Localization of the thiazide sensitive Na-Cl cotransporter, rTSC1, in the rat kidney

Plotkin, Matthew; Kaplan, Mark; Verlander, Jill W.; Lee, Wen Sen; Brown, Dennis; Poch, Esteban; Gullans, Steven R.; Hebert, Steven C.

doi:10.1038/ki.1996.300

Cited by 174 publications

(139 citation statements)

References 7 publications

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“…Immunocytochemical analysis revealed that hNCC was present in the apical domain of the cell, with virtually no staining at the basolateral side. This is in agreement with the observation that in rat, NCC is localized predominantly on apical microvilli of cells lining the DCT (12). The observed increase in thiazide-inhibitable Na ϩ uptake and transepithelial transport indicates that at least part of the cotransporter is functional and therefore present in the apical membrane.…”

Section: Discussionsupporting

confidence: 79%

“…This versatility in function reveals itself differences in patterns of tissue expression and cellular localization (11). NCC is present on the apical membranes of DCT (12), whereas NKCC2 is apically expressed in the thick ascending limb of the loop of Henle (13). Whole-animal studies have implicated the mineralocorticoid hormone aldosterone and the sex hormone estradiol in the regulation of NCC expression.…”

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confidence: 99%

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

Jong¹,

Willems²,

Heuvel³

et al. 2003

Journal of the American Society of Nephrology

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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.

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Section: Discussionsupporting

confidence: 79%

mentioning

confidence: 99%

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

Jong¹,

Willems²,

Heuvel³

et al. 2003

Journal of the American Society of Nephrology

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“…7 The two-dimensional structure of NCC is predicted to contain 12 transmembrane segments (S) with large intracellular amino (N)-and carboxy (C)-terminal regions, and a large hydrophilic extracellular loop between S7 and S8. 8 NCC localizes to the luminal membrane of the DCT segment of the nephron, 9 where it facilitates the cotransport of Na + and Cl À from the pro-urine to the intracellular compartment.…”

Section: Introductionmentioning

confidence: 99%

Novel NCC mutants and functional analysis in a new cohort of patients with Gitelman syndrome

et al. 2011

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Gitelman syndrome (GS) is an autosomal recessive disorder characterized by hypokalemic metabolic alkalosis in conjunction with significant hypomagnesemia and hypocalciuria. The GS phenotype is caused by mutations in the solute carrier family 12, member 3 (SLC12A3) gene that encodes the thiazide-sensitive NaCl cotransporter (NCC). We analyzed DNA samples of 163 patients with a clinical suspicion of GS by direct sequencing of all 26 exons of the SLC12A3 gene. In total, 114 different mutations were identified, 31 of which have not been reported before. These novel variants include 3 deletions, 18 missense, 6 splice site and 4 nonsense mutations. We selected seven missense mutations to investigate their effect on NCC activity and plasma membrane localization by using the Xenopus laevis oocyte expression system. The Thr392Ile mutant did not display transport activity (probably class 2 mutation), while the Asn442Ser and Gln1030Arg NCC mutants showed decreased plasma membrane localization and consequently function, likely due to impaired trafficking (class 3 mutation). Even though the NaCl uptake was hampered for NCC mutants Glu121Asp, Pro751Leu, Ser475Cys and Tyr489His, the transporters reached the plasma membrane (class 4 mutation), suggesting an effect on NCC regulation or ion affinity. The present study shows the identification of 38 novel mutations in the SLC12A3 gene and provides insight into the mechanisms that regulate NCC.

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“…This cotransporter mediates the apical reabsorption of Na ϩ with Cl Ϫ and is expressed predominantly in the distal convoluted tubule (9,10). Consequently, the expression of WNK4 and NCCT overlap in epithelial cells of the distal nephron.…”

mentioning

confidence: 99%

Molecular pathogenesis of inherited hypertension with hyperkalemia: The Na–Cl cotransporter is inhibited by wild-type but not mutant WNK4

Wilson

Kahle²,

Sabath³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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377

351

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Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl ؊ dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a ''mirror image'' of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCTmediated Na ؉ influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ( 22 Nainflux reduced 50%, P < 1 ؋ 10 ؊9 , surface expression reduced 75%, P < 1 ؋ 10 ؊14 in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.protein serine-threonine kinases ͉ hypertension ͉ thiazide-sensitive Na-Cl cotransporter ͉ ion transport ͉ medical genetics H ypertension is the most common disease in industrialized societies, affecting Ͼ20% of the adult population and contributing to morbidity and mortality from stroke, myocardial infarction, renal failure, and congestive heart failure (1). Its pathogenesis is largely unknown, resulting in empiric pharmacologic therapy. In recent years, genetic approaches investigating rare Mendelian forms of high and low blood pressure have provided fundamental insight into mechanisms that contribute to blood pressure variation (2). These have demonstrated the causal role of inherited variation in renal salt homeostasis in blood pressure variation, with mutations in many genes known to play a role in mediating or regulating renal salt reabsorption resulting in altered blood pressure.Pseudohypoaldosteronism type II (PHAII; Online Mendelian Inheritance in Man database no. 145260) is an autosomal dominant disease featuring hypertension with hyperkalemia despite normal glomerular filtration rate; renal tubular acidosis is a variable associated finding. The clinical features of this disease are chloride dependent and are also corrected with thiazide diuretics, specific a...

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Localization of the thiazide sensitive Na-Cl cotransporter, rTSC1, in the rat kidney

Cited by 174 publications

References 7 publications

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

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

Novel NCC mutants and functional analysis in a new cohort of patients with Gitelman syndrome

Molecular pathogenesis of inherited hypertension with hyperkalemia: The Na–Cl cotransporter is inhibited by wild-type but not mutant WNK4

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