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Shinichi Uchida

Publications and source records attributed to Shinichi Uchida.

At least 19 recordsLinked to original sources
PubMed2007

A case of nephrogenic diabetes insipidus with a novel missense mutation in the AVPR2 gene.

We describe a pediatric case of nephrogenic diabetes insipidus (NDI) with a novel missense mutation in the arginine vasopressin receptor 2 (AVPR2) gene. The patient, a 3-year-old boy, had polyuria (4357 ml/day, 7230 ml/m(2)/day) and polydipsia. Water deprivation testing demonstrated no decrease of urine volume, and urinary volume did not respond to subcutaneous injection of 0.1 U/kg pitressin. Molecular genetic analysis demonstrated that the patient had an AVPR2 missense mutation involving substitution of phenylalanine for tyrosine at position 205 (Y205F). It was also found that the patient's mother was heterozygous for this Y205F mutation. Analysis of the intermolecular interaction of the Tyr-205 hydrogen group by molecular modeling showed that Tyr-205 was located in transmembrane domain (TM) 5, and that its hydroxy group formed a hydrogen bond with Leu-169 main-chain =O located in TM 4. The mutation of Tyr-205 to phenylalanine would cause loss of this hydrogen bond and decrease or change the interaction between these TM coils, thus affecting the ability of AVP to bind to the receptor. According to this molecular model of AVPR2, the Y205F mutation would cause nephrogenic diabetes insipidus.

Base Sequence↗
PubMed2006

Morphologic and functional analysis of sperm and testes in Aquaporin 7 knockout mice.

OBJECTIVE: To investigate the functional and morphologic role of Aquaporin 7 (AQP7) in testis and sperm. DESIGN: Experimental laboratory study. SETTING: University and research institute units. ANIMAL(S): AQP7 knockout mice (C57BL/6J background). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Morphologic analysis of testis and epididymis, daily sperm production, sperm motility, in vitro fertilization. RESULT(S): There was no difference in the morphology of the testes and epididymis between AQP7 knockout and wild-type mice. The AQP7 knockout male mice and wild-type male mice had similar numbers of offspring. Analysis of the daily sperm production and motility of AQP7 knockout mice did not show any abnormalities. Similarly, the rate of in vitro fertilization using sperm from AQP7 knockout mice was not different from wild-type mice. CONCLUSION(S): Male AQP7 knockout mice were not sterile, and their sperm did not show any morphologic and functional abnormalities.

Animals↗
PubMed2006

Pathogenesis and treatment of autosomal-dominant nephrogenic diabetes insipidus caused by an aquaporin 2 mutation.

Frame-shift mutations within the C terminus of aquaporin 2 (AQP2) cause autosomal-dominant nephrogenic diabetes insipidus (AD-NDI). To identify the molecular mechanism(s) of this disease in vivo and to test possible therapeutic strategies, we generated a mutant AQP2 (763-772 del) knockin mouse. Heterozygous knockin mice showed a severely impaired urine-concentrating ability. However, they were able to slightly increase urine osmolality after dehydration. This milder phenotype, when compared with autosomal-recessive NDI, is a feature of AD-NDI in humans, thus suggesting successful establishment of an AD-NDI mouse model. Immunofluorescence of collecting duct cells in the AD-NDI mouse revealed that the mutant AQP2 was missorted to the basolateral instead of apical plasma membrane. Furthermore, the mutant AQP2 formed a heterooligomer with wild-type AQP2 and showed a dominant-negative effect on the normal apical sorting of wild-type AQP2 even under dehydration. Using this knockin mouse, we tested several drugs for treatment of AD-NDI and found that rolipram, a phosphodiesterase 4 inhibitor, was able to increase urine osmolality. Phosphodiesterase inhibitors may thus be useful drugs for the treatment of AD-NDI. This animal model demonstrates that a mutant monomer gains a dominant-negative effect that reverses the normal polarized sorting of multimers.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2006

Overexpression of human WNK1 increases paracellular chloride permeability and phosphorylation of claudin-4 in MDCKII cells.

