat-soluble vitamins. After excretion from hepatocytes into the bile canaliculus, most bile acids (~95%) are reabsorbed in the terminal ileum and return back to the liver via the portal vein (enterohepatic circulation, Figure). The influx and efflux of bile acids in organs is mediated by organ-specific transporters. In hepatocytes, bile acids are absorbed from the sinusoid by the Na+-taurocholate co-transporting polypeptide (NTCP, SLC10A1), and OATP1B1 and OATP1B3 (organic anion transporting peptide, SLCO1B1, SLCO1B3) and secreted into the bile canaliculus by the bile salt export pump (BSEP, ABCB11).
Other bile components are secreted by their corresponding transporters: phospholipids by multidrug resistance protein 3 (MDR3, ABCB4), organic anions by multidrug resistance-associated protein 2 (MRP2, ABCC2), and cholesterol by ABCG5/G8. In the terminal ileum, bile acids are absorbed from the intestinal lumen by the apical sodium-dependent bile acid transporter (ASBT, SLC10A2) and excreted into the portal vein by the organic solute transporters (OSTα/OSTβ, SLC51A/SLC51B) that facilitate bidirectional diffusion. Bile acids are detergents and are toxic to cells at high concentrations, therefore, their cellular concentration must be tightly regulated by refined feedback systems. Bile acids, now known as a signaling molecule, activate a nuclear receptor, farnesoid X-receptor (FXR, NR1H4), and the G protein-coupled bile acid receptor, TGR5, thereby triggering a number of physiological reactions. In hepatocytes, bile acids bind to FXR, which represses NTCP and prevents further bile acids uptake, down-regulates cholesterol 7α-hydroxylase (CYP7A1) to inhibit further bile acid synthesis, and activates BSEP to induce bile acid secretion into the bile canaliculus. All of these events ultimately result in a reduction in the concentration of intracellular bile acids.
（1）BSEP（bile salt export pump, ABCB11)
The human ABCB11 gene is located on chromosome 2 (2q24) and is translated into a protein comprising 1,321 amino acids, with a molecular mass of ~160 kDa. BSEP belongs to the ABC subfamily B, harboring 12 potential transmembrane segments and two sets of Walker A and B motifs that bind ATP. BSEP is exclusively expressed in hepatocytes, where it resides along the canalicular membranes and exports bile acids into the bile canaliculus in an ATP-dependent fashion. The rat Bsep receives N-linked glycosylation at four asparagine residues in the first extracellular loop, which sites are also present in human BSEP. These glycans are required for correct trafficking to the canalicular membrane; loss of two or more glycans results in rapid degradation at the proteasome (Mochizuki K, et al. AJP 2007).
There are two types of hereditary intrahepatic cholestatic diseases; progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC). PFIC patients progress to liver failure and require liver transplantation in childhood, whereas BRIC patients display intermittent and usually non-progressive jaundice. PFIC1 and BRIC1 are caused by mutations in the FIC1 (ATB8B1) gene, which encodes a P-type ATPase functioning as a flippase for phosphatidylserine, whereas PFIC3 is caused by mutations in the MDR3 gene. PFIC2 and BRIC2 are caused by mutations in BSEP, and more than 150 genetic abnormalities, including missense, nonsense, deletions, insertions, and splice-site mutations, have been identified. Some missense mutations and single nucleotide polymorphisms (SNPs) can cause aberrant pre-mRNA splicing, resulting in impaired BSEP function. PFIC2 is characterized by absent or much reduced canalicular BSEP expression as well as a markedly diminished concentration of biliary bile acid.
To elucidate the effects of these mutations and SNPs on BSEP function, we analyzed bile acids transport activity of BSEP in MDCKII cells. The activity of PFIC2 mutants (D482G, E297G, K461E, G982R, R1153C, R1268Q, and 3767–3768insC) was 0-30% of wild-type, whereas BRIC2 mutants (A570T and R1050C) exhibited 50-60% (Kagawa T, et al. AJP 2008). The reduced activity corresponded to the stability of synthesized BSEP protein. Thus, the difference in the severity of the clinical phenotype between PFIC2 and BRIC2 may be explained by the differences in transport activity of BSEP harboring corresponding mutations. Several patients who had clinical and histopathological characteristics of BRIC progressed to PFIC, suggesting a possible phenotypic progression between BRIC2 and PFIC2. Furthermore, the E297G mutation, which is responsible for PFIC2, is also found in BRIC2 patients. Therefore, although the BSEP genotype appears to play an important role in determining clinical severity, other precipitating factors, including viral infection and pregnancy, may also participate.
