Biomedical subjects

Ken-Ichi Inui

Publications and source records attributed to Ken-Ichi Inui.

30 records · Page 2Linked to original sources

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↗

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 cyclosporin A concentrations in the absorption phase using microemulsion preconcentrate formulation in rats with cisplatin-induced acute renal failure.

Cyclosporin A pharmacokinetics was studied in rats with cisplatin-induced acute renal failure (ARF) using microemulsion preconcentrate (MEPC) and completely dissolved formulations. Although the pharmacokinetics of cyclosporin A was unchanged after intravenous administration, maximum concentration of cyclosporin A in ARF rats was significantly reduced to 608+/-62 and 999+/-189 ng/ml compared with 1720+/-142 and 1832+/-250 ng/ml in controls after oral administration of MEPC and completely dissolved formulations, respectively. In an in situ intestinal loop sac study, the amount absorbed plus metabolized and the blood concentration of cyclosporin A were similar between control and ARF rats, and taurocholic acid, one of the bile acids, significantly increased absorption of cyclosporin A using the MEPC formulation in both control and ARF rats; the amount absorbed plus metabolized with taurocholic acid was increased to 137 and 186%, and simultaneously the blood concentration was increased to 155 and 158% of that without taurocholic acid in control and ARF rats, respectively. The bile flow in ARF rats was decreased compared with that in controls. These results suggested that renal dysfunction decreased the absorption of cyclosporin A in spite of the MEPC formulation, and the alternation of bile secretion partly affected the absorption rate of cyclosporin A in the gastrointestinal tract.

Absorption↗

PubMed2003

Intestinal absorption of drugs mediated by drug transporters: mechanisms and regulation.

The absorption of drugs from the gastrointestinal tract is one of the important determinants for oral bioavailability. Development of in vitro experimental techniques such as isolated membrane vesicles and cell culture systems has allowed us to elucidate the transport mechanisms of various drugs across the plasma membrane. Recent introduction of molecular biological techniques resulted in the successful identification of drug transporters responsible for the intestinal absorption of a wide variety of drugs. Each transporter exhibits its own substrate specificity, though it usually shows broad substrate specificity. In this review, we first summarize the recent advances in the characterization of drug transporters in the small intestine, classified into peptide transporters, organic cation transporters and organic anion transporters. In particular, peptide transporter (PEPT1) is the best-characterized drug transporter in the small intestine, and therefore its utilization to improve the oral absorption of poorly absorbed drugs is briefly described. In addition, regulation of the activity and expression levels of drug transporters seems to be an important aspect, because alterations in the functional characteristics and/or expression levels of drug transporters in the small intestine could be responsible for the intra- and interindividual variability of oral bioavailability of drugs. As an example, regulation of the activity and expression of PEPT1 is summarized.

Journal Article↗

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

C3435T polymorphism in the MDR1 gene affects the enterocyte expression level of CYP3A4 rather than Pgp in recipients of living-donor liver transplantation.

The bioavailability of structurally unrelated drugs is limited by active secretion via the multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) from enterocyte into lumen as well as intestinal metabolism by cytochrome P450 IIIA4 (CYP3A4). In the present study, we analyzed whether genetic polymorphism of the MDR1 had some influence on the intestinal expression levels of Pgp and CYP3A4 and the tacrolimus concentration/dose ratio over the first postoperative days in recipients of living-donor liver transplantation (LDLT). Genotyping assays were performed for the major 10 polymorphisms in the MDR1 gene by the polymerase chain reaction-restriction enzyme length polymorphism method. The allele frequencies of variations at five positions were almost comparable with those in the former studies in Caucasians and Japanese, but there was no variation at the other five positions. Although no polymorphism correlated with the intestinal expression of MDR1 mRNA or the tacrolimus concentration/dose ratio in the LDLT recipients, the C3435T polymorphism significantly affected the intestinal expression level of CYP3A4 mRNA as follows; 3435C/C>3435C/T (P < 0.05 vs. 3435C/C)>3435T/T (P < 0.01 vs. 3435C/C). Therefore, the identified polymorphisms including C3435T in the MDR1 gene were indicated to have no influence on the intestinal expression level of Pgp or the tacrolimus concentration/dose ratio in the recipients of LDLT. On the other hand, the C3435T polymorphism of MDR1 was suggested to correlate with the enterocyte expression of CYP3A4 rather than Pgp linking unknown genetic variation in CYP3A4 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

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

Transport mechanisms of nicotine across the human intestinal epithelial cell line Caco-2.

Ulcerative colitis is a disease more commonly seen in nonsmokers. Because nicotine was postulated to be a beneficial component of tobacco smoke for ulcerative colitis, various formulations of nicotine have been developed to improve the local bioavailability within the gastrointestinal tissue. In the present study, to characterize the disposition of nicotine in the intestines, we investigated intestinal nicotine transport using Caco-2 cells. Nicotine was predominantly transported across Caco-2 cell monolayers in a unidirectional mode, corresponding to intestinal secretion, by pH-dependent specific transport systems. The specific uptake systems appear to be distinct from organic cation transporters and the transport system for tertiary amines, in terms of its substrate specificity and the pattern of the interaction. These transport systems could play a role in the intestinal accumulation of nicotine from plasma and could also be responsible for the topical delivery of nicotine for ulcerative colitis therapy. These findings could provide useful information for the design of effective nicotine delivery.

