title: Hypotonicity stimulates potassium flux through the WNK-SPAK/OSR1 kinase cascade and the Ncc69 sodium-potassium-2-chloride cotransporter in the Drosophila renal tubule.
abstract: The ability to osmoregulate is fundamental to life. Adult Drosophila melanogaster maintain hemolymph osmolarity within a narrow range. Osmolarity modulates transepithelial ion and water flux in the Malpighian (renal) tubules of the fly, which are in direct contact with hemolymph in vivo, but the mechanisms causing increased transepithelial flux in response to hypotonicity are unknown. Fly renal tubules secrete a KCl-rich fluid. We have previously demonstrated a requirement for Ncc69, the fly sodium-potassium-2-chloride cotransporter (NKCC), in tubule K(+) secretion. Mammalian NKCCs are regulated by a kinase cascade consisting of the with-no-lysine (WNK) and Ste20-related proline/alanine-rich (SPAK)/oxidative stress response (OSR1) kinases. Here, we show that decreasing Drosophila WNK activity causes a reduction in K(+) flux. Similarly, knocking down the SPAK/OSR1 homolog fray also decreases K(+) flux. We demonstrate that a hierarchical WNK-Fray signaling cascade regulates K(+) flux through Ncc69, because (i) a constitutively active Fray mutant rescues the wnk knockdown phenotype, (ii) Fray directly phosphorylates Ncc69 in vitro, and (iii) the effect of wnk and fray knockdown is abolished in Ncc69 mutants. The stimulatory effect of hypotonicity on K(+) flux is absent in wnk, fray, or Ncc69 mutant tubules, suggesting that the Drosophila WNK-SPAK/OSR1-NKCC cascade is an essential molecular pathway for osmoregulation, through its effect on transepithelial ion flux and fluid generation by the renal tubule.
authors.affiliation: Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8856.
authors.forename: Yipin
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8856.
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Schellinger
authors.affiliation: Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8856.
authors.forename: Chou-Long
authors.initials: CL
authors.lastname: Huang
authors.affiliation: Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8856. Electronic address: aylin.rodan@utsouthwestern.edu.
authors.forename: Aylin R
authors.initials: AR
authors.lastname: Rodan
mesh_terms: D000818:Animals; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster; D007684:Kidney Tubules / Q000378:metabolism; D009994:Osmolar Concentration; D064587:Osmoregulation; D011188:Potassium / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; C577097:Ncc69 protein, Drosophila; D028021:Sodium-Potassium-Chloride Symporters; D017346:Protein Serine-Threonine Kinases; C000594845:WNK protein, Drosophila; C526279:fray protein, Drosophila; D011188:Potassium
keywords: Epithelial Ion Transport; Epithelium; Fray; Ion-sensitive Electrode; Malpighian Tubule; Na-K-Cl Cotransporter (NKCC); Potassium Transport; Renal Physiology; SLC12 Cotransporters; Serine/Threonine Protein Kinase
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