A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport

Am J Physiol Renal Physiol. 2012 Sep 15;303(6):F886-92. doi: 10.1152/ajprenal.00114.2012. Epub 2012 Jul 3.


Studying the molecular regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for understanding how the kidney contributes to blood pressure regulation. Until now, a native mammalian cell model to investigate this transporter remained unknown. Our aim here is to establish, for the first time, a primary distal convoluted tubule (DCT) cell culture exhibiting transcellular thiazide-sensitive Na(+) transport. Because parvalbumin (PV) is primarily expressed in the DCT, where it colocalizes with NCC, kidneys from mice expressing enhanced green-fluorescent protein (eGFP) under the PV gene promoter (PV-eGFP-mice) were employed. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to sort fluorescent PV-positive tubules from these kidneys, which were then seeded onto permeable supports. After 6 days, DCT cell monolayers developed transepithelial resistance values of 630 ± 33 Ω·cm(2). The monolayers also established opposing transcellular concentration gradients of Na(+) and K(+). Radioactive (22)Na(+) flux experiments showed a net apical-to-basolateral thiazide-sensitive Na(+) transport across the monolayers. Both hypotonic low-chloride medium and 1 μM angiotensin II increased this (22)Na(+) transport significantly by four times, which could be totally blocked by 100 μM hydrochlorothiazide. Angiotensin II-stimulated (22)Na(+) transport was also inhibited by 1 μM losartan. Furthermore, NCC present in the DCT monolayers was detected by immunoblot and immunocytochemistry studies. In conclusion, a murine primary DCT culture was established which expresses functional thiazide-sensitive Na(+)-Cl(-) transport.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin II / pharmacology
  • Angiotensin II Type 1 Receptor Blockers / pharmacology
  • Animals
  • Cells, Cultured
  • Female
  • Kidney Tubules, Distal / cytology
  • Kidney Tubules, Distal / drug effects
  • Kidney Tubules, Distal / metabolism*
  • Losartan / pharmacology
  • Mice
  • Mice, Knockout
  • Sodium Chloride / metabolism
  • Sodium Chloride Symporter Inhibitors / pharmacology
  • Sodium Chloride Symporters / genetics
  • Sodium Chloride Symporters / metabolism*
  • Thiazides / pharmacology*


  • Angiotensin II Type 1 Receptor Blockers
  • Sodium Chloride Symporter Inhibitors
  • Sodium Chloride Symporters
  • Thiazides
  • Angiotensin II
  • Sodium Chloride
  • Losartan