An association between BSEP SNP and acquired intrahepatic cholestasis has been reported. The C-allele frequency of ABCB11 c.1331T>C (p.V444A) (rs2287622) SNP was higher among patients with intrahepatic cholestasis of pregnancy (ICP) (67% in patients versus 54% in controls, P < 0.001). In a recent comprehensive study, two intronic SNPs (rs7577650 and rs3815676) were identified as significant risk alleles associated with ICP. The V444A SNP remained associated with the disease, but the association was driven by rs7577650, suggesting that the V444A SNP is not a causative variation. The effect of an amino acid substitution at position 444 on the BSEP function is controversial. Western blot analysis on normal liver tissues from patients undergoing liver resection revealed that canalicular BSEP expression was slightly, but not significantly, reduced in individuals carrying the 444A polymorphism. In another study that utilized a bank of human liver samples, BSEP mRNA, but not protein, expression was significantly attenuated in individuals with the 444A polymorphism. The bile acid transport activity of 444A BSEP was not reduced when expressed in Sf9 and HeLa cells, and was slightly reduced by up to 20% in MDCKII cells (Kagawa T, et al. Drug Metab Dispos 2015).
Impaired of BSEP function is also involved in drug-induced liver injury; its severity is associated with dual inhibition of BSEP and mitochondrial function. The association of BSEP V444A SNP in drug-induced cholestasis has been reported in European populations (76% in patients versus 57% in controls). However, this association was not reproducible among Japanese patients with drug-induced cholestasis (66% in patients versus 78% in controls) (Kagawa T, et al. Drug Metab Dispos 2015). Further study investigation is necessary to identify underlying causative risk alleles in the different populations.
Given that bile acids are the major driving force for bile excretion, drugs that up-regulate or activate BSEP are good candidates to treat intrahepatic cholestasis. UDCA is one of the drugs most commonly used for hepatobiliary diseases including PBC, PSC, cholestasis, and cholelithiasis. UDCA exerts a choleretic effect by targeting BSEP to the canalicular membrane (Kagawa T, et al. J Gastroenterol 2014) via activation of p38MAPK and a Ca2+-independent protein kinase C (PKC) isoform. Sodium 4-phenylbutyrate (4PBA) enhanced the cell surface expression and transport capacity of wild-type BSEP and BSEP carrying a PFIC2 mutation (E297G and D482G) in MDCKII cells. Administration of 4PBA also induced canalicular Bsep expression, accompanied by an increase in biliary excretion of taurocholic acids in rats. These effects may be achieved by decreasing short-chain ubiquitination-mediated Bsep degradation and by reducing AP2 adaptor complex-mediated clathrin-dependent endocytosis. In the clinical setting, 4PBA therapy improved serum liver tests, liver histology, and itching in patients with PFIC2 and BRIC2.
（2）OATP1B1, OATP1B3（organic anion transporting peptide, SLCO1B1, SLCO1B3）
OATP1B1 and OATP1B, exclusively localized to the sinusoidal membrane of zone 3 hepatocytes, are involved in the hepatic uptake of various drugs including statins, bosentan, olmesartan, and docetaxel, as well as endogenous substances such as bilirubin glucuronides, bile acids, and steroidal and thyroid hormones. Homozygous inactivation of both SLCO1B1 and SLCO1B3 causes Rotor syndrome, for which three disease-causing haplotypes linked to SLCO1B1 and SLCO1B3 are documented. We reported a Japanese population-specific haplotype: a homozygous c.1738C>T (p.R580X) nonsense mutation in SLCO1B1, and a homozygous insertion of a 6.5-kbp long interspersed element (LINE-1, L1) retrotransposon in intron 5 of SLOC1B3 that induced exon skipping and a premature stop codon that generated truncated proteins (ClinVar Variation ID: 977762, Kagawa T, et al. Hum Mutat 2015). We also demonstrated that deficiency of OATP1B3 causes constitutional indocyanine green (ICG) excretory defect, which manifests marked delay of ICG clearance (ClinVar Variation ID: 981013, Kagawa T, et al. Hepatology 2017)
2. Hepatocyte polarity
Mature hepatocytes become polarized, subsequently resulting in the formation of bile canaliculus between them. The molecules involved in the transport of bile constituents and drug metabolites into the bile canaliculus are trafficked to the right place. However, the molecular mechanism of polarity formation and intracellular trafficking remains largely unknown. We aim to elucidate these mechanisms by utilizing iPS-derived hepatocytes.
3. Mechanism of cyst growth in polycystic liver disease (PCLD)
PCLD is a rare disease which manifests many hepatic cysts. This disease sometimes causes symptoms such as abdominal fullness, edema, or jaundice by compressing other organs including vena cava and bile ducts. This disease occurs due to the genetic abnormalities in cilia-associated proteins (ciliopathy). However, such abnormalities are found in only half of the Asian patients. We aim to find novel genetic abnormalities and elucidate the mechanism of hepatic cyst enlargement by utilizing iPS-derived cholangiocytes.
4. Clinical study
(1) Cohort study of the patients with biopsy-proven fatty liver disease
(2) Clinical studies evaluating the effects of anti-diabetic or anti-hypertriglyceridemic drugs on hepatic fat accumulation in patients with fatty liver
(3) Efficacy and safety of denosumab treatment for osteoporosis in patients with primary biliary cholangitis; a randomized controlled trial with zoledronic acid (DELTA Study)
(4) Cohort study of patients with hepatocellular carcinoma receiving molecular targeting drugs or immune checkpoint inhibitors.