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Abstract: TH-PO503

Epithelial Depolarization Induces AQP2 Up-regulation via Non-Canonical Ser-269 Phospho-Regulation Independently of Any Hormonal and Chemical Stimulation

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Yui, Naofumi, Tokyo Medical and Dental University, Sagamihara-shi, Kanagawa-ken, Japan
  • Ando, Fumiaki, Tokyo Medical and Dental University, Sagamihara-shi, Kanagawa-ken, Japan
  • Sasaki, Sei, Tokyo Medical and Dental University, Sagamihara-shi, Kanagawa-ken, Japan
  • Uchida, Shinichi, Tokyo Medical and Dental University, Sagamihara-shi, Kanagawa-ken, Japan
Background

Aquaporin-2 (AQP2) has multiple vasopressin-sensitive phosphorylation sites in its C-terminal region. We previously examined how pS256-positive AQP2 is catalyzed by vasopressin or forskolin stimulation using a phospho-specific AQP2 immuno-isolation technique, and identified a combined phospho-regulation: pS256-pS261-AQP2 changed to pS256-pS261-pS269-AQP2, and then transformed to pS256-pS269-AQP2 in both MDCK cells and mouse kidney. This cascade translocates pS256-positive AQP2 to the apical plasma membrane. We defined this phospho-regulation as canonical in this study. We next applied phospho-specific AQP2 immuno-isolation to quantify phospho-AQP2 in the total-AQP2 population. In the process of assay establishment, we hypothesized that cell polarization status affects AQP2 phosphorylation.

Methods

In this study, we divided MDCK cells stably expressing rat-AQP2 into two groups: cells were grown to confluence for 3 days and 1) their medium was just replaced with fresh normal DMEM without trypsinization (polarized group), or 2) they were trypsinized and re-plated with the same DMEM (depolarized group). Cells were lysed 24 h later without any hormonal or chemical stimulation and subjected to western blotting.

Results

Wild-type AQP2 significantly increased in the depolarized group. Ser-256 and Ser-261 phosphorylation was not changed, whereas Ser-269 phosphorylation significantly increased in the depolarized group. In S269A-AQP2 cells, depolarization-induced AQP2 upregulation did not occur. Interestingly, S256A-AQP2 was significantly upregulated in the depolarized group with a significant increase of Ser-269 phosphorylation.

Conclusion

These results demonstrate the existence of polarity-induced and hormone-independent AQP2 regulation, indicating a novel rationale for AQP2 function, i.e. in wound healing, tubular regeneration, cyst formation. In addition, we identified non-canonical AQP2 Ser-269 phospho-regulation, which is independent of prior Ser-256 phosphorylation and is not accompanied by subsequent Ser-261 dephosphorylation. These findings provide a basis for subsequent studies to investigate multi-functional roles of AQP2 beyond the categorical flame of apical water channel in polarized epithelial cells.

Funding

  • Government Support - Non-U.S.