Yasuna Kobayashi

Transport mechanism and substrate specificity of human organic anion transporter 2 (hOat2 [SLC22A7])

Human organic anion transporter 2 (hOat2 [SLC22A7]) is highly expressed in the human liver. Although localization, gene expression, substrate specificity and transport mechanisms of other human Oat isoforms such as human Oat1 (hOat1), human Oat3 (hOat3) and human Oat4 (hOat4) have been elucidated, information concerning human Oat2 (hOat2) is less defined. The objective of this study was to provide further information on the transport mechanism and substrate specificity of hOat2. When expressed in Xenopus laevis oocytes, the transport of organic compounds mediated by hOat2 was not affected by the replacement of extracellular sodium with lithium, choline and mannitol. The uptake of estrone sulfate (ES) in hOat2‐expressing oocytes was significantly trans‐stimulated by preloading the oocytes with fumarate and succinate, but not glutarate. Moreover, we observed that hOat2 mediates the transport of bumetanide, ES, glutarate, dehydroepiandrosterone sulfate, allopurinol, prostaglandin E2, 5‐fluorouracil, paclitaxel and L‐ascorbic acid. These compounds are identified for the first time as hOat2 substrates. A wide range of structurally unrelated organic compounds inhibited the hOat2‐mediated uptake of tetracycline, except for sulfobromophthalein. All of these findings indicate that hOat2 is a sodium‐independent multi‐specific organic anion/dimethyldicarboxylate exchanger. Our present findings thus provide further insights into the role of hOat2 in hepatic drug transport.

FUNCTIONAL CHARACTERIZATION OF HUMAN MONOCARBOXYLATE TRANSPORTER 6 (SLC16A5)

Human monocarboxylate transporter 6 (MCT6) has recently been isolated, and its tissue distribution has been established at the mRNA level, but its functional properties remain unknown. The aim of this study is to investigate the transport properties of MCT6. When expressed in Xenopus laevis oocytes, MCT6 transported [3H]bumetanide in a pH- and membrane potential-sensitive but not proton gradient-dependent manner, with the Kt value of 84 μM. Furthermore, MCT6 transported various drugs such as probenecid and nateglinide. Neither [14C]l-lactic acid nor [3H]l-tryptophan, typical substrates of other MCT isoforms, was transported by MCT6. Four loop diuretics, i.e., furosemide, piretanide, azosemide, and torasemide, thiazides, probenecid, glibenclamide, and nateglinide inhibited the MCT6-mediated uptake of [3H]bumetanide. In contrast, short-chain carboxylic acids, such as l-lactic acid and succinic acid did not inhibit the MCT6-mediated uptake of bumetanide. These results suggest that the substrate specificity of MCT6 is distinct from those of other MCTs. Bumetanide would be a good tool for investigating the functional properties of MCT6. It is probable that MCT6 is involved in the disposition of various drugs, including bumetanide.

A Novel Method of Cryopreservation of Rat and Human Hepatocytes by Using Encapsulation Technique and Possible Use for Cell Transplantation

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37°C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.

Long-term maintenance of the drug transport activity in cryopreservation of microencapsulated rat hepatocytes.

Transplantation of isolated hepatocytes has been proposed to compensate for essential functions lacking in liver failure or for genetic defects that alter a specific liver metabolic pathway. Hepatocyte utilization for these purposes would be facilitated with a reliable, reproducible, and effective method of long-term hepatocyte storage. We have recently developed a simple new system for cryopreservation of hepatocytes that encapsulates alginate microspheres and maintains liver-specific function. The aim of this study was to elucidate the transport and drug-metabolizing enzyme activities of cryopreserved microencapsulated hepatocytes stored for a long time. Morphological examinations showed there is no apparent injury of the hepatocytes during cryopreservation processes. A drug-metabolizing enzyme (testosterone 6β-hydroxylase, a specific probe for CYP3A2) and drug transport activities [salicylate, allopurinol, and prostaglandin E2 (PGE2), typical substrates of rOat2] in cryopreserved microencapsulated hepatocytes were maintained up to 120 days. Our results thus demonstrate for the first time that cryopreservation of primary rat hepatocytes by the encapsulation technique allows long-term retention of drug metabolism and drug transport activities.

Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1).

We succeeded in isolating the cDNA-encoding mouse organic solute carrier protein 1 (mOscp1) from a mouse testis cDNA library. mOscp1 consisted of 1137 base pairs that encoded a 379-amino acid protein, and the amino acid sequence was 85% identical to that of human OSCP1 (hOSCP1). Northern blot analysis revealed that the gene coding for mOscp1 is highly expressed in the testis, but not in other tissues. When expressed in Xenopus laevis oocytes, mOscp1 mediated the high-affinity transport of p-aminohippurate (PAH) (Km = 18.8 ± 4.1 μM) with Na+ independence. mOscp1 transported various kinds of structurally dissimilar drugs and chemicals such as probenecid, dehydroepiandrosterone sulfate, and glutarate with some differences in substrate specificity compared with hOSCP1. Cyclophosphamide inhibited the mOscp1-mediated PAH uptake. Immunohistochemical analysis revealed that the mOscp1 protein is localized in the plasma membrane side of Sertoli cells in the testis. Our results indicate that isolated mOscp1 is a polyspecific organic solute carrier protein and may be a key molecule for the testicular handling of organic solutes.

Associations among Erythroferrone and Biomarkers of Erythropoiesis and Iron Metabolism, and Treatment with Long-Term Erythropoiesis-Stimulating Agents in Patients on Hemodialysis.

Background We aimed to identify associations between erythroferrone (ERFE), a regulator of hepcidin 25, and biomarkers of erythropoiesis and iron metabolism. We also aimed to determine the effects of erythropoiesis-stimulating agents (ESA), continuous erythropoietin receptor activator (CERA) and darbepoetin-α (DA) on ERFE production in patients on hemodialysis (HD). Methods Blood samples were obtained from 59 patients before HD sessions on day 0 (baseline). Twenty patients who were injected with either CERA (N = 10) or DA (N = 10) at the end of the dialysis week (day 0), who had ferritin ≥ 100 ng/mL and/or transferrin saturation ≥ 20%, and hemoglobin > 9 g/dL were selected from among the 59 patients. Blood was sampled serially before HD sessions on days 3, 5, 7 from patients on DA and on the same days plus day 14 from those on CERA. Results Levels of ERFE correlated inversely with those of hepcidin 25 and ferritin, and positively with those of soluble transferrin receptor. The hepcidin 25: ERFE ratio and hepcidin 25 levels positively correlated with ferritin levels. Levels of ERFE significantly increased from day 3 of treatment with DA and CERA and decreased by days 7 and 14, respectively. Erythropoiesis-stimulating agents concomitantly decreased levels of hepcidin 25 as those of ERFE increased. Conclusion We identified a novel association between ESA and ERFE in patients on HD. Both DA and CERA increased levels of ERFE that regulated hepcidin 25 and led to iron mobilization from body stores during erythropoiesis.

Involvement of testosterone in the induction of hepatic microsomal cytochrome P-450 2B1/2 (P-450 2B1/2) by 1-benzylimidazole in male and female rats: sex-differentiated induction of P-450 2B1/2 species.

