Biomedical subjects

Masahiro Okuda

Publications and source records attributed to Masahiro Okuda.

36 records · Page 2Linked to original sources

PubMed2004

Surgical resection deteriorates gemcitabine-induced leukopenia in pancreatic cancer.

BACKGROUND: Gemcitabine hydrochloride (GEM) is one of the most effective chemotherapeutic agents for pancreatic cancer; however, factors affecting GEM-induced leukopenia have not been clarified yet. In the present study, we analyzed the relationship between patients backgrounds and GEM-induced leukopenia. METHODS: Thirty-eight patients with pancreatic cancer were analyzed for correlation between the dose of GEM and the blood leukocyte number. Moreover, we compared leukopenia in resected and non-resected patients. RESULTS: The incidence of grade 3 or 4 leukopenia was 25% in the non-resected patients, whereas equivalent leukopenia was observed in 57% of the resected patients ( P = 0.048 by the chi(2) test). The relative decrease in blood leukocytes induced by GEM administration was more severe in resected patients (41.3 +/- 9.9%), as compared to non-resected patients (52.6 +/- 16.0%; P = 0.023 by t-test). CONCLUSION: In the present study, we found that the administration of GEM to patients after surgical resection caused more severe leukopenia, as compared to findings in non-resected patients. These data suggested that more frequent monitoring of the leukocyte count and prolonged intervals between GEM administrations are necessary for resected patients with pancreatic cancer.Gemcitabine hydrochloride (GEM) is one of the most effective chemotherapeutic agents for pancreatic cancer; however, factors affecting GEM-induced leukopenia have not been clarified yet. In the present study, we analyzed the relationship between patients' backgrounds and GEM-induced leukopenia.

Aged↗

PubMed2004

Expression levels of renal organic anion transporters (OATs) and their correlation with anionic drug excretion in patients with renal diseases.

PURPOSE: Because the urinary excretion of drugs is often decreased in renal diseases, dosage regimens are adjusted to avoid adverse drug reactions. The aim of present study was to clarify the alteration in the levels of renal drug transporters and their correlation with the urinary drug excretion in renal diseases patients. METHODS: We quantified the mRNA levels of human organic anion transporters (hOATs) by real-time polymerase chain reaction and examined the excretion of the anionic drug, cefazolin, in renal disease patients. Moreover, transport of cefazolin by hOAT1 and hOAT3 were examined using HEK293 transfectants. RESULTS: Among four hOATs, the level of hOAT1 mRNA was significantly lower in the kidney of patients with renal diseases than in the normal controls. The elimination constant of cefazolin showed a significant correlation with the values of phenolsulfonphthalein test and mRNA levels of hOAT3. The uptake study using HEK293 transfectants revealed that cefazolin and phenolsulfonphthalein were transported by hOAT3. CONCLUSIONS: These results suggest that hOAT3 plays an important role for anionic drug secretion in patients with renal diseases and that the expression levels of drug transporters may be related to the alteration of renal drug secretion.

Adult↗

PubMed2004

Creatinine transport by basolateral organic cation transporter hOCT2 in the human kidney.

PURPOSE: Creatinine is excreted into urine by tubular secretion in addition to glomerular filtration. The purpose of this study was to clarify molecular mechanisms underlying the tubular secretion of creatinine in the human kidney. METHODS: Transport of [14C]creatinine by human organic ion transporters (SLC22A) was assessed by HEK293 cells expressing hOCT1, hOCT2, hOCT2-A, hOAT1, and hOAT3. RESULTS: Among the organic ion transporters examined, only hOCT2 stimulated creatinine uptake when expressed in HEK293 cells. Creatinine uptake by hOCT2 was dependent on the membrane potential. The Michaelis constant (Km) for creatinine transport by hOCT2 was 4.0 mM, suggesting low affinity. Various cationic drugs including cimetidine and trimethoprim, but not anionic drugs, markedly inhibited creatinine uptake by hOCT2. CONCLUSION: These results suggest that hOCT2, but not hOCT1, is responsible for the basolateral membrane transport of creatinine in the human kidney.

1-Methyl-4-phenylpyridinium↗

PubMed2004

The diurnal rhythm of the intestinal transporters SGLT1 and PEPT1 is regulated by the feeding conditions in rats.

The intestinal Na(+)/glucose cotransporter 1 (SGLT1) and H(+)/peptide cotransporter 1 (PEPT1) play important roles in the absorption of carbohydrate and protein. Although they exhibit a diurnal rhythm in their expression and function, the factors responsible for this are unclear. In the present study, we examined the effects of various feeding conditions on the diurnal rhythm of intestinal SGLT1 and PEPT1. Rats were divided into 1 of 4 groups: group 1 was fed, group 2 was food deprived for 1-4 d, group 3 was food deprived for 4 d and then refed for 1 or 2 d, and group 4 was fed during the daytime (0900-1500 h) for 10 d. In fed rats, the SGLT1 protein level was significantly higher at 2000 h than at 0800 h. However, in rats deprived of food for 2-4 d, protein levels did not differ between 0800 and 2000 h. In contrast, the SGLT1 messenger RNA (mRNA) level was significantly higher at 2000 h than at 0800 h in rats deprived of food for 4 d. Refeeding for 2 d after 4 d of food deprivation returned the diurnal variation in SGLT1 and PEPT1 protein expressions to normal. Consuming food during the daytime only shifted the peaks of SGLT1 and PEPT1 mRNAs and protein expressions from the dark phase to the light phase. These findings suggest that food intake, rather than the light cycle, greatly affects the diurnal rhythm of SGLT1 and PEPT1 expressions.

Animals↗

PubMed2004

CYP3A5*1-carrying graft liver reduces the concentration/oral dose ratio of tacrolimus in recipients of living-donor liver transplantation.

OBJECTIVES: Tacrolimus is widely used for immunosuppressive therapy after organ transplantation, but its pharmacokinetics shows such great interindividual variation that control of its blood concentration is difficult. We have previously reported that an intestinal P-glycoprotein (MDR1) contributes to this variation as an absorptive barrier, but the role of hepatic metabolism is not clear. METHODS: In this study, we have evaluated the genotypes of MDR1 and cytochrome P450 (CYP) 3A in donor and recipient, and the influence of polymorphisms on mRNA expression and the tacrolimus concentration/dose (C/D) ratio in recipients of living-donor liver transplantation (LDLT). RESULTS: The expression level of MDR1 and tacrolimus C/D ratio were not affected by either MDR1 C3435T or G2677T/A. The CYP3A4*1B genotype was not detected, but the CYP3A5*3 genotype had an allelic frequency of 76.3%. The mRNA level of CYP3A5 was significantly reduced by the *3/*3 genotype, and the tacrolimus C/D ratio was decreased in recipients engrafted with partial liver carrying CYP3A5*1/*1 genotype. An analysis of the combination of intestinal MDR1 level and liver CYP3A5 genotype revealed that the tacrolimus C/D ratio was lower in the group with higher MDR1 levels regardless of CYP3A5 genotype during postoperative week 1. CONCLUSIONS: These results indicate that in recipients of LDLT, the pharmacokinetics of tacrolimus is influenced by flux via P-glycoprotein in the intestine during the first week; after that, it is mostly the hepatic metabolism that contributes to the excretion of tacrolimus, and carriers of the CYP3A5*1/*1 genotype require a high dose of tacrolimus to achieve the target concentration.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Gene expression variance based on random sequencing in rat remnant kidney.

BACKGROUND: Several examinations have been performed to identify the genes involved in chronic renal failure using 5/6 nephrectomized rats. Recently, many systematic techniques for examining molecular expression have been developed. They might also be effective in elucidating the molecular mechanism of progressive renal failure. In this study, digital expression profiling was carried out to construct a subtractive mRNA expression database for the 5/6 nephrectomized kidney. METHODS: One thousand clones were randomly sequenced from 5/6 nephrectomized and sham-operated rat kidney cDNA libraries, respectively, and defined by BLAST search. In silico subtractive analysis was performed to search for genes up- or down-regulated in the 5/6 nephrectomized kidney. RESULTS: The growth factor-related mRNAs and the mRNAs encoding cytoskeletal or membrane proteins were up-regulated, but the transporter-related mRNAs were down-regulated in the 5/6 nephrectomized kidney database. In silico subtraction revealed that 63 mRNAs were increased and 59 were decreased in the 5/6 nephrectomized kidney. To confirm whether the in silico subtractive database reflected the actual expression of mRNA or protein, 12 known genes were examined by Northern blotting or immunoblotting, respectively. The actual expression of the 12 genes was comparable with the results of in silico subtraction. In addition, we successfully isolated five unknown genes, two up-regulated and three down-regulated in the 5/6 nephrectomized kidney. CONCLUSION: We constructed a subtractive mRNA expression database for 5/6 nephrectomized kidney, which reflects the actual alterations in mRNA expression after subtotal nephrectomy. This database may be useful for elucidation of the molecular mechanism of progressive renal failure.

