title: Bilateral ureteral obstruction induces early downregulation and redistribution of AQP2 and phosphorylated AQP2.
abstract: Bilateral ureteral obstruction (BUO) is characterized by impairment of urine flow from the kidneys and altered expression of specific membrane proteins in the kidney involved in regulation of renal water and salt transport. Importantly, 24-h BUO reduces the abundance of the collecting duct water channel aquaporin-2 (AQP2) and AQP2 phosphorylated at serine 256 (AQP2pS256). To investigate the mechanism behind downregulation of AQP2 in BUO, rats were subjected to BUO and examined after 2, 6, 12, and 24 h. Q-PCR and immunoblotting showed significantly decreased AQP2 mRNA expression after 2-h BUO and decreased abundance of total AQP2 after 12 and 24 h. In parallel, immunohistochemistry showed weaker labeling of AQP2 at the apical surface of inner medullary collecting ducts (IMCD) compared with controls. The abundance of AQP2pS256 was significantly reduced from 6-h BUO and was confirmed by immunohistochemistry. Importantly, immunoblotting showed reduced abundance of AQP2pS261 after 12- and 24-h BUO mimicking total AQP2. Immunohistochemistry demonstrated early changed intracellular localization of AQP2pS261 in BUO, and colocalization studies showed redistribution from the apical membrane to early endosomes and lysosomes. In conclusion, BUO induces a very early regulation of AQP2 both at the level of abundance and on cellular localization. AQP2 and AQP2 phosphorylated at ser261 redistribute to more intracellular localizations and colocalize with the early endosomal marker EEA1 and the lysosomal marker cathepsin D, suggesting that early downregulation of AQP2 could in part be caused by degradation of AQP2 through a lysosomal degradation pathway.
authors.affiliation: The Water and Salt Research Center/Institute of Clinical Medicine, Aarhus University Hospital-Skejby, DK-8200 Aarhus, Denmark.
authors.forename: Lene
authors.initials: L
authors.lastname: Stødkilde
authors.forename: Rikke
authors.initials: R
authors.lastname: Nørregaard
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fenton
authors.forename: Guixian
authors.initials: G
authors.lastname: Wang
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
authors.forename: Jørgen
authors.initials: J
authors.lastname: Frøkiær
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000096:biosynthesis* / Q000235:genetics*; D015153:Blotting, Western; D015536:Down-Regulation / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D007536:Isomerism; D007668:Kidney / Q000378:metabolism; D007671:Kidney Concentrating Ability; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D008566:Membranes / Q000378:metabolism; D018613:Microscopy, Confocal; D009994:Osmolar Concentration; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014517:Ureteral Obstruction / Q000378:metabolism*; D014882:Water-Electrolyte Balance / Q000502:physiology
chemical_list: D051399:Aquaporin 2; D012333:RNA, Messenger