Pseudohypoaldosteronism type II (PHAII), an autosomal dominant disorder characterized by hypertension, hyperkalemia, and hyperchloremic acidosis, is reportedly due to mutations in WNK1 and WNK4 kinase genes. However, the pathogenesis of the disease remains unknown. Mutations in the WNK1 gene are the deletions in the first intron, which reportedly increases WNK1 mRNA expression. Thus, we generated WNK1 over-expressing stable cell lines using MDCKII cells to model the distal nephron of PHAII patients. Using these cell lines, we investigated whether increased WNK1 expression might affect paracellular chloride permeability and claudin phosphorylation, since we previously observed an increase in both with a disease-causing mutant WNK4. WNK1 expression in MDCKII cells increased chloride permeability two to threefold. Co-expression of wild-type WNK4 did not further increase WNK1-enhanced chloride permeability. WNK1 expression also induced phosphorylation of endogenous claudin-4 in MDCKII cells, as well as over-expressed claudin-4. Combined, these results suggest that increased WNK1 expression has the same effect on chloride permeability and claudin phosphorylation as the mutant WNK4. Thus, increased chloride shunt may be involved in the pathogenesis of PHAII caused by WNK1 mutations.

Animals↗
PubMed2006

Physiological roles of AQP7 in the kidney: Lessons from AQP7 knockout mice.

The aquaporin7 (AQP7) water channel is known to be a member of the aquaglyceroporins, which allow the rapid transport of glycerol and water. AQP7 is abundantly present at the apical membrane of the proximal straight tubules in the kidney. In this paper, we review the physiological functions of AQP7 in the kidney. To investigate this, we generated AQP7 knockout mice. The water permeability of the proximal straight tubule brush border membrane measured by the stopped flow method was reduced in AQP7 knockout mice compared to wild-type mice (AQP7, 18.0+/-0.4 x 10(-3 )cm/s vs. wild-type, 20.0+/-0.3 x 10(-3) cm/s). Although AQP7 solo knockout mice did not show a urinary concentrating defect, AQP1/AQP7 double knockout mice showed reduced urinary concentrating ability compared to AQP1 solo knockout mice, indicating that the contribution of AQP7 to water reabsorption in the proximal straight tubules is physiologically substantial. On the other hand, AQP7 knockout mice showed marked glycerol in their urine (AQP7, 1.7+/-0.34 mg/ml vs. wild-type, 0.005+/-0.002 mg/ml). This finding identified a novel pathway of glycerol reabsorption that occurs in the proximal straight tubules. In two mouse models of proximal straight tubule injury, the cisplatin-induced acute renal failure (ARF) model and the ischemic-reperfusion ARF model, an increase of urine glycerol was observed (pre-treatment, 0.007+/-0.005 mg/ml; cisplatin, 0.063+/-0.043 mg/ml; ischemia, 0.076+/-0.02 mg/ml), suggesting that urine glycerol could be used as a new biomarker for detecting proximal straight tubule injury.

Acute Kidney Injury↗
PubMed2006

Postnatal expression of KLF12 in the inner medullary collecting ducts of kidney and its trans-activation of UT-A1 urea transporter promoter.

Maturation of the inner medulla of the kidney occurs after birth and is vital for mammals to acquire maximal urinary concentrating ability. During this process, expression of several kidney transporters and channels involved in urine concentrating mechanisms is known to be regulated. We previously isolated KLF15 as a transcription factor that regulates the expression of the ClC-K1 chloride channel. We have now found that another KLF transcription factor, KLF12, is expressed in the kidney from around 15 days after birth. To gain insight into its involvement in the maturation process of the inner medulla, we first determined the expression site of KLF12 within the kidney by in situ hybridization. By comparing the AQP2 immunolocalization in sequential sections, KLF12 was found to be expressed in the collecting ducts. Because expression of the urea transporter UT-A1 and amiloride-sensitive epithelial sodium channels ENaC is known to be tightly regulated in the collecting ducts after birth, we tested whether KLF12 has a regulatory role in the promoter activities of these genes. KLF12 is able to increase UT-A1 but not ENaC promoter activity through the binding to CACCC motif. These results suggest that KLF12 is involved in the maturation processes of collecting ducts after birth, and that UT-A1 is a target gene of KLF12.

Animals↗
PubMed2006

Single-channel properties of volume-sensitive Cl- channel in ClC-3-deficient cardiomyocytes.