Amino Acid Sequence↗

PubMed2004

Methotrexate-loxoprofen interaction: involvement of human organic anion transporters hOAT1 and hOAT3.

Human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8) are responsible for renal tubular secretion of an antifolic acid methotrexate, and are considered to be involved in drug interaction of methotrexate with nonsteroidal anti-inflammatory drugs (NSAIDs). In our hospital, a delay of methotrexate elimination was experienced in a patient with Hodgkin's disease, who took loxoprofen, a commonly used NSAID in Japan, which suggested a cause. In this study, we examined the drug interaction via hOAT1 and hOAT3, using Xenopus laevis oocytes. hOAT1 and hOAT3 mediated the methotrexate transport with low affinity (K(m) of 724.0 muM) and high affinity (K(m) of 17.2 muM), respectively. Loxoprofen and its trans-OH metabolite, an active major metabolite, markedly inhibited the methotrexate transport by both transporters. Their inhibition concentrations (IC(50)) were in the range of the therapeutic levels. These findings suggest that loxoprofen retards the elimination of methotrexate, at least in part, by inhibiting hOAT1 and hOAT3.

Animals↗

PubMed2004

Decreased expression of glucose and peptide transporters in rat remnant kidney.

The loss of renal mass induces tubular hypertrophy as well as glomerular sclerosis and results in the end stage of renal disease. However, there is little information about adaptation of tubular glucose and peptide reabsorption under conditions of chronic renal failure. In the present study, we performed functional and molecular analyses focused on the tubular reabsorption of filtered glucose and small peptides using 5/6 nephrectomized rats at 16 weeks, as a model of chronic renal failure. Sixteen weeks after 5/6 nephrectomy or sham treatment, the brush-border membranes and total RNA were obtained from the renal cortex to evaluate the uptake of Na(+) gradient-dependent D-glucose and H(+) gradient-dependent glycylsarcosine. The amounts of SGLT and PEPT mRNA levels were quantified by competitive PCR. The urinary glucose/creatinine ratio was markedly higher in nephrectomized rats than in sham-operated controls. Na(+)-dependent glucose uptake by the isolated renal brush-border membrane vesicles was markedly decreased in nephrectomized rats compared with that in sham-operated controls. However, H(+)-dependent peptide transport, another secondary active transport system in the brush-border membranes, was maintained. In addition, kinetic analysis revealed that both SGLT1 (high-affinity type)- and SGLT2 (low-affinity type)-mediated Na(+)/glucose uptake had markedly decreased Vmax values, but not Km values. Furthermore, competitive PCR demonstrated that the mRNA expression levels of SGLT2, PEPT1 and PEPT2, but not SGLT1, were markedly depressed. These findings suggested that loss of SGLT2 during chronic renal failure implies a high risk of renal glucosuria.

Animals↗

PubMed2003

Common single nucleotide polymorphisms of the MDR1 gene have no influence on its mRNA expression level of normal kidney cortex and renal cell carcinoma in Japanese nephrectomized patients.

