title: Cellular localization of adenine receptors in the rat kidney and their functional significance in the inner medullary collecting duct.
abstract: The Gi-coupled adenine receptor (AdeR) binds adenine with high affinity and potentially reduces cellular cAMP levels. Since cAMP is an important second messenger in the renal transport of water and solutes, we localized AdeR in the rat kidney. Real-time RT-PCR showed higher relative expression of AdeR mRNA in the cortex and outer medulla compared with the inner medulla. Immunoblots using a peptide-derived and affinity-purified rabbit polyclonal antibody specific for an 18-amino acid COOH-terminal sequence of rat AdeR, which we generated, detected two bands between ∼30 and 40 kDa (molecular mass of native protein: 37 kDa) in the cortex, outer medulla, and inner medulla. These bands were ablated by preadsorption of the antibody with the immunizing peptide. Immunofluorescence labeling showed expression of AdeR protein in all regions of the kidney. Immunoperoxidase revealed strong labeling of AdeR protein in the cortical vasculature, including the glomerular arterioles, and less intense labeling in the cells of the collecting duct system. Confocal immunofluorescence imaging colocalized AdeR with aquaporin-2 protein to the apical plasma membrane in the collecting duct. Functionally, adenine (10 μM) significantly decreased (P < 0.01) 1-deamino-8-d-arginine vasopressin (10 nM)-induced cAMP production in ex vivo preparations of inner medullary collecting ducts, which was reversed by PSB-08162 (20 μM, P < 0.01), a selective antagonist of AdeR. Thus, we demonstrated the expression of AdeR in the renal vasculature and collecting ducts and its functional relevance. This study may open a new avenue for the exploration of autocrine/paracrine regulation of renal vascular and tubular functions by the nucleobase adenine in health and disease.
authors.affiliation: Nephrology Research (151M Veterans Affairs Salt Lake City Health Care System, 500 Foothill Drive, Salt Lake City, UT 84148. BK.Kishore@hsc.utah.edu.
authors.forename: Bellamkonda K
authors.initials: BK
authors.lastname: Kishore
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.forename: Haykanush
authors.initials: H
authors.lastname: Gevorgyan
authors.forename: Donald E
authors.initials: DE
authors.lastname: Kohan
authors.forename: Anke C
authors.initials: AC
authors.lastname: Schiedel
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
authors.forename: János
authors.initials: J
authors.lastname: Peti-Peterdi
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D003894:Deamino Arginine Vasopressin / Q000378:metabolism; D007679:Kidney Medulla / Q000378:metabolism* / Q000502:physiology; D007685:Kidney Tubules, Collecting / Q000378:metabolism* / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011983:Receptors, Purinergic / Q000378:metabolism* / Q000502:physiology
chemical_list: D012333:RNA, Messenger; D011983:Receptors, Purinergic; C459899:adenine receptor, rat; D000242:Cyclic AMP; D003894:Deamino Arginine Vasopressin
keywords: G protein-coupled receptor; adenine receptor; adenylyl cyclase; aquaporin-2; chronic kidney disease; collecting duct; purinergic receptors