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Small-molecule screening identifies modulators of aquaporin-2 trafficking

Authors

  • J. Bogum
  • D. Faust
  • K. Zühlke
  • J. Eichhorst
  • M.C. Moutty
  • J. Furkert
  • A. Eldahshan
  • M. Neuenschwander
  • J.P. von Kries
  • B. Wiesner
  • C. Trimpert
  • P.M. Deen
  • G. Valenti
  • W. Rosenthal
  • E. Klussmann

Journal

  • Journal of the American Society of Nephrology

Citation

  • J Am Soc Nephrol 24 (5): 744-758

Abstract

  • In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.


DOI

doi:10.1681/ASN.2012030295