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This Article Full Text Full Text (PDF) All Versions of this Article: 93/10/1147    most recent expphysiol.2008.042663v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Saito, T. Articles by Ishikawa, S.-e PubMed PubMed Citation Articles by Saito, T. Articles by Ishikawa, S.-e Related Collections Renal

¹ Department of Medicine, Jichi Medical University Saitama Medical Center, 1-847 Amanuma, Omiya-ku, Saitama 330-8503, Japan ² Department of Nephrology, Tokyo Medical and Dental University, 1-5-45, Yushima Bunkyo, Tokyo 113-8519, Japan

The present study was undertaken to determine whether hypotonicity regulates the aquaporin-2 (AQP-2) gene in vitro. The 5'-flanking region of the AQP-2 gene contains the tonicity-response enhancer (TonE) promoter located between –570 and –560 bp, and another distinct hypertonicity-responsive region between –6.1 and –4.3 kb of the AQP-2 gene. The 5'-flanking region of murine AQP-2 gene up to –9.5 kb was cloned into a luciferase (Luc) reporter plasmid. The constructs, which have TonE and/or the hypertonicity-responsive region, together with the murine AQP-2 gene, were co-transfected into murine IMCD₃ cells. When the cells were co-transfected with the construct containing more than 1.1 kb of the 5'-flanking region of murine AQP-2 gene (–9.5AQP2, –6.1AQP2 and –1.1AQP2) and the AQP-2 gene, 24 h exposure to 5 µmol l^–1 dibutyryl cAMP (DBcAMP) significantly increased the Luc activity by 2.3-fold in the isotonic medium (300 mosmol kg^–1). In the hypotonic medium (225 mosmol kg^–1), basal activity was not altered, and the response of Luc activity to 24 h exposure to 5 µmol l^–1DBcAMP was abolished. Similar findings were obtained in isosmotic, urea-supplemented medium (estimated tonicity, 225 mosmol kg^–1). The response of Luc activity to 5 µmol l^–1 DBcAMP in the hypotonic medium was not affected in cells either transfected with 0.36 kb of the 5'-flanking region of AQP-2 or co-transfected with –1.1AQP2 and a dominant-negative TonE binding protein (pDNTonEBP). Pre-incubation of cells with 1 µmol l^–1 SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), restored the response of Luc activity to 5 µmol l^–1 DBcAMP under hypotonic conditions. These findings may indicate that hypotonicity reduces the cAMP-induced AQP-2 promoter activity mediated via TonE by activating JNK kinase.

(Received 17 March 2008; accepted after revision 27 May 2008; first published online 30 May 2008)
Corresponding author S. Ishikawa: Department of Medicine, Jichi Medical University, Saitama Medical Center, 1-847 Amanuma Omiya-ku, Saitama, Saitama 330-8503, Japan.  Email: saneiskw{at}jichi.ac.jp