It is controversial whether the ClC-3 protein, which is one of the voltage-dependent chloride channel ClC family members, is a candidate for the volume-sensitive outwardly rectifying (VSOR) Cl(-) channel per se or its regulator. Here, for the first time, we examined the single-channel properties of the VSOR Cl(-) channel in ventricular myocytes isolated from ClC-3-deficient mice. The single-channel current induced by cell swelling exhibited Cl(-) selectivity, mild outward rectification, and an intermediate unitary conductance (around 38 pS). A Cl(-) channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), reversibly inhibited the outward current. These single-channel properties were identical with those in ClC-3 expressing wild-type ventricular myocytes. These results indicate that the single-channel activity of the VSOR Cl(-) channel is independent of the expression of ClC-3 proteins in mouse ventricular myocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2006

Intracellular localization of ClC chloride channels and their ability to form hetero-oligomers.

ClC chloride channels (ClCs) can be classified into two groups in terms of their cellular localizations: ClCs present in the plasma membranes and those residing in intracellular organelles. Members of the latter group, including ClC-3, ClC-4, ClC-5, ClC-6, and ClC-7, are often co-expressed in a variety of cell types in many organs. Although the localization of individual channels within cells has been investigated, the degree of overlap between the locations of different ClCs in the same cell has not been clarified. To address this question, different combinations of ClCs, engineered to encode specific epitope tags (FLAG or HA), were either transiently or stably transfected into HEK293 cells, and we then compared the intracellular localization of the expressed channel proteins by immunofluorescence microscopy. Immunofluorescence images of the alternatively labeled channels clearly showed significant co-localization between all pair-wise combinations of ClCs. In particular, ClC-3, ClC-4, and ClC-5 showed a high degree of co-localization. As a significant degree of co-localization between ClCs was observed, we used co-immunoprecipitation to evaluate oligomer formation, and found that each ClC tested could form homo-oligomers, and that any pair-wise combination of ClC-3, ClC-4, and ClC-5 could also form hetero-oligomers. Neither ClC-6 nor ClC-7 was co-precipitated with any other channel protein. These results suggest that within cells ClC-3, ClC-4, and ClC-5 may have combinatorial functions, whereas ClC-6 and ClC-7 are more likely to function as homo-oligomers.

Biomarkers↗
PubMed2005

WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1.

The WNK1 and WNK4 genes have been found to be mutated in some patients with hyperkalemia and hypertension caused by pseudohypoaldosteronism type II. The clue to the pathophysiology of pseudohypoaldosteronism type II was its striking therapeutic response to thiazide diuretics, which are known to block the sodium chloride cotransporter (NCC). Although this suggests a role for WNK1 in hypertension, the precise molecular mechanisms are largely unknown. Here we have shown that WNK1 phosphorylates and regulates the STE20-related kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1). WNK1 was observed to phosphorylate the evolutionary conserved serine residue located outside the kinase domains of SPAK and OSR1, and mutation of the OSR1 serine residue caused enhanced OSR1 kinase activity. In addition, hypotonic stress was shown to activate SPAK and OSR1 and induce phosphorylation of the conserved OSR1 serine residue, suggesting that WNK1 may be an activator of the SPAK and OSR1 kinases. Moreover, SPAK and OSR1 were found to directly phosphorylate the N-terminal regulatory regions of cation-chloride-coupled cotransporters including NKCC1, NKCC2, and NCC. Phosphorylation of NCC was induced by hypotonic stress in cells. These results suggested that WNK1 and SPAK/OSR1 mediate the hypotonic stress signaling pathway to the transporters and may provide insights into the mechanisms by which WNK1 regulates ion balance.

Amino Acid Motifs↗
PubMed2005

Polarized trafficking of the aquaporin-3 water channel is mediated by an NH2-terminal sorting signal.

Epithelial renal collecting duct cells express multiple types of aquaporin (AQP) water channels in a polarized fashion. AQP2 is specifically targeted to the apical cell domain, whereas AQP3 and AQP4 are expressed on the basolateral membrane. It is crucial that these AQP variants are sorted to their proper polarized membrane domains, because correct AQP sorting enables efficient water transport. However, the molecular mechanisms involved in the polarized targeting and membrane trafficking of AQPs remain largely unknown. In the present study, we have examined the polarized trafficking and surface expression of AQP3 in Madin-Darby canine kidney type II (MDCKII) cells in an effort to identify the molecular determinants of polarized targeting specificity. When expressed in MDCKII cells, the majority of the exogenous wild-type AQP3 was found to be targeted to the basolateral membrane, consistent with its localization pattern in vivo. A potential sorting signal consisting of tyrosine- and dileucine-based motifs was subsequently identified in the AQP3 NH2 terminus. When mutations were introduced into this signaling region, the basolateral targeting of the resulting mutant AQP3 was disrupted and the mutant protein remained in the cytoplasm. AQP2-AQP3 chimeras were then generated in which the entire NH2 terminus of AQP2 was replaced with the AQP3 NH2 terminus. This chimeric protein was observed to be mislocalized constitutively in the basolateral membrane, and mutations in the AQP3 NH2-terminal sorting signal abolished this effect. On the basis of these results, we conclude that an NH2-terminal sorting signal mediates the basolateral targeting of AQP3.

