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NDT Advance Access originally published online on January 25, 2005
Nephrology Dialysis Transplantation 2005 20(3):509-515; doi:10.1093/ndt/gfh677
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© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Original Article

Hypertonicity regulates the aquaporin-2 promoter independently of arginine vasopressin

Keizo Kasono1,3, Tomoyuki Saito1, Takako Saito1, Hiroyuki Tamemoto1, Chieko Yanagidate1, Shinichi Uchida2, Masanobu Kawakami1, Sei Sasaki2 and San-e Ishikawa1

1 Department of Medicine, Jichi Medical School, Omiya Medical Center, Saitama, 2 Department of Nephrology, Graduate School, School of Medicine, Tokyo Medical and Dental University, Tokyo and 3 Laboratory of Clinical Nutrition, Department of Clinical Dietetics and Human Nutrition, Faculty of Pharmaceutical Sciences, Josai University, Saitama, Japan

Correspondence and offprint requests to: San-e Ishikawa, MD, Department of Medicine, Jichi Medical School, Omiya Medical Center, 1-847 Amanuma, Omiya-ku, Saitama, Saitama 330-8503, Japan. E-mail: saneiskw{at}jichi.ac.jp

Background. Aquaporin-2 (AQP-2) is an arginine vasopressin (AVP)-regulated water channel in kidney collecting duct cells. The present study was undertaken to determine whether a change in tonicity could directly regulate the AQP-2 gene in an in vitro experiment.

Methods. Various fragments of the 5'-flanking region of the murine AQP-2 gene up to –9.5 kb were cloned into a luciferase (Luc) reporter plasmid, and they were transiently transfected into Madin–Darby canine kidney cells.

Results. Hypertonicity significantly increased the Luc activity of the constructs containing >6.1 kb of the 5'-flanking region of the AQP-2 gene (–6.1AQP2). However, promoter regions <4.3 kb in length containing the tonicity-responsive enhancer (TonE) at bp –570 to –560 were not stimulated by hypertonicity. The TonE-deleted construct which contains –9.5 to –1.1 kb of the 5' side of the AQP-2 gene, 8.4AQP2, was also stimulated by hypertonicity. Mitogen-activated protein (MAP) kinase inhibitors SB203580 and U0126 did not affect the Luc activity of –6.1AQP2 induced by hypertonicity. In addition, the vector expressing dominant-negative TonE-binding protein (TonEBP) did not affect the hypertonicity-induced Luc activity of –6.1AQP2. The Luc activity of –6.1AQP2 was stimulated by the overexpression of TonEBP. Hypertonicity further increased the Luc activity of –6.1AQP2 under the overexpression of TonEBP.

Conclusion. These findings indicate that hypertonicity regulates AQP-2 promoter activity via an AVP-independent mechanism, and that the tonicity-responsive element resides between the –6.1 and –4.3 kb 5'-flanking region of the AQP-2 gene, in which the structure and mechanism of response to hypertonicity could be distinct from those of TonE.

Keywords: aquaporin-2; gene regulation; hypertonicity; kidney; tonicity-responsive enhancer


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