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Abstract: TH-PO507

Phosphorylation of Ser261 and Dephosphorylation of Ser269 Is Important for Urinary Excretion of AQP2

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Sasaki, Sei, Tokyo Medical and Dental University, Bunkyo-ku, Japan
  • Sakai, Masaki, Meiji Pharmaceutical University, Tokyo, Japan
  • Mizumura, Hiroki, Meiji Pharmaceutical University, Tokyo, Japan
  • Matsumoto, Tomoki, Meiji Pharmaceutical University, Tokyo, Japan
  • Noda, Yumi, Tokyo Medical and Dental University, Bunkyo-ku, Japan
  • Yui, Naofumi, Tokyo Medical and Dental University, Bunkyo-ku, Japan
  • Ishibashi, Kenichi, Meiji Pharmaceutical University, Tokyo, Japan
Background

AQP2 a key membrane protein which determines water permeability of collecting ducts. AQP2 is regulated by phosphorylation at S256, S261 and S269. As a short-term regulation of vasopressin, intracellular amount S261 phosphorylated-AQP2 (261-P) decreases whereas 269-P increases with relatively stable 256-P. The decrease in 261-P can be explained by dephosphorylation, degradation, or even extrusion out of the cell. In this sence, it is intriguing whether phoshorylation is involved in the urinary excretion of exosomal AQP2 and if so, which phosphorylation site is critical.

Methods

Human urine samples were obtained from a central diabetes insipidus patient (CDI, 45 years old, female). DDAVP 0.25ug was administered to the nasal mucosa after 24 h withdrawal. Urine samples were collected pre and after the administration until 3 h. Urine exosomes were obtained by the differential centrifugation, and analyzed by Western blot using a usual and 256-P, 261-P and 269-P-specific antibodies. Endogenously AQP2-expressing cells (mpkCCD) and stably rat AQP2-transfected MDCK cells (AQP2-MDCK) were cultured on the permeable support and the culture medium in the apical side were analyzed

Results

1) In a CDI patient, DDAVP administration evoked a steady increase in urine osmolality until 3 h. Western blots of urine exosomal AQP2 excretion corrected for creatinine showed that total AQP2 rapidly increased until 1 h (8.5 times compared to the pre), then gradually increased until 3 h. 256-P progressively increased until 3 h. On the other hand, 261-P reached its peak increase at 0.5 h (2.4 times), and then, surprisingly decreased to the pre level at 2 h. 269-P was hardly visible throughout the study periods. 2) Western blots of apical culture medium of mpkCCD and AQP2-MDCK after vasopressin/forskolin stimulation, total, 256-P and 261-P were observed, but 269-P was minimally visible, although all these phosphorylated forms were observed in the cell lysates. 3) The LC-MS/MS phosphoproteomic analysis of human urine confirmed the presence of phosphorylation at S256 and S261, but not at S269.

Conclusion

These results indicate the importance of phoshorylation at S261 and dephoshphorylation at S269 in exosomal excretion of AQP2, highlighting the significant role of the endocytosis-exosome pathway in AQP2 metabolism in collecting ducts.

Funding

  • Government Support - Non-U.S.