Animals↗
PubMed2005

Water channel activities of Mimosa pudica plasma membrane intrinsic proteins are regulated by direct interaction and phosphorylation.

cDNAs encoding aquaporins PIP1;1, PIP2;1, and TIP1;1 were isolated from Mimosa pudica (Mp) cDNA library. MpPIP1;1 exhibited no water channel activity; however, it facilitated the water channel activity of MpPIP2;1 in a phosphorylation-dependent manner. Mutagenesis analysis revealed that Ser-131 of MpPIP1;1 was phosphorylated by PKA and that cooperative regulation of the water channel activity of MpPIP2;1 was regulated by phosphorylation of Ser-131 of MpPIP1;1. Immunoprecipitation analysis revealed that MpPIP1;1 binds directly to MpPIP2;1 in a phosphorylation-independent manner, suggesting that phosphorylation of Ser-131 of MpPIP1;1 is involved in regulation of the structure of the channel complex with MpMIP2;1 and thereby affects water channel activity.

Animals↗
PubMed2005

Apical localization of renal K channel was not altered in mutant WNK4 transgenic mice.

Missense mutations in the WNK4 gene have been postulated to cause pseudohypoaldosteronism type II, an autosomal-dominant disorder characterized by hyperkalemia and hypertension. A previous study using Xenopus oocytes showed that wild-type WNK4 expression inhibited surface expression of renal K channel (ROMK) and that a disease-causing mutant further decreased the surface expression. The decreased surface expression of ROMK caused by mutant WNK4 was postulated to be a mechanism for decreased potassium secretion in distal nephrons that would presumably lead to hyperkalemia. To determine if the mutant WNK4 had such an inhibitory effect on the apical localization of ROMK in vivo, we generated transgenic mice using the CLCNKB gene promoter that expressed a mutant WNK4 (D564A) in distal nephrons. In contrast to the tight junction localization of wild-type WNK4 described previously, the mutant WNK4 was present in the cytoplasm in the distal tubules and in the apical membranes in the thick ascending limb of Henle's loop. In both cell types, the apical localization of endogenous ROMK was not influenced by the co-expression of mutant WNK4. This result indicates that the mutant WNK4 does not have a dominant effect on the cellular localization of ROMK in vivo.

Animals↗
PubMed2005

Defective water and glycerol transport in the proximal tubules of AQP7 knockout mice.

The aquaporin-7 (AQP7) water channel is known as a member of the aquaglyceroporins, which facilitate the transport of glycerol as well as water. Although AQP7 is abundantly expressed on the apical membrane of the proximal straight tubules in the kidney, the physiological role of AQP7 is still unknown. To investigate this, we generated AQP7 knockout mice. The water permeability of the proximal tubule brush-border membrane measured by the stopped-flow method was slightly but significantly reduced in the AQP7 knockout mice compared with that of wild-type mice (AQP7, 18.0 +/- 0.4 x 10(-3) cm/s vs. wild-type, 20.0 +/- 0.3 x 10(-3) cm/s). Although AQP7 solo-knockout mice did not exhibit a urinary concentrating defect, AQP1/AQP7 double-knockout mice had a reduction in urinary concentrating ability compared with AQP1 solo-knockout mice, suggesting that the amount of water reabsorbed through AQP7 in the proximal straight tubules is physiologically substantial. On the other hand, AQP7 knockout mice showed marked glyceroluria (AQP7, 1.7 +/- 0.34 mg/ml vs. wild-type, 0.005 +/- 0.002 mg/ml). This identified a novel glycerol reabsorption pathway in the proximal straight tubules. In two mouse models of proximal straight tubule injury, the cisplatin-induced acute renal failure (ARF) model and the ischemic ARF model, an increase in urine glycerol was observed (pretreatment, 0.007 +/- 0.005 mg/ml; cisplatin, 0.063 +/- 0.043 mg/ml; ischemia, 0.076 +/- 0.02 mg/ml), suggesting that urine glycerol could be used as a new biomarker for detecting proximal straight tubule injury.