In this study, we have quantified the mRNA expression levels of multidrug resistance gene 1 (MDR1) in the normal kidney cortex and renal cell carcinoma (RCC) segments from 24 Japanese nephrectomized patients by real-time polymerase chain reaction (PCR). The mRNA expression level of MDR1 in RCC segments was significantly decreased in comparison with each normal segment (P=0.0042, by Student's paired t-test). In addition, the ten common single nucleotide polymorphisms (SNPs) of the MDR1 gene in the patients were assessed using the PCR-restriction enzyme fragment length polymorphism method to investigate the influence of these SNPs on its mRNA expression levels. The allele frequencies of these SNPs were comparable with our previous report in the Japanese recipients of living-donor liver transplantation (Goto et al., Pharmacogenetics 12:451-457; 2002). MDR1 expression levels in the normal kidney cortex were independent on the five SNPs, which were polymorphic in the Japanese population. Furthermore, the effect of the SNPs on expression levels of MDR1 mRNA in RCC segments was not recognized. These findings suggest that the common SNPs in the MDR1 gene have no influence on the expression of its transcript in RCC segments as well as in the normal kidney cortex.

Adult↗

PubMed2003

Decreased activity of basolateral organic ion transports in hyperuricemic rat kidney: roles of organic ion transporters, rOAT1, rOAT3 and rOCT2.

We investigated organic anion and cation transport activity and the expression of several organic ion transporters in hyperuricemic rat kidney. Feeding oxonic acid, an inhibitor of uric acid metabolism, and uric acid for 10 days significantly increased plasma uric acid level. Plasma creatinine and blood urea nitrogen concentrations also increased in hyperuricemic rats, indicating impaired renal function. The accumulation of organic anions, p-aminohippurate (PAH) and methotrexate, and cations, tetraethylammonium (TEA) and cimetidine, into renal slices was markedly decreased, suggesting decreased transport activity for organic anions and cations at the basolateral membrane in the kidney. The expression levels of basolateral organic anion transporters rOAT1 and rOAT3, and organic cation transporter, rOCT2, significantly decreased in hyperuricemic rat kidney as assessed by mRNA and protein levels. In contrast, the expression of rOCT1 was unaltered by hyperuricemia at both mRNA and protein levels. Moreover, the mRNA expression of kidney-specific organic anion transporters, OAT-K1 and OAT-K2, and organic anion transporting polypeptide (oatp) 1, which localize at the brush-border membrane in the kidney, was unchanged in hyperuricemic rats. In conclusion, we showed decreased basolateral organic anion and cation transport activity, accompanied by a specific decrease in rOAT1, rOAT3 and rOCT2 expression in hyperuricemic rat kidney. These phenomena partly contribute to the changed renal disposition of organic anions and cations in hyperuricemia.

Animals↗

PubMed2003

Altered diurnal rhythm of intestinal peptide transporter by fasting and its effects on the pharmacokinetics of ceftibuten.

We previously demonstrated that H+/peptide cotransporter PEPT1 shows a diurnal rhythm in the rat small intestine. In the present study, we examined the effect of food intake on the diurnal rhythm of intestinal PEPT1 using fed and fasted rats and also determined whether such variation affected the pharmacokinetics of peptide-like drugs. In fed rats, PEPT1 protein level was significantly higher at 8:00 PM than at 8:00 AM. However, during fasting for 2 to 4 days, the differences of PEPT1 protein levels between 8:00 AM and 8:00 PM gradually disappeared. Intestinal absorption of an oral antibiotic ceftibuten (CETB), a pharmacological substrate for PEPT1, was also greater at 8:00 PM than at 8:00 AM in fed rats, but not different in 4-day fasted rats. In contrast to PEPT1 protein levels, PEPT1 mRNA levels retained a diurnal rhythm after 4 days of fasting. Pharmacokinetic analyses of CETB after intraintestinal administration demonstrated that both Cmax and area under the plasma concentration-time curve from 0 to 3 h were greater at 8:00 PM than at 8:00 AM in fed rats. In contrast, pharmacokinetic parameters showed no significant difference between 8:00 AM and 8:00 PM for intraintestinal administration in 4-day fasted rats and for intravenous administration in fed and 4-day fasted rats. These findings suggested that the diurnal rhythm of intestinal PEPT1 transport activity was disrupted by fasting and that diurnal variation of intestinal PEPT1 functionality could influence the pharmacokinetics of peptide-like drugs such as CETB.

Animals↗

PubMed2003

Na(+)-dependent fructose transport via rNaGLT1 in rat kidney.

