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

Kerim Mutig, Thomas Kahl, Turgay Saritas, Michael Godes, Pontus Persson, James Bates, Hajamohideen Raffi, Luca Rampoldi, Shinichi Uchida, Carsten Hille, Carsten Dosche, Satish Kumar, Maria Castañeda-Bueno, Gerardo Gamba, Sebastian Bachmann

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)30200-30210
Number of pages11
JournalJournal of Biological Chemistry
Volume286
Issue number34
DOIs
Publication statusPublished - Aug 26 2011

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Member 1 Solute Carrier Family 12
Uromodulin
Bumetanide
Chlorides
Chemical activation
Phosphorylation
Extremities
Vasopressin Receptors
Arginine Vasopressin
Complementary RNA
Active Biological Transport

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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Activation of the bumetanide-sensitive Na +,K +, 2Cl -Cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner. / Mutig, Kerim; Kahl, Thomas; Saritas, Turgay; Godes, Michael; Persson, Pontus; Bates, James; Raffi, Hajamohideen; Rampoldi, Luca; Uchida, Shinichi; Hille, Carsten; Dosche, Carsten; Kumar, Satish; Castañeda-Bueno, Maria; Gamba, Gerardo; Bachmann, Sebastian.

In: Journal of Biological Chemistry, Vol. 286, No. 34, 26.08.2011, p. 30200-30210.

Research output: Contribution to journalArticle

Mutig, K, Kahl, T, Saritas, T, Godes, M, Persson, P, Bates, J, Raffi, H, Rampoldi, L, Uchida, S, Hille, C, Dosche, C, Kumar, S, Castañeda-Bueno, M, Gamba, G & Bachmann, S 2011, 'Activation of the bumetanide-sensitive Na +,K +, 2Cl -Cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner', Journal of Biological Chemistry, vol. 286, no. 34, pp. 30200-30210. https://doi.org/10.1074/jbc.M111.222968
Mutig, Kerim ; Kahl, Thomas ; Saritas, Turgay ; Godes, Michael ; Persson, Pontus ; Bates, James ; Raffi, Hajamohideen ; Rampoldi, Luca ; Uchida, Shinichi ; Hille, Carsten ; Dosche, Carsten ; Kumar, Satish ; Castañeda-Bueno, Maria ; Gamba, Gerardo ; Bachmann, Sebastian. / Activation of the bumetanide-sensitive Na +,K +, 2Cl -Cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 34. pp. 30200-30210.
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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.",
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AU - Saritas, Turgay

AU - Godes, Michael

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AU - Bates, James

AU - Raffi, Hajamohideen

AU - Rampoldi, Luca

AU - Uchida, Shinichi

AU - Hille, Carsten

AU - Dosche, Carsten

AU - Kumar, Satish

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AU - Gamba, Gerardo

AU - Bachmann, Sebastian

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