We examined the effect of 1-benzylimidazole on the induction of cytochrome P-450 1A1/2 (P-450 1A1/2) and cytochrome P-450 2B1/2 (P-450 2B1/2) in normal, castrated, ovariectomized and hypophysectomized male rats, and in castrated rats treated with testosterone. 1-Benzylimidazole markedly increased P-450 content in male and female rats. Parallel to the dose-dependent increase in P-450 content, 1-benzylimidazole produced a significant increase in P-450 2B1/2 in male rats, but not in female rats. 1-Benzylimidazole failed to induce P-450 2B1/2 in castrated male and ovariectomized female rats. Treatment of castrated male rats with testosterone restored the induction of P-450 2B1/2 by 1-benzylimidazole. Treatment of ovariectomized female rats with 1-benzylimidazole or phenobarbital led to the increase in P-450 content, accompanying by the induction of P-450 2B1/2 by the latter treatment, but not the former. In hypophysectomized male rats, 1-benzylimidazole was able to induce P-450 2B1/2 in contrast to castrated male rats. Neonatal male and female rats responded well to the induction of P-450 2B1/2 by 1-benzylimidazole. The present findings suggest that P-450 2B1/2 induction by 1-benzylimidazole would be coupled with circulating testosterone regulated by hypophysis-testis axis. 1-Benzylimidazole produced sex-differentiated induction of P-450 2B1/2 in pubertal rats, but not in neonatal animals. The present findings would be provide information on a unique effect of 1-benzylimidazole on P-450 2B1/2 induction in rats.

Induction of hepatic microsomal P450 by 4-phenylalkylpyridines in rat: chain length-dependent and sex-related differential induction of P450s

The effects of 4-phenylalkylpyridines (chain length of 0-5, 7, 9 and 11 carbon atoms) on the induction of hepatic microsomal P450 and its multiple forms (1A1/2 (1A1, 1A2), 3A2, 2B1/2, 2C6 and 2E1) in the male and female rat have been investigated. P450 induction gradually declined with increasing chain length of the 4-phenylalkylpyridines. Immunoblot analysis revealed that three pyridine compounds having methylene units of 0, 1 and 2 only induced P4501A2, whereas those having 4, 5 or 7 methylene units only induced 1A1 in the male rat. In the female rat, however, we could not observe such a chain length-dependent differential induction of the P4501A subfamily. Induced levels of P4503A2 and 2E1 were dependent on total P450 specific content, but 2C6 was increased in a chain length-dependent manner in both sexes. These results provide new information on the differential effects of pyridine-containing compounds on P450 induction in the rat.

Effects of Long-Term Erythropoiesis-Stimulating Agents on Iron Metabolism in Patients on Hemodialysis.

Continuous erythropoietin receptor activator (CERA) and darbepoetin‐α (DA) might differently affect iron metabolism and erythropoiesis in patients on hemodialysis (HD). This prospective study examined a cohort of patients on HD who had received either intravenous CERA every 2 or 4 weeks (N = 25) or DA once each week (N = 47). Blood was sampled before HD sessions on days 0, 2, 4, 7 and 14, and on days 0, 3, 5, 7 and 14 from patients who were injected with ESA at the beginning and end of the dialysis week, respectively. Changes in factors indicating erythropoiesis and biomarkers of iron metabolism were examined. Hemoglobin levels were maintained in the target range between 10.0 and 11.0 g/dL and ferritin levels at baseline and during the study period were similar between the DA and CERA groups. Levels of hepcidin 25 decreased from days 2–3 to day 5 and returned to the baseline at day 7 in the DA group, whereas those and transferrin saturation were serially suppressed from days 2–3 to day 14 in the CERA group. Levels of soluble transferrin receptor and reticulocyte counts were significantly elevated from days 4–5 to day 14 by CERA. Both DA and CERA stabilized erythropoiesis, but CERA might mobilize iron from body stores more effectively than DA in patients on HD.

Gene polymorphism and frequencies of the NPC1L1 Gene (rs2072183, rs217434 and rs217428) in Japanese patients with dyslipidemia

Niemann‐Pick C1‐Like 1 (NPC1L1) plays a pivotal role in intestinal cholesterol absorption. Ezetimibe is known as an inhibitor for NPC1L1 and decreases concentration of low‐density lipoprotein cholesterol (LDL‐C) in blood. Responses of the decrease of serum LDL‐C levels to ezetimibe have been reported to be different among NPC1L1 variants. However, there are still limited data concerning the genetic variation in the NPC1L1 gene, specifically, in Japanese patients with dyslipidemia. The purpose of this study is to elucidate genotype and allele frequencies of the NPC1L1 gene in Japanese patients with dyslipidemia.