title: Autophagic degradation of aquaporin-2 is an early event in hypokalemia-induced nephrogenic diabetes insipidus.
abstract: Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.
authors.affiliation: Department of Pediatrics, Faculty of Medicine, Thammasat University Klong Luang, Pathumthani, 12120, Thailand.
authors.forename: Sookkasem
authors.initials: S
authors.lastname: Khositseth
authors.affiliation: Department of Physiology, Faculty of Medicine Siriraj Hospital, Mahidol University Bangkok, 10700, Thailand.
authors.forename: Panapat
authors.initials: P
authors.lastname: Uawithya
authors.affiliation: Systems Biology Center, Research Affairs, Faculty of Medicine, Chulalongkorn University 1873 Rama 4 Road, Pathumwan, Bangkok, 10330, Thailand.
authors.forename: Poorichaya
authors.initials: P
authors.lastname: Somparn
authors.affiliation: Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University Bangkok, 10700, Thailand.
authors.forename: Komgrid
authors.initials: K
authors.lastname: Charngkaew
authors.affiliation: Department of Pediatrics, Faculty of Medicine, Thammasat University Klong Luang, Pathumthani, 12120, Thailand.
authors.forename: Nattakan
authors.initials: N
authors.lastname: Thippamom
authors.affiliation: National Institute of Diabetes and Digestive and Kidney, Bethesda MD 20892, United States.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Hoffert
authors.affiliation: Department of Electrical &Computer Engineering and Department of Computer Science, Western Michigan University Kalamazoo, 49008, United States.
authors.forename: Fahad
authors.initials: F
authors.lastname: Saeed
authors.affiliation: Systems Biology Center, Research Affairs, Faculty of Medicine, Chulalongkorn University 1873 Rama 4 Road, Pathumwan, Bangkok, 10330, Thailand.
authors.forename: D
authors.initials: D
authors.lastname: Michael Payne
authors.affiliation: Department of Chemistry, National Cheng Kung University, Tainan City, 701, Taiwan.
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Chen
authors.affiliation: Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, Aarhus, 8000, Denmark.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fenton
authors.affiliation: Systems Biology Center, Research Affairs, Faculty of Medicine, Chulalongkorn University 1873 Rama 4 Road, Pathumwan, Bangkok, 10330, Thailand.|Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, Aarhus, 8000, Denmark.|Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, Bethesda MD 20892, United States.
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D001343:Autophagy; D002853:Chromatography, Liquid; D018500:Diabetes Insipidus, Nephrogenic / Q000378:metabolism* / Q000503:physiopathology; D007008:Hypokalemia / Q000378:metabolism* / Q000503:physiopathology; D015151:Immunoblotting; D007679:Kidney Medulla / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000378:metabolism / Q000648:ultrastructure; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008297:Male; D016253:Microscopy, Immunoelectron; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism / Q000648:ultrastructure; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D051399:Aquaporin 2; D051907:Lysosomal Membrane Proteins; D008869:Microtubule-Associated Proteins; D020543:Proteome; C526109:Anxa3 protein, rat; C489117:Lamp1 protein, rat; D052118:Lysosomal-Associated Membrane Protein 1