Animals↗
PubMed2005

Identification of a novel aquaporin, AQP12, expressed in pancreatic acinar cells.

Members of the aquaporin (AQP) water channel family are widely distributed in various tissues and contribute to the water permeability of epithelial and endothelial cells. Currently 11 members of the AQP family (AQP0-10) have been reported in mammals. Here we report the identification of AQP12, which we found by performing a BLAST program search. Northern blot analysis revealed that AQP12 was specifically expressed in the pancreas. Further analysis by in situ hybridization and RT-PCR studies showed that AQP12 was selectively localized in the acinar cells of the pancreas. To analyze the cellular localization and function of AQP12, we expressed AQP12 in Xenopus oocytes and cultured mammalian cells. Immunocytochemistry revealed that AQP12 was not targeted to the plasma membrane. The selective localization of AQP12 in pancreatic acinar cells and possibly in the intracellular organelles suggests a role of AQP12 in digestive enzyme secretion such as maturation and exocytosis of secretory granules.

Amino Acid Sequence↗
PubMed2005

Regulation of apical localization of the thiazide-sensitive NaCl cotransporter by WNK4 in polarized epithelial cells.

Missense mutations in the WNK4 gene have been postulated to cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder characterized by hyperkalemia and hypertension. Previous reports using Xenopus oocytes showed that wild-type WNK4 expression inhibited surface expression of the thiazide-sensitive NaCl cotransporter (NCC), while a disease-causing mutant lost the inhibitory effect on NCC surface expression. To determine if these changes observed in oocytes really occur in polarized epithelial cells, we generated stable MDCK II cell lines expressing NCC alone or NCC plus wild-type WNK4 or a disease-causing (D564A) WNK4. In contrast to the apical localization of NCC without co-expression of WNK4, immunofluorescence microscopy and biotin surface labeling revealed that this apical localization was equally decreased by both the wild-type and the mutant WNK4 expression. Apical localizations of two PHAII-unrelated apical transporters, sodium-independent amino acid transporter, BAT1 and bile salt export pump, Bsep, were also found to be decreased by both wild-type and mutant WNK4 expression. These results indicate that the regulation of NCC was not related to the disease-causing mutation and not restricted to the PHAII-related specific transporters. The regulation of intracellular localization of NCC by WNK4 might not be involved in the pathogenesis of PHAII.

Animals↗
PubMed2005

Progressive adipocyte hypertrophy in aquaporin-7-deficient mice: adipocyte glycerol permeability as a novel regulator of fat accumulation.

Aquaporin-7 (AQP7) is a water/glycerol transporting protein expressed in adipocyte plasma membranes. We report here remarkable age-dependent hypertrophy in adipocytes in AQP7-deficient mice. Wild type and AQP7 null mice had similar growth at 0-16 weeks as assessed by body weight; however, by 16 weeks AQP7 null mice had 3.7-fold increased body fat mass. Adipocytes from AQP7 null mice of age 16 weeks were greatly enlarged (diameter 118 mum) compared with wild type mice (39 mum). Adipocytes from AQP7 null mice also accumulated excess glycerol (251 versus 86 nmol/mg of protein) and triglycerides (3.4 versus 1.7 mumol/mg of protein). In contrast, at age 4 weeks, adipocyte volume and body fat mass were comparable in wild type and AQP7 null mice. To investigate the mechanism(s) responsible for the progressive adipocyte hypertrophy, glycerol permeability and fat metabolism were studied in adipocytes isolated from the younger mice. Plasma membrane glycerol permeability measured by [(14)C]glycerol uptake was 3-fold reduced in AQP7-deficient adipocytes. However, adipocyte lipolysis, measured by free fatty acid release and hormone-sensitive lipase activity, and lipogenesis, measured by [(14)C]glucose incorporation into triglycerides, were not affected by AQP7 deletion. These data suggest that adipocyte hypertrophy in AQP7 deficiency results from defective glycerol exit and consequent accumulation of glycerol and triglycerides. Increasing AQP7 expression/function in adipocytes may reduce adipocyte volume and fat mass in obesity.

Adipocytes↗