Abstract: FR-OR129

Proteomics and Water Channel Function of AQP2-Rich Extracellular Vesicles in Human Urine

Session Information

Category: Fluid, Electrolytes, and Acid-Base

  • 702 Water/Urea/Vasopressin, Organic Solutes

Authors

  • Miyazawa, Yuko, Meiji Pharmaceutical University, Kiyose-shi, Tokyo, Japan
  • Mikami, Saki, Meiji Pharmaceutical University, Kiyose-shi, Tokyo, Japan
  • Sakai, Masaki, Meiji Pharmaceutical University, Kiyose-shi, Tokyo, Japan
  • Saito, Tatsuya, Meiji Pharmaceutical University, Kiyose-shi, Tokyo, Japan
  • Ishibashi, Kenichi, Meiji Pharmaceutical University, Kiyose-shi, Tokyo, Japan
  • Sasaki, Sei, Meiji Pharmaceutical University, Kiyose-shi, Japan
Background

AQP2 water channel is a key membrane protein which determines urine concentrating ability. AQP2 is excreted in urine in a form of extracellular vesicles (EVs), mostly in exosomes and urine AQP2 is now measured as a useful biomarker for diagnosis and treatment of water-balance disorders. However, proteomic and functional analysis of AQP2-bearing EVs have not been performed.

Methods

Urine EVs were obtained from heathy volunteers by the differential centrifugation. Membrane-disrupted (freeze-thaw) AQP2-bearing EVs were obtained by immunoprecipitation with an AQP2-specific antibody and the proteins were digested with trypsin and applied to LC-MS/MS proteomic analysis. The proteins were also analyzed by Western blots. Osmotic water permeability (Pf) of the AQP2-enriched EVs was measured by a stopped flow method monitoring 90 degree scattered light intensity in response to outwardly directed osmotic gradient created by glycerol.

Results

1) The MS analysis of the EVs co-immunoprecipitated with the AQP2 antibody identified 137 proteins, surprisingly, 103 of these 137 proteins have been shown to express in collecting duct cells, suggesting that our co-immunoprecipitation successfully gather AQP2-bearing EVs membranes. Pathway analysis show the presence of late endosome and multiple vesicular body-related proteins such as TSG101, ALIX, CHMP1-6, VPS4,7,9,25. MS analysis of urine EVs showed the phosphorylation of AQP2 at Ser256 and Ser261. Western blot analysis confirmed the presence of these proteins and S256, S261, and S269 phosphorylated AQP2. 2) Pf of 160,000 xg EVs was 4.75 ± 0.38 × 10-4 cm/s which was inhibited by 63% by 0.3 mM HgCl2 pretreatment. The activation energy was 3.51 kcal/mol which is consistent with a water channel activity. Pf was not detectable when EVs membranes were disrupted by extensive sonication. The measured Pf values of EVs samples were proportional to the protein amounts of AQP2 (r=0.95, p<0.014). A positive correlation was also observed between the EVs Pf and osmolality of the urine from which urine EVs were prepared (r=0.879, p<0.049).

Conclusion

Urine AQP2 excretion is mediated by late endosome-multiple vesicular body pathway, and AQP2 at EVs preserve the original water channel function.

Funding

  • Government Support - Non-U.S.