title: E3 ubiquitin-protein ligases in rat kidney collecting duct: response to vasopressin stimulation and withdrawal.
abstract: The E3 ubiquitin (Ub)-protein ligases (E3s) play a role as regulators of protein trafficking and degradation. We aimed to integrate the profile of E3s in rat kidney and examine the changes in protein abundance of the selected E3s in response to 1-deamino-8-D-arginine vasopressin (dDAVP) stimulation/withdrawal. Sprague-Dawley rats were infused with vehicle (n = 13), dDAVP for 5 days (n = 13), or dDAVP was withdrawn for periods (15 min, 30 min, 1, 3, 6, 12, or 24 h) after 5-day infusion (n = 46). Total RNA was isolated from the inner medulla (IM) for transcriptome analysis. Plasma membrane (PM)- or intracellular vesicle (ICV)-enriched fractions of whole kidney were immunoisolated for liquid chromatography-tandem mass spectrometry analysis. dDAVP infusion for 5 days (D5d) significantly increased urine osmolality, which was maintained during 3-h withdrawal of dDAVP after 5-day infusion (D5d-3h). Consistent with this, aquaporin-2 (AQP2) expression in the PM fractions of D5d and D5d-3h increased, whereas AQP2 expression in the ICV fractions of D5d-3h was further increased, indicating internalization of AQP2. Transcriptome analysis revealed 86 genes of E3s and LC-MS/MS analysis demonstrated 16 proteins of E3s. Among these, seven E3s (BRCA1, UBR4, BRE1B, UHRF1, NEDD4, CUL5, and FBX6) were shared. RT-PCR demonstrated mRNA expressions of the seven identified E3s in the kidney, and immunoblotting demonstrated changes in protein abundance of the selected E3s (BRE1B, NEDD4, and CUL5) in response to dDAVP stimulation/withdrawal or lithium-induced nephrogenic diabetes insipidus. The rate of AQP2 degradation was retarded in mpkCCDc14 cells with small interfering RNA-mediated knockdown of NEDD4 or CUL5. Taken together, identified E3s could be involved in the degradation of proteins associated with vasopressin-induced urine concentration.
authors: Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea.
authors: Yu-Jung
authors: YJ
authors: Lee
authors: Jeong-Eun
authors: JE
authors: Lee
authors: Hyo-Jung
authors: HJ
authors: Choi
authors: Jung-Suk
authors: JS
authors: Lim
authors: Hyun Jun
authors: HJ
authors: Jung
authors: Moon-Chang
authors: MC
authors: Baek
authors: Jørgen
authors: J
authors: Frøkiær
authors: Søren
authors: S
authors: Nielsen
authors: Tae-Hwan
authors: TH
authors: Kwon
mesh_terms: D000818:Animals; D050034:Antidiuretic Agents / Q000494:pharmacology*; D051399:Aquaporin 2 / Q000096:biosynthesis; D002460:Cell Line; D044842:Cullin Proteins / Q000378:metabolism; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology*; D018500:Diabetes Insipidus, Nephrogenic / Q000139:chemically induced / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D020869:Gene Expression Profiling; D007685:Kidney Tubules, Collecting / Q000187:drug effects* / Q000201:enzymology*; D008094:Lithium / Q000633:toxicity; D008297:Male; D000075702:Nedd4 Ubiquitin Protein Ligases; D051381:Rats; D017207:Rats, Sprague-Dawley; D017483:Receptors, Vasopressin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D028761:Withholding Treatment
chemical_list: D050034:Antidiuretic Agents; C496025:Aqp2 protein, rat; D051399:Aquaporin 2; C094547:Cul5 protein, rat; D044842:Cullin Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D017483:Receptors, Vasopressin; D008094:Lithium; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618484:Nedd4 protein, rat; D044767:Ubiquitin-Protein Ligases; D003894:Deamino Arginine Vasopressin