title: A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport.
abstract: 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.
authors: Department of Physiology, Radboud University Nijmegen Medical Centre, The Netherlands.
authors: Nicolas
authors: N
authors: Markadieu
authors: Pedro
authors: P
authors: San-Cristobal
authors: Anil V
authors: AV
authors: Nair
authors: Sjoerd
authors: S
authors: Verkaart
authors: Ellen
authors: E
authors: Lenssen
authors: Kukiat
authors: K
authors: Tudpor
authors: Femke
authors: F
authors: van Zeeland
authors: Johannes
authors: J
authors: Loffing
authors: René J M
authors: RJ
authors: Bindels
authors: Joost G J
authors: JG
authors: Hoenderop
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019808:Losartan / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D012965:Sodium Chloride / Q000378:metabolism; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology; D049992:Sodium Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D049971:Thiazides / Q000494:pharmacology*
chemical_list: D047228:Angiotensin II Type 1 Receptor Blockers; D049993:Sodium Chloride Symporter Inhibitors; D049992:Sodium Chloride Symporters; D049971:Thiazides; D000804:Angiotensin II; D012965:Sodium Chloride; D019808:Losartan