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Biomedical subjects

K Fushimi

Publications and source records attributed to K Fushimi.

At least 55 records · Page 3Linked to original sources
PubMed1996

Role of vasopressin V2 receptor in acute regulation of aquaporin-2.

Aquaporin-2 (AQP-2) has been shown to be a vasopressin-sensitive water channel in collecting duct (CD) cells of the kidney. To prove the role of the vasopressin V2 receptor (V2R) in the regulation of intracellular AQP-2 shuttling, we examined the acute effects of vasopressin and V2R antagonist on the distribution of AQP-2 in the cells. Normal Wistar rats were given continuous infusions of vasopressin, vasopressin V2R antagonist (OPC31260), or both. The kidneys were then processed for immunofluorescent studies with an affinity-purified specific antibody to AQP-2. One hour after the infusion of the V2R antagonist, AQP-2 staining was diffusely distributed in the CD cells from the cortex to the inner medulla. This tendency was not changed by the concomitant infusion with vasopressin. Vasopressin infusion without antagonist, however, induced intensified AQP-2 staining of the apical membrane in the CD cells. The ratio of the fluorescence intensity of the apical to subapical region was determined by confocal laser microscopy. In the inner medulla, this ratio was significantly increased in the vasopressin treatment group (2.26 +/- 0.76) as compared to the V2R antagonist group (1.03 +/- 0.34) and the combined treatment group (0.84 +/- 0.43). The increase in the ratio was also demonstrated in the cortex and the outer medulla in the vasopressin-treated group. In addition, Northern blotting studies clearly revealed that mRNA of AQP-2 in the vasopressin-treated group was increased when compared to the combined treatment animals. Our present results reveal that localization and gene expressions of AQP-2 are acutely regulated via vasopressin V2R.

Animals↗
PubMed1996

Immortalization of human cells and its mechanisms.

One of the contributions of in vitro neoplastic transformation studies on human cells is the confirmation that cells become malignant by a multistep process. In this review, we use the term "neoplastic transformation" to indicate that the cells become capable of forming a histologically malignant tumor, that is, one that invades and/or erodes normal tissue and is serially transplantable in a susceptible animal host. Phenotypically, the cells first become immortalized and then neoplastic. If normal human cells do not overcome aging by becoming immortalized, they cannot be neoplastically transformed. However, normal human cells are stringently destined to cellular aging and very rarely become immortalized by oncogenic DNA viruses (SV40, papillomavirus types 16 and 18, adenovirus types 5 and 12), chemicals, X-rays, or spontaneously. Once human cells get immortalized, they relatively easily become neoplastic. Evidence shows that immortalization is a critical step in the neoplastic transformation process of human cells. Therefore, investigation of the mechanisms of immortalization is indispensable in understanding the multistep carcinogenesis of human cells. Although the precise mechanisms of immortalization are unknown at present, data indicate that the p53 cascade appears to be important for the immortalization of human cells. The loss of functions of normal p53 may induce genomic instability, resulting in mutations of some other unknown genes that are more directly involved in the immortalization of human cells. These unknown genes, which are presumably located at chromosomes 1, 4, 6, 7, 9, 11, 13, 17, 18, and X are now under extensive investigation.

Cell Transformation, Neoplastic↗
PubMed1995

Isolation of human aquaporin 3 gene.

Human aquaporin 3 (AQP3) gene was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the human genome and to comprise six exons distributing over 7 kilobases. The sizes of the exons are 171, 127, 138, 119, 218, and 1035 base pairs, and those of introns are approximately 3530, 300, 350, 330, and 90 base pairs, respectively. The initiation site of transcription was identified to locate 64 base pairs upstream of the first ATG codon by primer extension analysis and ribonuclease protection assay. The 5'-flanking region has a TATA box, two Sp1 sequences, and some consensus sequences including AP2 sites. With luciferase assay, the 5'-flanking region was demonstrated to have a promoter activity, which is up-regulated 4-fold by phorbol ester. These findings about the genomic clone of human AQP3 will contribute to elucidate the molecular mechanism of transcriptional regulation of AQP3.