Biological Transport↗

PubMed2002

Thyroid hormone regulates the activity and expression of the peptide transporter PEPT1 in Caco-2 cells.

An oligopeptide transporter (PEPT1) in the small intestine plays an important role in the absorption of small peptides and peptide-like drugs. We examined the effect of thyroid hormone 3,5,3'-L-triiodothyronine (T(3)) on the activity and expression of PEPT1 in human intestinal Caco-2 cells. Treatment of Caco-2 cells with T(3) inhibited [(14)C]glycylsarcosine uptake in a time- and dose-dependent manner. [(14)C]glycylsarcosine uptake was reduced by pretreatment of the cells with 100 nM T(3) for 4 days (67% of control value), whereas methyl-alpha-D-[U-(14)C]glucopyranoside and [(3)H]threonine uptake were not decreased. Kinetic analysis showed that T(3) treatment significantly decreased the maximum uptake (V(max)) value for [(14)C]glycylsarcosine uptake but had no effect on the K(m) value. Moreover, T(3) treatment caused a significant decrease in the amount of PEPT1 mRNA (25% of the control). Western blotting indicated that the amount of PEPT1 protein in the apical membrane was decreased (70% of the control). These findings indicate that T(3) treatment inhibits the uptake of [(14)C]glycylsarcosine by decreasing the transcription and/or stability of PEPT1 mRNA.

Blotting, Western↗

PubMed2002

Diurnal rhythm of H+-peptide cotransporter in rat small intestine.

In mammals, most physiological, biochemical, and behavioral processes show a circadian rhythm. In the present study, we examined the diurnal rhythm of the H+-peptide cotransporter (PEPT1), which transports small peptides and peptide-like drugs in the small intestine and kidney, using rats maintained in a 12-h photoperiod with free access to chow. The transport of [14C]glycylsarcosine (Gly-Sar), a typical substrate for PEPT1 by in situ intestinal loop and everted intestine, was greater in the dark phase than the light phase. PEPT1 protein and mRNA levels varied significantly, with a maximum at 2000 and minimum at 800. Similar functional and expressional diurnal variations were observed in the intestinal Na+-glucose cotransporter (SGLT1). In contrast, renal PEPT1 and SGLT1 showed little diurnal rhythmicity in protein and mRNA expression. These findings indicate that the intestinal PEPT1 undergoes diurnal regulation in its activity and expression, and this could affect the intestinal absorption of dietary protein.

Absorption↗

PubMed2002

Gene expression levels and immunolocalization of organic ion transporters in the human kidney.

Renal excretion of organic anions and cations is mediated by the organic ion transporter family (SLC22A). In this study, the mRNA levels of the organic ion transporters were quantified by real-time PCR in normal parts of renal tissues from seven nephrectomized patients with renal cell carcinoma, and the distributions and localization of human (h)OAT1, hOAT3, and hOCT2 proteins were investigated by immunohistochemical analyses in the human kidney. The expression level of hOAT3 mRNA was the highest among the organic ion transporter family, followed by that of hOAT1 mRNA. The hOCT2 mRNA level was the highest in the human OCT family, and the level of hOCTN2 mRNA was higher than that of hOCTN1. hOCT1 mRNA showed the lowest level of expression in organic ion transporter family. hOAT1, hOAT3, and hOCT2 proteins were detected in crude membranes from the kidney of all patients by Western blot analyses, whereas hOCT1 protein could not be detected. Immunohistochemical analyses showed that both hOAT1 and hOAT3 were localized to the basolateral membrane of the proximal tubules in the cortex, and hOCT2 was localized to the basolateral membrane of the proximal tubules in both the cortex and medullary ray. Immunohistochemical analyses of serial sections indicated that hOAT1, hOAT3, and hOCT2 were coexpressed in a portion of the proximal tubules. These results suggest that hOAT1, hOAT3, and hOCT2 play predominant roles in the transport of organic ions across the basolateral membrane of human proximal tubules.

Aged↗

PubMed2002

Rat renal organic anion transporter rOAT1 mediates transport of urinary-excreted cephalosporins, but not of biliary-excreted cefoperazone.

Most cephalosporin antibiotics are excreted into urine via glomerular filtration and active tubular secretion by renal organic anion transporters. In this study, we investigated the interaction of cephalosporins with rat organic anion transporter rOAT1, mainly expressed at the basolateral membrane of the renal proximal tubules, using Xenopus laevis oocytes, to assess the roles of rOAT1 in renal excretion of cephalosporin antibiotics. The expression of rOAT1 significantly stimulated the uptake of cefazolin, cefotiam and cephalexin into oocytes, but not of cefoperazone. The inhibition constants of these cephalosporins to rOAT1-mediated p-aminohippurate (PAH) uptake were 72 microM for cefazolin, 298 microM for cefoperazone, 718 microM for cefotiam and 6 mM for cephalexin. Eadie-Hofstee plot analysis revealed that cefoperazone as well as cefotiam inhibited rOAT1-mediated PAH uptake competitively. These results suggest that rOAT1 mediates basolateral uptake of cephalosporin antibiotics in the renal tubules. Furthermore, it is suggested that a minor contribution of the kidney to cefoperazone excretion could be related to the finding that cefoperazone is a poor substrate of rOAT1.

Journal Article↗