We found a system of Na(+)-dependent uptake of fructose by rat renal brush-border membrane vesicles. It consisted of two saturable components, and was thought to involve at least two transporters. rNaGLT1, a novel glucose transporter in rat kidney, showed fructose uptake as well as alpha-methyl-D-glucopyranoside uptake by transfected HEK293 cells. The features of the lower affinity type of fructose transporter in the brush-border membranes, such as affinity and substrate recognition, were very comparable with those of rNaGLT1-transfected HEK293 cells. These results indicated that rNaGLT1 is a primary fructose transporter in rat renal brush-border membranes.

Animals↗

PubMed2003

Cloning and characterization of a novel Na+-dependent glucose transporter (NaGLT1) in rat kidney.

To identify novel transporters in the kidney, we have constructed an mRNA data base composed of 1000 overall clones by random sequencing of a male rat kidney cDNA library. After a BLAST search, approximately 40% of the clones were unknown and/or unannotated and were screened by measuring the uptake of various compounds using Xenopus oocytes. One clone stimulated the uptake of alpha-methyl-d-glucopyranoside and therefore was termed rat Na(+)-dependent glucose transporter 1 (rNaGLT1). The rNaGLT1 cDNA (2173 bp) has an open reading frame encoding a 484-amino acid protein, showing <22% homology to known SGLT and GLUT glucose transporters. alpha-Methyl-d-glucopyranoside uptake by rNaGLT1 cRNA-injected oocytes showed saturability, with an apparent K(m) of 3.7 mm and a coupling ratio of 1:1 with Na(+). rNaGLT1 mRNA was expressed predominantly in the kidney upon Northern blot analysis and reverse transcription-PCR. Reverse transcription-PCR in microdissected nephron segments revealed that rNaGLT1 mRNA was primarily localized in the proximal tubules. A clear signal corresponding to rNaGLT1 protein was recognized in the brush-border (but not basolateral) membrane fraction by immunoblot analysis. The rNaGLT1 mRNA level in the kidney was significantly higher than rat SGLT1 and SGLT2 mRNA levels. These findings suggest that rNaGLT1 is a novel Na(+)-dependent glucose transporter with low substrate affinity that mediates tubular reabsorption of glucose.

Amino Acid Sequence↗

PubMed2003

A new method of purifying fibrinogen with both biological and immunological activity from human plasma.

Using a combination of Cohn ethanol fractionation, virus inactivation, glycine and sodium chloride precipitation, and lysine-Sepharose affinity chromatography, a unique and rapid simplified method was developed to obtain highly purified fibrinogen for diagnostic use with both biological (Clauss method) and immunological (Jacobsson method) activity. Yield was 0.66 g of fibrinogen per liter of starting pooled plasma, and the purified product showed good agreement in activity with the starting material. The purified fibrinogen solution contained over 95% clottable protein and had a clear appearance. No degradation was observed after urokinase treatment and the preparation provided good precision in fibrinogen measurement compared to pooled plasma. The simplified method was, thus, shown to result in a high-purity fibrinogen preparation, suitable for in vitro diagnostic use, as well as for use to prepare a fibrinogen reference material and to perform fibrinogen quality control using an automated coagulation analyzer.

Algorithms↗

PubMed2003

Decreased function of genetic variants, Pro283Leu and Arg287Gly, in human organic cation transporter hOCT1.

We have evaluated the functional consequences of genetic variations in human organic cation transporter hOCT1 (SLC22A1). Three coding single nucleotide polymorphisms (cSNPs) resulted in the amino acid changes Pro283Leu, Arg287Gly and Pro341Leu were assessed. Uptake experiments with transient expression system using HEK293 cells revealed that the variants Pro283Leu and Arg287Gly had completely diminished transport activity. The other variant Pro341Leu had a significantly, but not completely, decreased transport activity. Western blot analysis showed that the expression levels of all three variant proteins in the crude membranes of HEK293 cells were comparable to those of wild type hOCT1. Moreover, the expression of variant proteins at the plasma membrane was confirmed by indirect immunofluorescence, indicating that these SNPs did not affect the membrane localization of hOCT1. Present results suggest that the amino acid residues Pro283 and Arg287 have a substantial role in substrate recognition of hOCT1.

