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Abstract: PO1398

The Phosphorylated States of Human Aquaporin 2 Revealed by Liquid Chromatography Coupled to Tandem Mass Spectrometry Phosphoproteomic Analysis of Urinary Exosomes

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

  • 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Ishibashi, Kenichi, Meiji Pharmceutical university, Kiyose, Tokyo, Japan
  • Sasaki, Sei, Meiji Pharmceutical university, Kiyose, Tokyo, Japan
  • Yamamoto, Tadashi, Biofluid Biomarker Center, Niigata University, Niigata, Niigata, Japan
  • Yamamoto, Keiko, Biofluid Biomarker Center, Niigata University, Niigata, Niigata, Japan
  • Tanaka, Yasuko, Meiji Pharmceutical university, Kiyose, Tokyo, Japan
Background

Aquaporin-2 (AQP2) is a key water channel to enhance water permeability of collecting ducts. Multiple phosphorylation sites at the C-terminal of AQP2 have been identified including S256 (serine at the 256 residue), S261, S264 and S/T269. Moreover, the phosphorylation profile induced by vasopressin (VP) seems to affect AQP2 trafficking to and from the plasma membrane in rodent AQP2, with limited studies in human AQP2 whose residue at 269 is T instead of S. As AQP2 is abundantly excreted in urinary exosomes, they will be useful to examine the phosphoprotein profile of human AQP2 modulated by VP.

Methods

We examined the phosphorylation profile of urinary exosomal human AQP2 to obtain the insight into the mechanism of AQP2 trafficking by VP. Human urinary exosomes were digested with trypsin or glutamyl endopeptidase (Glu-C) to apply for the liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) phosphoproteomic analysis, in parallel with immunoblots by commercially available phosphoryation-site specific anti-human AQP2 antibodies.

Results

The most dominant phosphorylated AQP2 peptide was phosphorylated at S256 (pS256), followed by pS261, less pS264 and much less pT269. The results were confirmed by the western blot analysis using antibodies specific to each phosphorylated AQP2. To document the time-course of urinary exosomal AQP2 phosphorylation by VP, a VP analogue was administered to a patient with central diabetes insipidus. It induced total exosomal AQP2 urinary excretion plateauing at 60 min with a transient increase (peaking at 30~60 min) of pS261 and a progressive increase of pS256. All four corresponding phosphorylation sites of human AQP2 including T269 were phosphorylated and the phosphorylation sites at S256 and S261 were tightly linked to toal exosomal AQP2 urinary excretion.

Conclusion

We conclude that human AQP2 is predominantly phosphorylated at S256 and moderately at S261 in urinary exosomes. T269-phosphorylation may not be needed for exosomal AQP2 excretion in human urine.

Funding

  • Government Support - Non-U.S.