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Author Notes:

Address for reprint requests and other correspondence: R. S. Hoover, Departments of Physiology and Medicine, Emory University, 615 Michael Street, Room 601, Atlanta, GA 30322; Email: robert.hoover@emory.edu

Author contributions: B.K., A.C.M., and R.S.H. conception and design of research; B.K., A.C.M., L.N.H., R.M., L.L.C., and B.K.H. performed experiments; B.K., A.C.M., and R.S.H. analyzed data; B.K., A.C.M., L.N.H., R.M., L.L.C., B.K.H., P.N.C., and R.S.H. interpreted results of experiments; B.K., A.C.M., L.N.H., L.L.C., B.K.H., and R.S.H. prepared figures; B.K. and R.S.H. drafted manuscript; B.K. and R.S.H. edited and revised manuscript; B.K. and R.S.H. approved final version of manuscript.

The authors thank Eugene Chang and Mark Musch for advice and discussions.

The authors thank Sheri Wanderling and Dan Levy for help with the immunohistochemistry.

The authors also thank David Ellison for the t53 NCC phosphoantibody.

No conflicts of interest, financial or otherwise, are declared by the author(s).

Subjects:

Research Funding:

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-085097 (to R. S. Hoover), K08-DK-081728 (to B. Ko), and R01-DK-080863 (to P. Cunningham) as well as by the Research Service, Atlanta Veterans Affairs Medical Center, Decatur, GA (to R. S. Hoover).

Keywords:

  • sodium-chloride cotransporter
  • STE20/SPS-1-related proline/alanine-rich kinase
  • with-no-lysine kinase 4

A new model of the distal convoluted tubule

Tools:

Journal Title:

AJP - Renal Physiology

Volume:

Volume 303, Number 5

Publisher:

, Pages F700-F710

Type of Work:

Article | Post-print: After Peer Review

Abstract:

The Na+-Cl− cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney is a key determinant of Na+ balance. Disturbances in NCC function are characterized by disordered volume and blood pressure regulation. However, many details concerning the mechanisms of NCC regulation remain controversial or undefined. This is partially due to the lack of a mammalian cell model of the DCT that is amenable to functional assessment of NCC activity. Previously reported investigations of NCC regulation in mammalian cells have either not attempted measurements of NCC function or have required perturbation of the critical without a lysine kinase (WNK)/STE20/SPS-1-related proline/alanine-rich kinase regulatory pathway before functional assessment. Here, we present a new mammalian model of the DCT, the mouse DCT15 (mDCT15) cell line. These cells display native NCC function as measured by thiazide-sensitive, Cl−-dependent 22Na+ uptake and allow for the separate assessment of NCC surface expression and activity. Knockdown by short interfering RNA confirmed that this function was dependent on NCC protein. Similar to the mammalian DCT, these cells express many of the known regulators of NCC and display significant baseline activity and dimerization of NCC. As described in previous models, NCC activity is inhibited by appropriate concentrations of thiazides, and phorbol esters strongly suppress function. Importantly, they display release of WNK4 inhibition of NCC by small hairpin RNA knockdown. We feel that this new model represents a critical tool for the study of NCC physiology. The work that can be accomplished in such a system represents a significant step forward toward unraveling the complex regulation of NCC.

Copyright information:

© 2012 The American Physiological Society

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