title: Hypercalcemia induces targeted autophagic degradation of aquaporin-2 at the onset of nephrogenic diabetes insipidus.
abstract: Hypercalcemia can cause renal dysfunction such as nephrogenic diabetes insipidus (NDI), but the mechanisms underlying hypercalcemia-induced NDI are not well understood. To elucidate the early molecular changes responsible for this disorder, we employed mass spectrometry-based proteomic analysis of inner medullary collecting ducts (IMCD) isolated from parathyroid hormone-treated rats at onset of hypercalcemia-induced NDI. Forty-one proteins, including the water channel aquaporin-2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the downregulated proteins were associated with cytoskeletal protein binding, regulation of actin filament polymerization, and cell-cell junctions. Targeted LC-MS/MS and immunoblot studies confirmed the downregulation of 16 proteins identified in the initial proteomic analysis and in additional experiments using a vitamin D treatment model of hypercalcemia-induced NDI. Evaluation of transcript levels and estimated half-life of the downregulated proteins suggested enhanced protein degradation as the possible regulatory mechanism. Electron microscopy showed defective intercellular junctions and autophagy in the IMCD cells from both vitamin D- and parathyroid hormone-treated rats. A significant increase in the number of autophagosomes was confirmed by immunofluorescence labeling of LC3. Colocalization of LC3 and Lamp1 with aquaporin-2 and other downregulated proteins was found in both models. Immunogold electron microscopy revealed aquaporin-2 in autophagosomes in IMCD cells from both hypercalcemia models. Finally, parathyroid hormone withdrawal reversed the NDI phenotype, accompanied by termination of aquaporin-2 autophagic degradation and normalization of both nonphoshorylated and S256-phosphorylated aquaporin-2 levels. Thus, enhanced autophagic degradation of proteins plays an important role in the initial mechanism of hypercalcemic-induced NDI.
authors.affiliation: Department of Pediatrics, Faculty of Medicine, Thammasat University, Pathumthani, Thailand. Electronic address: Sookkasem@yahoo.com.
authors.forename: Sookkasem
authors.initials: S
authors.lastname: Khositseth
authors.affiliation: Department of Pathology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Komgrid
authors.initials: K
authors.lastname: Charngkaew
authors.affiliation: Systems Biology Center, Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Chatikorn
authors.initials: C
authors.lastname: Boonkrai
authors.affiliation: Systems Biology Center, Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Poorichaya
authors.initials: P
authors.lastname: Somparn
authors.affiliation: Department of Physiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Panapat
authors.initials: P
authors.lastname: Uawithya
authors.affiliation: Department of Pathology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Nusara
authors.initials: N
authors.lastname: Chomanee
authors.affiliation: Systems Biology Center, Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
authors.forename: D Michael
authors.initials: DM
authors.lastname: Payne
authors.affiliation: Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, Aarhus, Denmark.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fenton
authors.affiliation: Systems Biology Center, Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA. Electronic address: pisitkut@nhlbi.nih.gov.
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D001343:Autophagy; D002853:Chromatography, Liquid; D018500:Diabetes Insipidus, Nephrogenic / Q000209:etiology / Q000378:metabolism / Q000503:physiopathology*; D004097:Dihydrotachysterol / Q000633:toxicity; D004195:Disease Models, Animal; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D006207:Half-Life; D006801:Humans; D006934:Hypercalcemia / Q000139:chemically induced / Q000150:complications*; D007365:Intercellular Junctions / Q000378:metabolism / Q000648:ultrastructure; D007685:Kidney Tubules, Collecting / Q000378:metabolism / Q000503:physiopathology*; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008297:Male; D016253:Microscopy, Immunoelectron; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology; D010766:Phosphorylation; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D051399:Aquaporin 2; D004097:Dihydrotachysterol; D051907:Lysosomal Membrane Proteins; D008869:Microtubule-Associated Proteins; D010281:Parathyroid Hormone; C496025:Aqp2 protein, rat; C526109:Anxa3 protein, rat; C489117:Lamp1 protein, rat; D052118:Lysosomal-Associated Membrane Protein 1
keywords: autophagy; hypercalcemia; hypercalciuria; parathyroid hormone; proteomic analysis; vitamin D