Disappearance of perihepatic lymph node enlargement after hepatitis C viral eradication with direct-acting antivirals.

Perihepatic lymph node enlargement (PLNE) which has been shown to be negatively associated with hepatocellular carcinoma (HCC) occurrence is frequently observed in chronic liver disease; however, changes in the state of perihepatic lymph nodes after eradication of hepatitis C virus (HCV) have not been investigated yet. We aimed to evaluate this issue. We enrolled 472 patients with chronic HCV infection who achieved viral eradication with direct-acting antivirals (DAA). We investigated whether the status of perihepatic lymph nodes changed before and after HCV eradication (primary endpoint). We also evaluated the association between PLNE and clinical findings such as liver fibrosis or hepatocellular injury before HCV eradication (secondary endpoint). Perihepatic lymph node enlargement was detected in 164 of 472 (34.7%) patients before DAA treatment. Surprisingly, disappearance of PLNE was observed in 23.8% (39 patients) of all PLNE-positive patients after eradication of HCV. Disappearance of PLNE was not associated with baseline clinical parameters or changing rates of clinical findings before and after DAA treatment. At baseline, presence of PLNE was significantly associated with a lower serum HCV-RNA level (P = .03), a higher serum AST level (P = .004) and a higher ALT level (P < .001) after adjustment for sex and age. In conclusion, PLNEs became undetectable after DAA treatment in 23.8% of PLNE-positive patients. Further study with a longer follow-up period is needed to clarify the clinical importance of this phenomenon especially in relationship with the risk of HCC development.

Human liver-specific organic anion transporter, LST-1, mediates uptake of pravastatin by human hepatocytes.

Involvement of LST-1 (a human liver-specific transporter, also called OATP2) as the major transporter in the uptake of pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, by human liver was demonstrated. The hepatic uptake of pravastatin evaluated using human hepatocytes was Na(+)-independent and reached saturation with a Michaelis constant (K(m)) of 11.5 +/- 2.2 microM. The uptake of pravastatin was temperature-dependent and was inhibited by estradiol-17beta-D-glucuronide, taurocholic acid, bromosulfophthalein, and simvastatin acid, but not by p-aminohippurate. Estradiol-17beta-D-glucuronide competitively inhibited pravastatin uptake with an inhibition constant comparable to the K(m) value for estradiol-17beta-D-glucuronide transport, indicating that a common transporter mediates the transport of pravastatin and estradiol-17beta-D-glucuronide in human hepatocytes. The results obtained with human hepatocytes agreed with those obtained with LST-1 expressing Xenopus oocytes. Oocytes microinjected with human liver polyadenylated mRNA showed Na(+)-independent uptake of pravastatin and estradiol-17beta-D-glucuronide. A simultaneous injection of LST-1 antisense oligonucleotides completely abolished this uptake. Expression of LST-1 was immunohistochemically demonstrated in the human hepatocytes, but not in Hep G2 cells, which showed very low uptake of pravastatin. Therefore, LST-1 was regarded as a key molecule for pravastatin in liver-specific inhibition of cholesterol synthesis, making pravastatin accessible to the target enzyme, which would otherwise not be inhibited by this hydrophilic drug.

LST-2, a human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers.

BACKGROUND & AIMS: One approach to the development of targeted cancer chemotherapy exploits increased uptake of the agent into neoplastic cells. In this scenario, higher concentrations of the agent in cancer cells are responsible for differential killing, whereas the low concentration in normal human cells decreases side effects. The aim of this study was to isolate an organic anion transporter that is weak in normal cells, but abundantly expressed in cancer cells, to deliver the anticancer drugs to the cells. METHODS: A human liver complementary DNA (cDNA) library was screened with liver-specific transporter (LST)-1 cDNA as a probe. Northern blot analyses were performed using the isolated cDNA (termed LST-2). An LST-2-specific antibody was raised, and immunohistochemical analyses including immunoelectron microscopy were performed. Xenopus oocyte expression system was used for functional analysis. We also established a permanent cell line that consistently expresses LST-2 to examine the relationship between methotrexate uptake and sensitivity. RESULTS: The isolated cDNA, LST-2, has 79.7% of overall homology with human LST-1. LST-2 exclusively expressed in the liver under normal conditions and its immunoreactivity was highest at the basolateral membrane of the hepatocytes around the central vein. Although its weak expression in the liver, LST-2 is abundantly expressed in the gastric, colon, and pancreatic cancers. On the other hand, the LST-1 was only detected in a hepatic cell line. LST-2 transports methotrexate in a saturable and dose-dependent manner. Furthermore, introduction of the LST-2 gene into mammalian cells potentiates sensitivity to methotrexate. CONCLUSIONS: LST-2 is one of the prime candidate molecules for determining methotrexate sensitivity and may be a good target to deliver anticancer drugs to the gastrointestinal cancers.

Molecular identification of a rat novel organic anion transporter moat1, which transports prostaglandin D(2), leukotriene C(4), and taurocholate.

