Functional Expression of the Human Thiazide-Sensitive NaCl Cotransporter in Madin-Darby Canine Kidney Cells

Abstract

Abstract. The thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC), which is expressed on the apical membrane of epithelial cells lining the distal convoluted tubule, is responsible for the reabsorption of 5% to 10% of filtered Na ϩ and Cl Ϫ . To date, functional studies on the structural and regulatory requirements for localized trafficking and ion-transporting activity of NCC have been hampered by lack of a suitable cell system expressing this cotransporter. Reported here is the functional expression of human NCC (hNCC) in a polarized mammalian cell of renal origin-that is, the high-resistance Madin-Darby canine kidney (MDCK) cell. Western blot testing revealed that the cells predominantly expressed the complex glycosylated (approximately 140 kD) form of hNCC. hNCC was present primarily in the apical part of the cell. The functionality of hNCC was demonstrated by the gain of thiazide-sensitive Na ϩ uptake and transepithelial transport activity. Na ϩ uptake was significantly increased after short-term (15 min) treatment with forskolin, whereas cyclic guanosine monophosphate, wortmannin, phorbol 12-myriatate 13-acetate, and staurosporine were without effect. This indicates that hNCC activity is regulated through cyclic adenosine monophosphate, rather than via cyclic guanosine monophosphate, phosphoinositide 3-kinases or protein kinase C. Aldosterone did not alter Na ϩ uptake in the short term (15 min) but significantly increased the transport activity in the long term (16 h). The latter effect of aldosterone was due to an effect on the cytomegalovirus promoter/enhancer driving the expression of hNCC. hNCC-MDCK cells are a good model for the study of the regulation of apical trafficking and ion-transporting activity of hNCC.

Molecular Physiology and Pathophysiology of Electroneutral Cation-Chloride Cotransporters

“…This expression system has shown to be an excellent tool to obtain clean and reproducible TSC expression, whereas transfection in mammalian cells has not been successful. The best results obtained to date using wild-type TSC cDNA transfected into mammalian cells (MDCK cells) consisted of a small increase over background not Ͼ25% (74). Thus basically all TSC functional characterization has been performed using X. laevis oocytes as expression system.…”

Transport of iron chelators and chelates across MDCK cell monolayers: implications for iron excretion during chelation therapy

“…While the variation is impacted upon by the lipophilicities of chelators (compare log D values of the more highly permeable CP363, deferiprone, and deferasirox with those of CP502 and DFO in Table 1), other determining factors may also be present. The bidirectional transepithelial transport of chelators and chelates was investigated with MDCK cell monolayers in a Transwell system, as this has proven to be a useful in vitro model of defined aspects of tubular transport [44,45]. Chelators were used at a concentration of 500 lM.…”

Gitelman’s syndrome: towards genotype-phenotype correlations?

“…It must be noted that expression studies using stably transfected Madin-Darby canine kidney (MDCK) cells, which express predominantly the complex glycosylated NCCT, may be an alternative to investigate the regulation of the apical trafficking of the transporter. However, the latter system appears to be limited by the relatively low expression levels obtained (the increase over background is below 25%) [27].…”