Amino Acid Sequence↗
PubMed1995

Urinary excretion of aquaporin-2 in patients with diabetes insipidus.

BACKGROUND: Urine-concentrating ability is regulated by vasopressin. Recently, the specific water-channel protein of the renal collecting duct, known as aquaporin-2, was cloned. However, it is not certain whether this molecule is responsive to vasopressin. METHODS: We measured the urinary excretion of aquaporin-2 and its response to vasopressin in 11 normal subjects and 9 patients with central or nephrogenic diabetes insipidus. The urine samples were collected during periods of dehydration and hydration and after the administration of vasopressin. Urine samples were analyzed for aquaporin-2 by the Western blot assay and immunogold labeling, and the amount of aquaporin-2 was determined by radioimmunoassay. RESULTS: Aquaporin-2 was detectable in the urine in both soluble and membrane-bound forms. In the five normal subjects tested, the mean (+/- SE) urinary excretion of aquaporin-2 was 11.2 +/- 2.2 pmol per milligram of creatinine after a period of dehydration, and it decreased to 3.9 +/- 1.9 pmol per milligram of creatinine (P = 0.03) during the second hour after a period of hydration. In the six other normal subjects, an infusion of desmopressin (1-desamino-8-D-arginine vasopressin) increased the urinary excretion of aquaporin-2 from 0.8 +/- 0.3 to 11.2 +/- 1.6 pmol per milligram of creatinine (P < 0.001). The five patients with central diabetes insipidus also had increases in urinary excretion of aquaporin-2 in response to the administration of vasopressin, but the four patients with X-linked or non-X-linked nephrogenic diabetes insipidus did not. CONCLUSIONS: Aquaporin-2 is detectable in the urine, and changes in the urinary excretion of this protein can be used as an index of the action of vasopressin on the kidney.

Adult↗
PubMed1995

cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes.

Among water channel proteins (aquaporins), aquaporin-collecting duct (AQP-CD) is the vasopressin-regulated water channel. Vasopressin causes cAMP production in the renal collecting duct cells, and this is believed to lead to exocytic insertion of water channel into the apical membrane (shuttle hypothesis). AQP-CD contains a consensus sequence for cAMP-dependent protein kinase, residues at positions 253-256 (Arg-Arg-Gln-Ser). To determine the role of this site, Ser-256 was substituted for Ala, Leu, Thr, Asp, or Glu by site-directed mutagenesis. In Xenopus oocytes injected with wild-type or mutated AQP-CD cRNAs, osmotic water permeability (Pf) was 4.8-7.7 times higher than Pf of water-injected oocytes. Incubation with cAMP plus forskolin or direct cAMP injection into the oocytes increased Pf of wild-type, but not mutated, AQP-CD-expressing oocytes, whereas the amounts of AQP-CD expression were similar in wild and mutated types as identified by Western blot analysis. In vitro phosphorylation studies of AQP-CD proteins expressed in oocyte showed that cAMP-dependent protein kinase phosphorylated wild-type, but not mutated, AQP-CD proteins. Phosphoamino acid analysis revealed that this phosphorylation occurred at the serine residue. Moreover, phosphorylation of AQP-CD protein in intact rat kidney medulla tissues was stimulated by incubation with cAMP. Our data suggest that cAMP stimulates water permeability of AQP-CD by phosphorylation. This process may contribute to the vasopressin-regulated water permeability of collecting duct in addition to the apical insertion of AQP-CD by exocytosis.

Animals↗
PubMed1995

Water channels.

The aquaporin water channels are expressed in various fluid-transporting epithelia. Physiological and genetic investigations have revealed that aquaporin channel-like intrinsic protein is expressed in numerous tissues, but its significance in water transport physiology is unclear. It has been shown that aquaporin-collecting duct is a vasopressin-responsive water channel, and that it is regulated by a membrane shuttle mechanism. Three unique models for a water pore have been presented but further studies will be required to verify them. New aquaporin members have been isolated and their discrete localization may reflect their specific physiological roles.

Animals↗
PubMed1995

Vasopressin increases AQP-CD water channel in apical membrane of collecting duct cells in Brattleboro rats.