We have isolated a rat novel multispecific organic anion transporter, moat1. The isolated clones were originated by alternative splicing of the moat1 mRNA. The nucleotide sequences predict a protein of 682 amino acids with moderate sequence similarity to LST-1, the oatp family, and the prostaglandin transporter. Northern blot analysis of rat moat1 identified a predominant transcript of 4.4 kilonucleotides in all tissues. Northern blot and in situ hybridization analyses of rat brain further indicated that moat1 mRNA is widely distributed in neuronal cells of the central nervous system, especially in the hippocampus and cerebellum. moat1 transports prostaglandin D(2) (K(m); 35.5 nM), leukotriene C(4) (K(m); 3.2 microM) and taurocholate (K(m); 17.6 microM) in a sodium-independent manner. moat1 also transports prostaglandin E(1), E(2), thromboxane B(2), and iloprost but not dehydroepiandrosterone sulfate and digoxin, of which the substrate specificity is similar, but definitively different from those of any other organic anion transporters.

Molecular characterization and functional regulation of a novel rat liver-specific organic anion transporter rlst-1.

BACKGROUND & AIMS: Recently, we isolated a new complementary DNA (cDNA) encoding human liver-specific organic anion transporter (LST-1), representing the multispecificity of human liver. The aim of this study was to isolate a rat counterpart of human LST-1 and examine the expression regulation of its messenger RNA (mRNA) to clarify the molecular basis of cholestasis. METHODS: A rat liver cDNA library was screened with human LST-1 cDNA as a probe. Xenopus oocyte expression system was used for functional analysis. Northern blot analyses were performed using the isolated cDNA (termed rlst-1). The bile duct ligation model and the cecum ligation and puncture model were used for expression analyses. RESULTS: rlst-1 encodes 652 amino acids, predicting at least 11 transmembrane regions. The overall homology with human LST-1 was 60.2%, which is the highest among all known organic anion transporters. rlst-1 also belongs to the same new gene family as human LST-1, located between the organic anion transporter family and the prostaglandin transporter. rlst-1 preferably transports taurocholate (K(m), 9.45 micromol/L) in an Na(+)-independent manner. The rlst-1 mRNA is exclusively expressed in the liver. In both the bile duct ligation model and the cecum ligation and puncture model, mRNA expression levels of rlst-1 were down-regulated. CONCLUSIONS: rlst-1 is a counterpart of human LST-1 and is one of the important transporters in rat liver for the clearance of bile acid. The expression of rlst-1 may be under feedback regulation of cholestasis by biliary obstruction and/or sepsis.

Pravastatin, an HMG-CoA reductase inhibitor, is transported by rat organic anion transporting polypeptide, oatp2.

PURPOSE: We previously demonstrated the HMG-CoA reductase inhibitor, pravastatin, is actively taken up into isolated rat hepatocytes through multispecific organic anion transporters. The present study examined whether a newly cloned organic anion transporting polypeptide (oatp2) transports pravastatin. METHODS: We investigated functional expression of oatp2 in Xenopus laevis oocytes, to examine [14C] pravastatin uptake. RESULTS: [14C] Pravastatin (30 microM) uptake into oatp2 cRNA-injected oocytes was 40 times higher than that of water-injected control oocytes. The oatp2-mediated pravastatin uptake was Na+-independent and saturable. The Michaelis-Menten constant was 37.5+/-9.9 microM, a level comparable to that obtained in isolated rat hepatocytes in our previous study. As is the case with rat hepatocytes, the uptake of pravastatin (30 microM) was inhibited by 300 microM concentrations of taurocholate, cholate, bromosulfophthalein, estradiol-17beta-glucuronide, and simvastatin acid, but not by para-aminohippurate. On the other hand, [14C] simvastatin acid (30 microM) uptake of oatp2 cRNA-injected oocytes was not significantly different from that of water-injected oocytes. CONCLUSIONS: The cloned oatp2 was identified as the transporter responsible for the active hepatocellular pravastatin uptake.

Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1.

We have isolated a novel liver-specific organic anion transporter, LST-1, that is expressed exclusively in the human, rat, and mouse liver. LST-1 is a new gene family located between the organic anion transporter family and prostaglandin transporter. LST-1 transports taurocholate (Km = 13.6 microM) in a sodium-independent manner. LST-1 also shows broad substrate specificity. It transports conjugated steroids (dehydroepiandrosterone sulfate, estradiol-17beta-glucuronide, and estrone-3-sulfate), eicosanoids (prostaglandin E2, thromboxane B2, leukotriene C4, leukotriene E4), and thyroid hormones (thyroxine, Km = 3.0 microM and triiodothyronine, Km = 2.7 microM), reflecting hepatic multispecificity. LST-1 is probably the most important transporter in human liver for clearance of bile acids and organic anions because hepatic levels of another organic anion transporter, OATP, is very low. This is also the first report of the human molecule that transports thyroid hormones.

Immunohistochemical distribution and functional characterization of an organic anion transporting polypeptide 2 (oatp2).

The rabbit polyclonal antibody against rat organic anion transporting polypeptide 2 (oatp2) was raised and immunoaffinity-purified. Western blot analysis for oatp2 detected two bands ( 74 and 76 kDa) in rat brain and a single band (76 kDa) in the liver. By immunohistochemical analysis, the oatp2 immunoreactivity was specifically high at the basolateral membrane of rat hepatocytes. Functionally, the oatp2-expressing oocytes were found to transport dehydroepiandrosterone sulfate, delta1 opioid receptor agonist [D-Pen2,D-Pen5]enkephalin, Leuenkephalin, and biotin significantly, as well as the substrates previously reported. These data reveal the exact distribution of the rat oatp2 at the protein level in the liver, and that oatp2 appears to be involved in the multispecificity of the uptaking substrates in the liver and brain.