Journal Article↗

PubMed2002

cDNA cloning, functional characterization, and tissue distribution of an alternatively spliced variant of organic cation transporter hOCT2 predominantly expressed in the human kidney.

A cDNA coding a novel organic cation transporter, hOCT2-A, was isolated from human kidney. The hOCT2-A cDNA is an alternatively spliced variant of hOCT2 with an insertion of 1169 bp. The open reading frame encodes a 483-amino acid protein that has 81% amino acid identity with hOCT2. From hydropathy analysis, hOCT2-A is predicted to have nine transmembrane domains. hOCT2-A mRNA is expressed mainly in kidney and weakly in brain, liver, colon, skeletal muscle, bone marrow, spinal cord, testis, and placenta. When expressed in HEK293 cells, hOCT2-A stimulated the uptake of tetraethylammonium (TEA) in an electrogenic manner. The transport of TEA by hOCT2-A-transfected cells was saturable with the apparent Km value of 63 microM. hOCT2-A stimulated the uptake of TEA, 1-methyl-4-phenylpyridinium, and cimetidine as well as did hOCT2. The uptake of guanidine and choline by hOCT2-transfected cells also increased markedly but not that by hOCT2-A-transfected cells. The uptake of TEA mediated by hOCT2-A but not by hOCT2 was inhibited significantly by organic cations such as procainamide, N-acetylprocainamide, and levofloxacin, indicating that hOCT2-A differs from hOCT2 in its affinity for several compounds. These findings suggested that hOCT2-A contributes to the renal clearance of endogenous and exogenous organic cations.

Alternative Splicing↗

PubMed2002

Expression of calcium-sensing receptor in rat colonic epithelium: evidence for modulation of fluid secretion.

The calcium-sensing receptor (CaSR) is activated by extracellular calcium (Ca2+(o)) and mediates many of the known effects of extracellular divalent minerals on body cells. Both surface and crypt cells express CaSR transcripts and protein on both apical and basolateral surfaces. Raising Ca2+(o) elicited increases in intracellular calcium (Ca2+(o)) in both surface and crypt cells with an EC50 of 2 mM. The Ca-induced increase in Ca2+(i) was associated with increases in inositol 1,4,5-trisphosphate and eliminated by U-73129, an inhibitor of phosphatidylinositol-phospholipase C, as well as by thapsigargin. Other CaSR agonists, Gd3+ and neomycin, mimicked these Ca2+(o)-induced responses. Both luminal and bath Ca2+(o), Gd3+, and neomycin induced increases in Ca2+(i) in isolated perfused crypts. The stimulatory effect of forskolin on net fluid secretion in perfused crypts was abolished by increasing Ca2+(o) in either luminal or bath perfusates. Thus both apical and basolateral CaSR on crypt cells are functional and provide pathways modulating net intestinal fluid transport that may have important implications for the prevention and treatment of certain diarrheal diseases associated with elevated cAMP.

Animals↗

PubMed2002

Involvement of specific transport system of renal basolateral membranes in distribution of nicotine in rats.

We measured the nicotine concentrations in tissues after a bolus i.v. administration of [(3)H]nicotine to rats to characterize the distribution profile of nicotine. The kidney showed the greatest distribution of nicotine compared to other tissues including liver, lung, heart, brain, and intestine. We also performed an HPLC assay for the determination of nicotine and its major metabolite, cotinine, and found that cotinine was negligible in the distribution of almost all tissues, except for the kidney and lung. In the kidney, cotinine was detected at a lower level than nicotine, while cotinine tended to be distributed in the lung compared to nicotine. [(3)H]Nicotine was accumulated in renal slices in a concentration dependent fashion, suggesting that the nicotine uptake in the renal tubules could be mediated by a specific transport system. Unlabeled nicotine, cotinine, and quinidine showed potent inhibitory effects on [(3)H]nicotine uptake by renal slices. In contrast, tetraethylammonium (TEA), cimetidine, and N(1)-methylnicotinamide (NMN), which were substrates of renal organic cation transporters, had no effects on the uptake. These findings suggested that a specific transporter was involved in nicotine transport at the basolateral membranes of rat renal tubules, which could mediate the high accumulation of nicotine from blood into the kidney.

Journal Article↗