The effect of vasopressin on subcellular localization of AQP-CD and AQP3 water channels was examined in thirsted Brattleboro rats by immunohistochemistry and immunoelectron microscopy. AQP-CD was mainly present in the cytoplasm of the collecting duct cells in association with cytoplasmic vesicles but was sparse in the apical membrane in control vehicle-injected rats. In rats given vasopressin 15 min before death, the number of immunogold particles for AQP-CD in the apical membrane increased significantly (P < 0.002) from 1.8 +/- 0.2 to 10.0 +/- 0.4/microns with a significant decrease (P < 0.05) of cytoplasmic labeling from 32.6 +/- 6.4 to 24.6 +/- 5.6/microns 2, indicating that AQP-CD is the vasopressin-regulated water channel predicted by the "shuttle" hypothesis. In contrast, AQP3 was restricted to the basolateral membrane of the collecting duct cells, and the labeling density of AQP3 was unchanged by vasopressin treatment, indicating that AQP3 is constitutively expressed and may maintain high water permeability of the basolateral membrane.

Amino Acid Sequence↗
PubMed1995

Role of water channel AQP-CD in water retention in SIADH and cirrhotic rats.

We determined whether aquaporin of collecting duct (AQP-CD) is involved in pathogenesis of water retention in rats with experimental models of syndrome of inappropriate secretion of antidiuretic hormone (SIADH) and liver cirrhosis. SIADH rats were made by administering 1-desamino-8-D-arginine vasopressin (DDAVP) subcutaneously and providing them with a liquid diet. Serum Na levels decreased to < 120 meq/l on day 2, and hyponatremia persisted throughout the rest of observation period. Six hours after the DDAVP infusion, the expression of AQP-CD mRNA significantly increased by 198%, followed by > 144% increases in its expression during the 14-day observation period. On day 7, the increased expression of AQP-CD mRNA was abolished after the administration of an antidiuretic, nonpeptide arginine vasopressin (AVP) antagonist, OPC-31260, which was closely related to a marked diuresis and a prompt normalization of serum Na levels in SIADH rats. Rats were made cirrhotic by injecting a mixture of carbon tetrachloride and olive oil subcutaneously for 3 mo. The expression of AQP-CD mRNA was increased by 164% in the decompensated cirrhotic rats. The blockade of AVP action by OPC-31260 significantly diminished its expression. These results indicate that water channel AQP-CD plays an important role in water retention in pathological states of SIADH and liver cirrhosis.

Animals↗
PubMed1995

Human AQP2 and MIP genes, two members of the MIP family, map within chromosome band 12q13 on the basis of two-color FISH.

The human AQP2 (collecting duct water channel, aquaporin 2) gene encodes a 271 amino acid protein and is a member of the MIP (major intrinsic protein of lens fiber) gene family. Using two-color fluorescence in situ hybridization on high-resolution R-banded chromosomes and human genomic DNA clones for AQP2 and MIP as probes, we found that both genes mapped closely within the human chromosome region 12q13.

Animals↗
PubMed1995

Human fibroblasts (KMST-6/RAS cell line) transformed with 60Co gamma-rays and c-Ha-ras oncogene produce a large amount of granulocyte colony-stimulating factor (G-CSF); production is enhanced by cAMP, theophylline, and butyrate.

Human fibroblasts (KMST-6/RAS cell line), which was malignantly transformed in vitro with 60Co gamma-rays and the c-Ha-ras oncogene, produced a large amount of granulocyte colony-stimulating factor (G-CSF). The production was greater during the logarithmic growth phase than during the stationary phase. cAMP and theophylline, alone or in combination, and butyrate significantly enhanced G-CSF production, but dexamethasone or 5-azacytidine did not. Enhanced production of G-CSF by these agents was regulated at the posttranscriptional level. Neither the expression of the ras oncogene nor the tumorigenicity of the cells correlated with the production of G-CSF.

Butyrates↗
PubMed1995

Importance of impairment of the airway epithelium for ozone-induced airway hyperresponsiveness in guinea pigs.

