Hypertonicity regulates the aquaporin-2 promoter independently of arginine vasopressin.
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.


CONCLUSIONS
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.
Kasono
Keizo
K
Department of Medicine, Jichi Medical School, Omiya Medical Center, 1-847 Amanuma, Omiya-ku, Saitama, Saitama 330-8503, Japan.
Saito
Tomoyuki
T
Saito
Takako
T
Tamemoto
Hiroyuki
H
Yanagidate
Chieko
C
Uchida
Shinichi
S
Kawakami
Masanobu
M
Sasaki
Sei
S
Ishikawa
San-e
SE
D024506:5' Flanking Region / Q000187:drug effects; D000818:Animals; D051399:Aquaporin 2; D020346:Aquaporins / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D001127:Arginine Vasopressin / Q000494:pharmacology; D018929:Cell Culture Techniques; D003563:Cyclic CMP / Q000031:analogs & derivatives* / Q000502:physiology; D004285:Dogs; D017930:Genes, Reporter; D005822:Genetic Vectors; D007668:Kidney / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012076:Renal Agents / Q000494:pharmacology; D012462:Saline Solution, Hypertonic / Q000494:pharmacology*; D014162:Transfection
C496024:Aqp2 protein, mouse; D051399:Aquaporin 2; D020346:Aquaporins; D012076:Renal Agents; D012462:Saline Solution, Hypertonic; D001127:Arginine Vasopressin; D003563:Cyclic CMP; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate; D008156:Luciferases