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      Gene Regulation of Renal-Osmotic Stress-Induced Na-Cl Organic Solute Cotransporter

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          Abstract

          Background: Na- and Cl-dependent organic solute cotransporters participate in transporting neurotransmitters in the brain and organic osmolytes in the kidney. Methods: We examined the intranephron localization and regulation of the renal osmotic-stress-induced Na-Cl organic solute cotransporter (ROSIT) mRNA expression using microdissected nephron segments from control and dehydrated rats and RT-PCR. To further know the mechanisms of gene regulation of ROSIT, microdissected proximal straight tubules (PST) were incubated in isotonic (290 mosm/kg H<sub>2</sub>O) or hyperosmotic (490– 1,090 mosm/kg H<sub>2</sub>O) solution. Results: ROSIT mRNA was expressed predominantly in PST and to a lesser extent in cortical thick ascending limbs and cortical collecting ducts in control rats, and dehydration caused an increase in the expression in whole nephron segments. ROSIT mRNA in PST was decreased with time by incubation in isotonic solution. Incubation of PST in hypertonic solution by adding NaCl increased mRNA expression as early as 15 min (1.5- and 3-fold at 15 and 30 min, respectively). This stimulating effect of NaCl was largest at 890 mosm/kg H<sub>2</sub>O. Hypertonicity by mannitol or myoinositol also increased ROSIT mRNA expression. In contrast, hyperosmolality by urea reduced ROSIT mRNA expression. GAPDH mRNA expression, an internal standard, did not change by incubation in NaCl or mannitol solution. Conclusion: In summary, ROSIT mRNA expression was most abundant in PST in control and it was stimulated in whole nephron segments by dehydration. ROSIT mRNA expression in PST was stimulated by hypertonicity but not by urea. These data suggest that ROSIT may participate in the transport of amino acid under control conditions and organic osmolytes in dehydration.

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          Most cited references 5

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          Electrical conductivity measurements from the GISP2 and GRIP Greenlandice cores

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            Activity of the TonEBP/OREBP transactivation domain varies directly with extracellular NaCl concentration.

            Hypertonicity-induced binding of the transcription factor TonEBP/OREBP to its cognate DNA element, ORE/TonE, is associated with increased transcription of several osmotically regulated genes. Previously, it was found that hypertonicity rapidly causes nuclear translocation and phosphorylation of TonEBP/OREBP and, more slowly, increases TonEBP/OREBP abundance. Also, the C terminus of TonEBP/OREBP was found to contain a transactivation domain (TAD). We have now tested for tonicity dependence of the TAD activity of the 983 C-terminal amino acids of TonEBP/OREBP. HepG2 cells were cotransfected with a reporter construct and one of several TAD expression vector constructs. The reporter construct contained GAL4 DNA binding elements, a minimal promoter, and the Photinus luciferase gene. TAD expression vectors generate chimeras comprised of the GAL4 DNA binding domain fused to (i) the 983 C-terminal amino acids of TonEBP/OREBP, (ii) 17 glutamine residues, (iii) the TAD of c-Jun, or (iv) no TAD. All TAD-containing chimeras were functional at normal extracellular osmolality (300 mosmol/kg), but the activity only of the chimera containing the 983 C-terminal amino acids of TonEBP/OREBP varied with extracellular NaCl concentration, decreasing by >80% at 200 mosmol/kg and increasing 8-fold at 500 mosmol/kg. The chimera containing the 983 C-terminal amino acids of TonEBP/OREBP was constitutively localized to the nucleus and showed tonicity-dependent posttranslational modification consistent with phosphorylation. The activity at 500 mosmol/kg was reduced by herbimycin, a tyrosine kinase inhibitor and by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a protein kinase CK2 inhibitor. Thus, the 983 C-terminal amino acids of TonEBP/OREBP contain a TAD that is regulated osmotically, apparently by tonicity-dependent phosphorylation.
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              The TonE/TonEBP pathway mediates tonicity-responsive regulation of UT-A urea transporter expression.

              The rat renal urea transporter UT-A includes four isoforms. UT-A1, UT-A3, and UT-A4 are transcribed from a single initiation site at the 5'-end of the gene; a distinct internal initiation site is used for UT-A2 transcription. We cloned 1.3 kilobases (kb) of the 5'-flanking region upstream of the transcription start site of UT-A1, UT-A3, and UT-A4. This region contains three CCAAT sequences but lacks a TATA motif. A tonicity-responsive enhancer (TonE) was identified at -377bp. The 1.3-kb full fragment subcloned into pGL3 vector induced luciferase activity in Madin-Darby canine kidney cells and in mouse inner medullary collecting duct cells in isotonic medium. Luciferase activity was increased significantly in hypertonic medium, whereas deletion or mutation of the TonE sequence abolished this response. Electrophoretic mobility shift assay using the 5' UT-A TonE sequence as DNA probe showed formation of a specific DNA-protein complex with nuclear extracts from cells exposed to hypertonic medium and was weakly detectable in isotonic controls. A supershift in the mobility of the DNA-protein complex was observed with antiserum targeted to the TonE-binding protein (TonEBP). Co-transfection with dominant-negative TonEBP abolished the luciferase activity induced by the UT-A 1.3-kb construct under hypertonic and isotonic conditions. These data suggest that the TonE/TonEBP pathway mediates tonicity-responsive transcriptional regulation of UT-A1, UT-A3, and UT-A4 expression.
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                Author and article information

                Journal
                NEE
                Nephron Exp Nephrol
                10.1159/issn.1660-2129
                Cardiorenal Medicine
                S. Karger AG
                1660-2129
                2004
                March 2004
                17 November 2004
                : 96
                : 3
                : e89-e96
                Affiliations
                Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, Honjo, Kumamoto, Japan
                Article
                76750 Nephron Exp Nephrol 2004;96:e89–e96
                10.1159/000076750
                15056985
                © 2004 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 7, References: 21, Pages: 1
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/76750
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