We examined the relationship between ozone (O3)-induced airway hyperresponsiveness (AHR) and inflammation in guinea pigs. Inhalation of methacholine (MCh) was adopted in the time course study of AHR that was assessed by measuring pulmonary inflation pressure after O3 exposure (3 ppm, for 2 hr) because the degree of AHR detected by inhalation of MCh was greater than that detected by i.v. administration. AHR was detected up to 5 hr after O3 exposure and was not observed at 24 and 48 hr. In the bronchoalveolar lavage (BAL) study, the numbers of neutrophils, eosinophils, lymphocytes and macrophages in BAL fluid (BALF) reached maximum at 24 hr or later. On the other hand, the number of airway epithelial cells in the BALF significantly increased at 2 and 5 hr. In the histological study, disorder and impairment of the airway epithelium in the trachea and lung were observed at 2 and 5 hr. Changes in the airway epithelium were recovered at 48 hr, although an increase in leukocytes was observed in the lung. These results indicate that O3-induced AHR in guinea pigs is most probably associated with impairment of the epithelium rather than with infiltration of inflammatory cells in the airway.

Administration, Inhalation↗
PubMed1995

Increased gene expression of water channel in cirrhotic rat kidneys.

In patients with liver cirrhosis, impaired water and sodium excretion has been incriminated in the pathogenesis of ascites formation. Increased reabsorption of water in the distal nephron has been shown to play an important role in water retention in cirrhotic rat kidneys. Recently, a complementary DNA (cDNA) for the vasopressin-regulated water channel (the aquaporin of the apical membrane of the kidney collecting duct [AQP-CD]) has been cloned. It is suggested that AQP-CD plays an important role in renal water handling. Therefore, in the present study, to investigate the pathogenic role of the water channel in water retention in liver cirrhosis, gene expression of AQP-CD in the kidney was evaluated in cirrhotic rats. Liver cirrhosis was induced by an intraperitoneal administration of carbon tetrachloride twice a week for 12 weeks in 14 rats. Messenger RNA expression of AQP-CD in whole kidney homogenates determined by Northern blot hybridization was significantly increased in cirrhotic rats (147%; P < .01) and dehydrated rats (206%; P < .0001) compared with control rats. Protein expression of AQP-CD in the homogenates of kidney medulla determined by Western blot analysis was significantly increased in cirrhotic rats (203%; P < .03) compared with control rats. Furthermore, mRNA expression of AQP-CD in the kidney showed a significant correlation with the volume of ascites in cirrhotic rats (r = .62, P < .02). No significant difference was observed in water intake, urinary volume, serum osmolality, serum sodium, and creatinine clearance between control and cirrhotic rats, suggesting that dehydration was unlikely in cirrhotic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Isolation of human aquaporin-CD gene.

The human gene encoding aquaporin-CD (AQP-CD) was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the human genome and comprises four exons distributing over 5 kilobases. The size range of exons is 81-761 base pairs, and that for introns is approximately 3000 to approximately 250 base pairs. The exon-intron boundaries of human AQP-CD gene are identified at identical positions in other related genes, the human AQP-CHIP gene and the human major intrinsic protein gene. The major transcription initiation sites were identified to positions 93 and 94 base pairs upstream of the ATG initiation codon by primer extension and ribonuclease protection assay. The 5'-flanking region of the hAQP-CD gene was characterized by a TATA box, two GATA consensus sequences, an AP-1 site, an AP-2 site, three E-boxes, and a cyclic AMP-responsive element. These structural features will lead to a better understanding of the mechanisms of tissue-specific expression and the regulation by dehydration in AQP-CD gene and will also be of help in search for possible genetic disorders in human AQP-CD gene.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and expression of a member of the aquaporin family with permeability to glycerol and urea in addition to water expressed at the basolateral membrane of kidney collecting duct cells.

