Activation of the bumetanide-sensitive Na+,K+,2Cl- cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner

J Biol Chem. 2011 Aug 26;286(34):30200-10. doi: 10.1074/jbc.M111.222968. Epub 2011 Jul 7.

Abstract

Active transport of NaCl across thick ascending limb (TAL) epithelium is accomplished by Na(+),K(+),2Cl(-) cotransporter (NKCC2). The activity of NKCC2 is determined by vasopressin (AVP) or intracellular chloride concentration and includes its amino-terminal phosphorylation. Co-expressed Tamm-Horsfall protein (THP) has been proposed to interact with NKCC2. We hypothesized that THP modulates NKCC2 activity in TAL. THP-deficient mice (THP(-/-)) showed an increased abundance of intracellular NKCC2 located in subapical vesicles (+47% compared with wild type (WT) mice), whereas base-line phosphorylation of NKCC2 was significantly decreased (-49% compared with WT mice), suggesting reduced activity of the transporter in the absence of THP. Cultured TAL cells with low endogenous THP levels and low base-line phosphorylation of NKCC2 displayed sharp increases in NKCC2 phosphorylation (+38%) along with a significant change of intracellular chloride concentration upon transfection with THP. In NKCC2-expressing frog oocytes, co-injection with THP cRNA significantly enhanced the activation of NKCC2 under low chloride hypotonic stress (+112% versus +235%). Short term (30 min) stimulation of the vasopressin V2 receptor pathway by V2 receptor agonist (deamino-cis-D-Arg vasopressin) resulted in enhanced NKCC2 phosphorylation in WT mice and cultured TAL cells transfected with THP, whereas in the absence of THP, NKCC2 phosphorylation upon deamino-cis-D-Arg vasopressin was blunted in both systems. Attenuated effects of furosemide along with functional and structural adaptation of the distal convoluted tubule in THP(-/-) mice supported the notion that NaCl reabsorption was impaired in TAL lacking THP. In summary, these results are compatible with a permissive role for THP in the modulation of NKCC2-dependent TAL salt reabsorptive function.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antidiuretic Agents / pharmacology
  • Cell Line, Transformed
  • Chlorides / metabolism*
  • Deamino Arginine Vasopressin / pharmacology
  • Kidney Tubules, Distal / metabolism*
  • Mice
  • Mice, Knockout
  • Osmotic Pressure
  • Phosphorylation / drug effects
  • Phosphorylation / physiology
  • Rabbits
  • Receptors, Vasopressin / genetics
  • Receptors, Vasopressin / metabolism
  • Sodium-Potassium-Chloride Symporters / genetics
  • Sodium-Potassium-Chloride Symporters / metabolism*
  • Solute Carrier Family 12, Member 1
  • Uromodulin / genetics
  • Uromodulin / metabolism*
  • Xenopus laevis

Substances

  • Antidiuretic Agents
  • Chlorides
  • Receptors, Vasopressin
  • Slc12a1 protein, mouse
  • Sodium-Potassium-Chloride Symporters
  • Solute Carrier Family 12, Member 1
  • Umod protein, mouse
  • Uromodulin
  • Deamino Arginine Vasopressin