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.

Phosphorylation Decreases Ubiquitylation of the Thiazide-sensitive Cotransporter NCC and Subsequent Clathrin-mediated Endocytosis

“…Previous studies of NCC trafficking have been performed using immortalized DCT cell lines (33)(34)(35)(36), stably transfected NCC-expressing MDCKII cell lines (26,37), or transiently transfected cells such as COS and HEK (10,11,38). However, these cell lines are not ideal for studying trafficking events of NCC due to, for example, a lack of polarized delivery of NCC, absence of NCC complex glycosylation, and in the case of immortalized DCT cells the inability to assess the roles of sitedirected mutations for NCC activity.…”

Mutations in PCBD1 Cause Hypomagnesemia and Renal Magnesium Wasting

“…Parvalbumin-eGFP-positive tubules were isolated as described previously. 39 In short, mice ages 4-6 weeks were anesthetized and perfused transcardially with ice cold buffer of 145 mmol/L NaCl, Per mouse, 4000 eGFP-fluorescent tubules were collected; 4000 tubules were pooled on a microcolumn for RNA extraction according to the manufacturer's protocol. Subsequently, reverse transcription of the RNA by M-MLV reverse transcription (Invitrogen) was performed for 1 hour at 37°C.…”

Harvest and primary culture of the murine aldosterone-sensitive distal nephron

“…Existing methodologies that isolate DCT or CCD include laser capture microdissection (12,51) and transgenic mice expressing green fluorescent protein with Complex Object Parametric Analyzer and Sorter (CO-PAS) technology (36,39). These techniques are not easily accessible to most laboratories and require technical expertise and/or specialized mouse strains.…”