Water transport in highly water-permeable membranes is conducted by water-selective pores--namely, water channels. The recent cloning of water channels revealed the water-selective characteristics of these proteins when expressed in Xenopus oocytes or reconstituted in liposomes. Currently, it is assumed that the function of water channels is to transport only water. We now report the cloning of a member of the water channel that also transports nonionic small molecules such as urea and glycerol. We named this channel aquaporin 3 (AQP3) for its predominant water permeability. AQP3 has amino acid sequence identity with major intrinsic protein (MIP) family proteins including AQP-channel-forming integral membrane protein, AQP-collecting duct, MIP, AQP-gamma tonoplast intrinsic protein, nodulin 26, and glycerol facilitator (33-42%). Thus, AQP3 is an additional member of the MIP family. Osmotic water permeability of Xenopus oocytes measured by videomicroscopy was 10-fold higher in oocytes injected with AQP3 transcript than with water-injected oocytes. The increase in osmotic water permeability was inhibited by HgCl2, and this effect was reversed by a reducing agent, 2-mercaptoethanol. Although to a smaller degree, AQP3 also facilitated the transport of nonionic small solutes such as urea and glycerol, while the previously cloned water channels are permeable only to water when expressed in Xenopus oocytes. AQP3 mRNA was expressed abundantly in kidney medulla and colon. In kidney, it was exclusively immunolocalized at the basolateral membrane of collecting duct cells. AQP3 may function as a water and urea exit mechanism in antidiuresis in collecting duct cells.

Amino Acid Sequence↗
PubMed1994

Functional characterization and cell immunolocalization of AQP-CD water channel in kidney collecting duct.

Vasopressin-regulated water permeability of the kidney collecting duct is a key component of the urine concentration machinery. Recently, a cDNA for AQP-CD, the vasopressin-regulated water channel, initially reported as WCH-CD, has been isolated (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). AQP-CD was expressed in oocyte membrane using a Xenopus expression vector, and functional characteristics of AQP-CD were examined. Osmotic water permeability (Pf) of oocytes expressing AQP-CD was 138 +/- 19 microns/s (mean +/- SE), 12 times greater than the control (11 +/- 3 microns/s), 90% inhibited by 0.3 mM HgCl2, and weakly temperature dependent (energy of activation for Pf was 4.0 kcal/mol). Urea influx measured from 15-min [14C]urea uptake by oocytes injected with AQP-CD/expression vector 1 cRNA was 86 +/- 17% of the control. Two-electrode voltage-clamp experiments revealed insignificant ion conductance of AQP-CD. Immunoblots of membranes from rat kidney medulla and oocytes expressing AQP-CD using anti-AQP-CD COOH-terminal antibody showed a 29-kDa protein and 35- to 50-kDa high-molecular-mass forms. Immunohistochemistry showed apical and subapical localization of AQP-CD in the collecting duct principal cells. Our results indicated that AQP-CD is a 29-kDa protein, a selective water channel, distinct from a urea channel, and localized to the membranes of vasopressin-sensitive components in kidney collecting duct principal cells.

Amino Acid Sequence↗
PubMed1994

Cloning, characterization, and chromosomal mapping of human aquaporin of collecting duct.

We recently cloned a cDNA of the collecting duct apical membrane water channel of rat kidney, which is important for the formation of concentrated urine (Fushima, K., S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. 1993. Nature [Lond.]. 361:549-552). Since urine concentrating ability varies among mammalian species, we examined whether an homologous protein is present in human kidney. By screening a human kidney cDNA library, we isolated a cDNA clone, designated human aquaporin of collecting duct (hAQP-CD), that encodes a 271-amino acid protein with 91% identity to rat AQP-CD. mRNA expression of hAQP-CD was predominant in the kidney medulla compared with the cortex, immunohistochemical staining of hAQP-CD was observed only in the collecting duct cells, and the staining was dominant in the apical domain. Functional expression study in Xenopus oocytes confirmed that hAQP-CD worked as a water channel. Western blot analysis of human kidney medulla indicated that the molecular mass of hAQP-CD is 29 kD, which is the same mass expected from the amino acid sequence. Chromosomal mapping of the hAQP-CD gene assigned its location to chromosome 12q13. These results could be important for future studies of the pathophysiology of human urinary concentration mechanisms in normal and abnormal states.

Amino Acid Sequence↗