Control of aquaporin 2 expression in collecting ducts of quail kidneys q Keith K. Lau a,c , Yimu Yang a , George A. Cook b , Robert J. Wyatt c , Hiroko Nishimura a, * a Department of Physiology, University of Tennessee Health Science Center, College of Medicine, 894 Union Avenue, Memphis, TN 38163, USA b Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN 38163, USA c Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN 38103, USA article info Article history: Received 25 March 2008 Revised 25 November 2008 Accepted 7 December 2008 Available online 24 December 2008 Keywords: Avian aquaporin AQP2 Water channel Urine concentration Medullary collecting duct Coturnix quail ADH abstract Birds and mammals are the only vertebrates that can concentrate urine. Avian kidneys contain structur- ally primitive loopless nephrons and also more advanced looped nephrons, in the cortical and medullary regions, respectively. We have identified the gene sequence of an aquaporin 2 (AQP2)-homologue water channel in collecting ducts of kidneys from adult quail, Coturnix japonica. Although immunoreactive quail AQP2 (qAQP2) was found in both types of nephrons, the expression is enhanced more clearly in the med- ullary regions after water deprivation. We therefore hypothesized that regulation of qAQP2 expression in quail kidneys via antidiuretic hormone (ADH) may require more advanced nephron structure. In this study, we determined the expression of qAQP2 mRNA in tissues isolated from the cortical and medullary regions before and after water deprivation, by conventional reverse transcriptase-polymerase chain reac- tion (RT-PCR) and quantitative real-time PCR. In both normally hydrated and water-deprived groups, qAQP2 mRNA levels in the medullary regions were significantly higher (P < 0.01) than in the cortical regions. In medullary areas, qAQP2 mRNA levels (real-time PCR normalized with 18S) were significantly higher (P < 0.01, ANOVA) after water deprivation (1.09 ± 0.10) than in normally hydrated controls (0.46 ± 0.08). In cortical areas, qAQP2 mRNA levels were also higher after water deprivation (0.37 ± 0.05) than in controls (0.11 ± 0.02). qAQP2 mRNA signals determined by in situ hybridization of digoxigenin-labeled riboprobe were also enhanced after water deprivation in both cortical and medullary collecting ducts. The results suggest that, contrary to our hypothesis, the endogenous production of ADH by water deprivation stimulates qAQP2 mRNA in both loopless and looped nephrons. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction The ability to concentrate urine and conserve body water is essential for adaptation to terrestrial environments. Among verte- brates, however, only birds and mammals are able to form urine hyperosmolar to plasma (Schmidt-Nielsen, 1979; Braun and Dant- zler, 1997). The avian kidney consists of both primitive loopless nephrons (reptilian type) and more advanced looped nephrons (mammalian type) (Dantzler and Braun, 1980; Braun and Reimer, 1988; Goldstein and Braun, 1989). While the looped nephrons are capable of conserving water by a countercurrent mechanism (Nishimura et al., 1989; Nishimura 1993), hyposmotic–isosmotic urine is formed in cortical loopless nephrons. Urine-concentrating capacity is in general lower in birds than in mammals (Dantzler, 1989). We have identified aquaporin (AQP) 2 from medullary cones of Japanese quail that shows molecular and functional characteris- tics of a water channel (Yang et al., 2004). Immunoreactive AQP2 increased after in vivo water deprivation; the increase was more clearly observed in the medullary collecting duct (CD) cells than in cortical CDs (Yang et al., 2004). We therefore hypothesized that water homeostasis via control of AQP2 water channels by endoge- nous antidiuretic hormone (ADH) has evolved preferentially in the structurally more advanced looped nephrons in the quail kidneys. To test whether the effects of water deprivation on the expression of quail AQP2 (qAQP2) mRNA are stronger in medullary looped nephrons than in cortical loopless nephrons, we used three meth- ods: (1) conventional reverse transcriptase-polymerase chain reac- tion (RT-PCR), (2) quantitative real-time PCR, and (3) in situ hybridization of riboprobes. 2. Materials and methods 2.1. Animals and maintenance Fertilized eggs of Coturnix japonica quail were purchased from G.Q.F. Manufacturing (Savannah, GA) and hatched in a temperature- and humidity-controlled incubator in our laboratory (Nishimura et al., 1989, 1996). Hatched birds (Japanese quail take 17 days to 0016-6480/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2008.12.007 q Preliminary studies were presented at the Annual Meetings of the Federation of American Societies for Experimental Biology, 2006. Dr. Keith K. Lau’s present address is Department of Pediatrics, University of California, Davis, Sacramento, CA 95817. * Corresponding author. Fax: +1 901 448 7126. E-mail address: nishimur@physio1.utmem.edu (H. Nishimura). General and Comparative Endocrinology 160 (2009) 288–294 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen