Functional Expression of the Human Thiazide-Sensitive NaCl Cotransporter in Madin-Darby Canine Kidney Cells
Joke C. de Jong*,
Peter H.G.M. Willems,
Lambertus P.W.J. van den Heuvel,
Nine V.A.M. Knoers and
René J.M. Bindels*
Departments of *Physiology, Biochemistry, Pediatrics, and Human Genetics, University Medical Centre Nijmegen, Nijmegen, The Netherlands.
Correspondence to Dr. René J.M. Bindels, 160 Cell Physiology, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, Netherlands. Phone: +31-24-3614211; Fax: +31-24-3616413;
ABSTRACT. The thiazide-sensitive Na+-Cl- cotransporter (NCC),which is expressed on the apical membrane of epithelial cellslining the distal convoluted tubule, is responsible for thereabsorption of 5% to 10% of filtered Na+ and Cl-. To date,functional studies on the structural and regulatory requirementsfor localized trafficking and ion-transporting activity of NCChave been hampered by lack of a suitable cell system expressingthis cotransporter. Reported here is the functional expressionof human NCC (hNCC) in a polarized mammalian cell of renal origin—thatis, the high-resistance Madin-Darby canine kidney (MDCK) cell.Western blot testing revealed that the cells predominantly expressedthe complex glycosylated (approximately 140 kD) form of hNCC.hNCC was present primarily in the apical part of the cell. Thefunctionality of hNCC was demonstrated by the gain of thiazide-sensitiveNa+ uptake and transepithelial transport activity. Na+ uptakewas significantly increased after short-term (15 min) treatmentwith forskolin, whereas cyclic guanosine monophosphate, wortmannin,phorbol 12-myriatate 13-acetate, and staurosporine were withouteffect. This indicates that hNCC activity is regulated throughcyclic adenosine monophosphate, rather than via cyclic guanosinemonophosphate, phospho-inositide 3-kinases or protein kinaseC. Aldosterone did not alter Na+ uptake in the short term (15min) but significantly increased the transport activity in thelong term (16 h). The latter effect of aldosterone was due toan effect on the cytomegalovirus promoter/enhancer driving theexpression of hNCC. hNCC-MDCK cells are a good model for thestudy of the regulation of apical trafficking and ion-transportingactivity of hNCC. E-mail r.bindels@ncmls.kun.nl
The mammalian thiazide-sensitive Na+-Cl+ cotransporter (NCC)is expressed in the distal convoluted tubule (DCT), where itis responsible for the reabsorption of 5% to 10% of filteredNa+ and Cl- (1). NCC is the site of action of the thiazide diuretics,which are of particular therapeutic relevance for the treatmentof hypertension (2,3). These antihypertensive drugs act by inhibitingNCC transport activity, thus promoting the excretion of Na+and Cl-. Loss-of-function mutations in NCC have been shown tocause Gitelman syndrome (OMIM 263800), a disease characterizedby salt wasting, hypokalemic metabolic alkalosis, hypomagnesemia,and hypocalciuria (4). Mutations include missense, frameshift,nonsense, and splice-site mutations. Gitelman syndrome showsconsiderable phenotypic variability. Thus far, no correlationbetween specific mutations and particular phenotypes has beenreported.
NCC belongs to the family of electroneutral cation-chloridecotransporters (CCC) (5,6), which also comprises two Na+-K+-2Cl-cotransporters (NKCC1 and NKCC2) (7,8) and at least four K+-Cl-cotransporters (KCC1 to 4) (9). Genes encoding the various CCCshow substantial sequence homology and hydropathy analysis revealeda common predicted topology of 12 membrane-spanning domains,a large extracellular loop with potential N-linked glycosylationsites, and amino and carboxy termini located within the cytoplasm(10). The CCC perform a wide variety of physiologic functions,including cellular volume regulation, vectorial movement ofCl-, and cations across epithelia and control of the extra-and intracellular ionic environments of neurons. This versatilityin function reveals itself differences in patterns of tissueexpression and cellular localization (11). NCC is present onthe apical membranes of DCT (12), whereas NKCC2 is apicallyexpressed in the thick ascending limb of the loop of Henle (13).
Whole-animal studies have implicated the mineralocorticoid hormonealdosterone and the sex hormone estradiol in the regulationof NCC expression. Evidence for a regulatory role of aldosteronecomes from several observations. Thus, dietary NaCl restriction,which is known to potently stimulate aldosterone secretion,markedly increased thiazide-sensitive Na+ reabsorption by theDCT (14). Furthermore, treatment with aldosterone significantlyincreased the number of thiazide-binding sites in renal corticalmembrane (15,16). Finally, NCC protein expression was stronglyupregulated in rats treated with aldosterone for 10 d (17).A role of sex hormones in the regulation of NCC expression wassuggested by the observation that the thiazide receptor densitywas twofold higher in female rats than in male rats (18). Morerecently, it was demonstrated that ovariectomy decreased NCCexpression and that estradiol replacement fully restored thiseffect (19).
In addition to the whole-animal studies discussed above, regulationof NCC function and expression was also studied in Xenopus laevisoocytes. By use of this expression system, it was found thatneither cyclic adenosine monophosphate (cAMP) nor cyclic guanosinemonophosphate (cGMP) affected Na+ uptake mediated by flounderNCC (11). On the other hand, the phorbol ester phorbol 12-myriatate13-acetate (PMA), which is a potent activator of protein kinaseC (PKC), markedly decreased Na+ uptake by oocytes expressingeither flounder (20) or rat (21) NCC. It was not investigated,however, whether PKC decreased the activity or the plasma membraneexpression of NCC. Although these studies provide importantnew insights into the regulation of NCC expression and function,a major drawback of the oocyte expression system is that itlacks the physiologic background and polarized structure ofthe native epithelial kidney cell. This urged us to stably expresshuman NCC (hNCC) in polarized Madin-Darby canine kidney (MDCK)cells.
Thus far, NKCC1 is the only one of the CCC that has been studiedin a polarized cell. To this end, human NKCC1 was stably expressedin the low K+-resistant mutant MDCK cell line LK-C1 (22). Regulationof human NKCC1 proved to be similar to that in the native secretorycell, as it was activated by cell shrinkage, calyculin A, andlow Cl- (23). The data presented here show that we successfullygenerated a MDCK cell line stably expressing hNCC at the apicalmembrane. The cells were used to assess the involvement of severalsignaling pathways in the regulation of membrane localizationand transport activity of this Na+-Cl- cotransporter.
Contructs
To obtain DNA encoding hNCC, the oocyte-expression vector pT7Ts-NCCwas digested with BamHI and SpeI. The generation of the pT7Ts-hNCCvector has been described previously (24). Subsequently, theBamHI/SpeI fragment was ligated into the BglII/XbaI sites ofthe mammalian vector pCB6. Transcription of hNCC is driven bythe cytomegalovirus promoter.
Culturing, Transfection, and Selection of Clones
High-resistance MDCK (MDCK-HRS) and MDCK-AQP2-EK were grownin DMEM supplemented with 5% (vol/vol) FCS at 37°C in 5%CO2. The MDCK-AQP2-EK cell line was provided by PM Deen, CellPhysiology, University Medical Centre Nijmegen. MDCK-HRS cellswere transfected with 25 to 30 µg of circular pCB6-hNCCby means of calcium-phosphate precipitation technique as describedin detail elsewhere (25). Twenty-four hours after transfection,the cells were trypsinized, divided over 6 to 8 petri dishesof 57 cm2, and cultured in DMEM containing 800 µg/ml G418(Life Technologies Europe, Breda, The Netherlands). Ten to 14d after transfection, individual colonies were selected by meansof cloning rings and expanded. After eight passages after selectionof a clone, G418 was omitted from the medium.
Immunoblotting
The cells were grown to confluence in a humidified atmosphereof 95% air, 5% CO2 at 37°C, and lysed in 200 µl Laemmlibuffer containing 50 mM Tris/HCl (pH 6.8), 2% (w/v) SDS, 12%(vol/vol) glycerol, 0.01% (w/v) bromophenol blue, and 25 mMdithiothreitol (DTT) for 30 min. Proteins were separated ona 6% (w/v) SDS-polyacrylamide gel and transferred electrophoreticallyto PVDF membranes (Millipore, Bedford, MA). Blots were incubatedwith either rabbit anti-hNCC antibody A857 (26) or affinity-purifiedrabbit AQP2 (27) diluted 1:10,000 or 1:3000 in PBS buffer supplementedwith 5% (w/v) nonfat dried milk, respectively. Subsequently,blots were incubated with sheep horseradish peroxidase conjugatedto anti-rabbit IgG (Sigma, St. Louis, MO) diluted 1:5000. Finally,proteins were visualized by enhanced chemiluminescence (Pierce,Rockford, IL).
Immunocytochemistry
Cells were seeded at a density of 3 x 105 cells/cm2 on 0.33cm2 filters and grown to confluence for 2 d. Next, the cellswere rinsed with ice-cold PBS and fixed in PBS containing 3%(w/v) paraformaldehyde at room temperature. After three washeswith PBS, the cells were incubated in PBS containing 50 mM NH4Clfor 15 min. Cells were washed with PBS containing 1 mM MgCl2and 0.1 mM CaCl2 and incubated in PBS with 0.1% (w/v) BSA (blockingbuffer) for 20 min. Subsequently, cells were washed in PBS,and filters were cut from their plastic support. Filters wereincubated overnight with 25 to 30 µl anti-hNCC antibodyA857 (26) diluted 1:6000 in PBS containing 0.05% (w/v) saponin.After three washes with PBS, the filters were incubated with30 µl goat anti-rabbit IgG Alexa 488 conjugate (MolecularProbes, Eugene, OR) diluted 1:250 in PBS for 30 min in the dark.Finally, filters were washed another three times with PBS andmounted on slides in Vectashield (Vector Laboratories, Burlingame,CA). Horizontal and vertical images were obtained with a Bio-RadMRC-100 laser scanning confocal imaging system (Bio-Rad, Hercules,CA).
22Na+ Uptake
Cells were seeded in 24-well plates at a density of 3 x 105cells/cm2 and grown for 2 d. On the second day, cells were transferredto serum-free medium, followed by an incubation in Cl--freeKrebs-Henseleit bicarbonate (KHB) medium containing 5 mM Hepes/Tris(pH 7.4), 96 mM sodium gluconate, 2 mM potassium gluconate,1.8 mM calcium gluconate, 1 mM Mg(NO3)2, 2.5 mM sodium pyruvate,and 5 mg/dl gentamycin for 1 to 3 h. Next, Cl--depleted cellswere transferred to 500 µl uptake medium containing 20mM Hepes/Tris (pH 7.4), 72 mM N-methyl-D-glucosamine–HCl,48 mM NaCl, 5 mM KCl, 2 mM Na2H2PO4, 1 mM CaCl2, 1 mM MgSO4,0.5 mM ouabain, 100 µM amiloride, 100 µM bumetanide,and 1 µCi per milliliter of 22Na+ and incubated for 15min at room temperature with or without 100 µM hydrochlorothiazide(HCT). Ouabain was added to prevent Na+ exit via the Na+-K+-ATPase,bumetanide to inhibit the Na+-K+-2Cl- cotransporter, amilorideto block the Na+-H+ antiporter and epithelial Na+ channels,and HCT to inhibit NCC. The uptake reaction was stopped after15 min by washing the cells 4 times with ice-cold uptake medium.The cells were lysed in 500 µl of 0.1% (w/v) SDS, andradioactivity was counted in a liquid scintillation counter.
22Na+ Transport
Cells were seeded on 0.33 cm2 filters (3 x 105 cells/cm2) andgrown for 2 d to form a confluent monolayer. Monolayers werewashed with KHB medium containing 110 mM NaCl, 5 mM KCl, 2 mMNaH2PO4, 1.2 mM MgSO4, 1 mM CaCl2, 10 mM sodium acetate, 1 mML-lactate, 10 mM D-glucose, 4 mM L-alanine, and 20 mM Hepes/Tris(pH 7.4). Next, 100 µl KHB medium containing 100 µMamiloride, 100 µM bumetanide, and 0.5 µCi/ml 22Na+was added at the apical side of the filter. Where indicated,100 µM HCT was included. At the basolateral side of thecells, 600 µl KHB medium containing 100 µM amilorideand 100 µM bumetanide was added. Na+ transport form theapical to the basolateral side was measured by sampling (10µl) from the basolateral side. Radioactivity was countedin a liquid scintillation counter.
Statistical Analyses
Values are expressed as mean ± SEM. Statistical significancewas determined by t test. Differences in mean with P < 0.05were considered statistically significant.
Generation of a MDCK Cell Line Stably Expressing hNCC
To generate a polarized renal epithelial cell line stably expressinghNCC, MDCK cells were transfected with pCB6-hNCC by means ofthe calcium phosphate precipitation procedure. Transfectionwas performed in the absence and presence of the specific inhibitorof NCC, hydrochlorothiazide (1 µM HCT). The inhibitorwas added to protect the cells from possible deleterious effectsas a result of increased Na+ uptake. Transcription of hNCC isdriven by a cytomegalovirus promoter. G418-resistant colonieswere selected at 10 to 14 d after transfection and tested forhNCC expression by Western blot analysis of total cell lysate.Figure 1 shows seven G418-resistant colonies of MDCK cells,four of which express hNCC. Colonies 1 and 2 were cultured inthe absence of HCT, whereas colonies 6 and 7 were cultured inthe continuous presence of the inhibitor. Cell viability andgrowth were not different between cells cultured in the absenceor presence of HCT (data not shown).
Figure 1. MDCK cell lines stably expressing human Na+-Cl+ cotransporter. High-resistance MDCK cells were transfected with pCB6-hNCC and cultured in the presence of G418 and the absence (clones 1 to 5) or presence (clones 6 and 7) of 1 µM hydrochlorothiazide (HCT). After 10 to 14 d, G418-resistant colonies were selected, and total cell lysates were subjected to Western blot testing with rabbit anti-hNCC antibody A857. Clones 1, 2, 6, and 7 display a major band of approximately 120 to 140 kD, whereas clones 2 and 7 show an additional minor band of approximately 110 kD. No positive signal was observed with nontransfected cells (nt).
Glycosylation State of hNCC Stably Expressed in MDCK Cells Figure 1 shows a broad band of high molecular weight (approximately120 to 140 kD) and a single band (2 and 7) of lower molecularweight (approximately 110 kD). To assess the glycosylation stateof the NCC comprising these bands, total cell lysate of clone2 was incubated with either endoglycosidase H (EndoH) or N-glycosidaseF (PGNaseF). The broad band of approximately 120 to 140 kD completelydisappeared after treatment with PGNaseF, leaving a nonglycosylatedprotein of approximately 100 kD (Figure 2). On the other hand,treatment with EndoH did not affect this broad band. This indicatesthat hNCC stably expressed in MDCK cells is predominantly complexglycosylated. Lane 4 shows that EndoH treatment resulted inthe disappearance of the approximately 110 kD band and the appearanceof an approximately 100-kD band. This suggests that the smallband of approximately 110 kD reflects the high-mannose glycosylatedform of the transporter. The apparent molecular sizes of theNCC expressed in MDCK cells are in good agreement with thosereported in oocytes and rat kidney (28,29). Because with increasingpassage number the ratio between high mannose and complex glycosylatedhNCC changed in favor of the high mannose form, we only usedlow passage numbers in this study.
Figure 2. Glycosylation pattern of human Na+-Cl- cotransporter (hNCC) stably expressed in MDCK cells. Total cell lysate of hNCC-MDCK cells (Figure 1, clone 2) was subjected to digestion with either endoglycosidase H (EndoH) or N-glycosidase F (PGNaseF). PGNaseF completely removed the major band of approximately 120 to 140 kD, whereas it caused the appearance of a band of approximately 100 to 110 kD. In contrast, EndoH removed the small band of approximately 110 kD and caused the formation of band of approximately 100 kD. EndoH did not affect the predominant approximately 120 to 140 kD form of hNCC. nt indicates nontransfected cells.
Plasma Membrane Localization of hNCC Stably Expressed in MDCK Cells
For immunolocalization studies, cells were grown on permeablesupports until they formed a confluent monolayer. After mildpermeabilization and fixation, cells were stained with anti-hNCCantibody A857 (26). Figure 3 depicts monolayers of untransfectedMDCK cells (A) and cells of clone 2 expressing hNCC (B). Thefigure shows that expression of hNCC is clearly heterogeneous.The Z-scan shows a predominant expression to the apical domainof the cell.
Figure 3. Localization of human Na+-Cl- cotransporter (hNCC) stably expressed in MDCK cells. Nontransfected MDCK cells (A) and hNCC-MDCK cells (B; Figure 1, clone 2) were grown to confluence on permeable supports. Immunocytochemistry was performed with rabbit anti-hNCC antibody A857 as a primary antibody and goat anti-rabbit IgG Alexa 488 conjugate as the secondary antibody. XY (A and B, top) and XZ (B, bottom) images were obtained by confocal laser scanning microscopy. Nontranfected monolayers show background staining only (A), whereas hNCC-MDCK monolayers show a heterogeneous expression pattern (B, top). The cross section reveals that hNCC is largely expressed in the apical part of the cell (B, bottom).
Thiazide-Sensitive Na+ Uptake in hNCC-MDCK Cells
The functionality of NCC stably expressed in MDCK cells wasdetermined by measuring thiazide-sensitive 22Na+ uptake. Figure 4Ashows that Na+ uptake was significantly increased in cellsexpressing hNCC as compared with the corresponding untransfectedcells (P < 0.05). Importantly, HCT (1 µM) reduced Na+uptake in hNCC-MDCK cells to the level observed in untransfectedcells. Similarly, hNCC-MDCK cells cultured on permeable supportsexhibited net apical to basolateral Na+ transport that was inhibitedby thiazides (Figure 4B). These data demonstrate that the hNCCis functionally expressed at the cell surface. UntransfectedMDCK cells displayed a relatively high Na+ uptake (Figure 4A).However, the presence of bumetanide and amiloride in the mediumexcluded the involvement of the Na+-K+-2Cl- cotransporter, theNa+-H+ antiporter, and epithelial Na+ channels. At present,we have no explanation for the relatively high Na+ uptake valuein untransfected MDCK cells.
Figure 4. Thiazide-sensitive Na+ uptake in human Na+-Cl- cotransporter (hNCC)–MDCK cells. (A) Nontransfected MDCK cells and MDCK cells stably expressing hNCC (Figure 1, clone 2) were cultured to subconfluence in 24-well plates. 22Na+ uptake was measured in the absence and presence of 100 µM hydrochlorothiazide (HCT). The uptake values were corrected for the number of cells present in the well. 22Na+ uptake was significantly (*P < 0.05) increased in hNCC cells, and this increase was completely blocked by the inhibitor. The values presented are the mean ± SEM of 20 wells. (B) MDCK cells stably transfected with hNCC (clone 2) were cultured to confluence on 0.33-cm (2) filters. Net apical to basolateral 22Na+ transport was measured in the absence (control) and presence of 1 µM HCT as a function of time (mean of two filters).
Thiazide-Sensitive Na+ Uptake in hNCC-MDCK Cells is Increased with Forskolin
To determine possible effects of aldosterone, phospho-inositide3-kinases (PI3Ks) or protein kinases A (PKA), G (PKG), and C(PKC) on thiazide-sensitive Na+ uptake, hNCC-MDCK cells wereincubated in Cl--free medium containing aldosterone (1 µM,15 min), wortmannin (100 nM, 1 h), forskolin (30 µM, 30min), cGMP (100 µM, 45 min), and either PMA (1 µM,15 min) or staurosporine (1 µM, 15 min), respectively.Figure 5 demonstrates that only forskolin evoked a significantchange in Na+ uptake. At the concentration used, forskolin readilyincreases cAMP, leading to rapid activation of PKA (30). NCCactivity was not altered by short-term stimulation with aldosteroneor short-term activation of PKG or PKC (PMA). Neither was italtered by short-term inhibition of PI3K (wortmannin) or PKC(staurosporine).
Figure 5. Short-term regulation of thiazide-sensitive Na+ uptake in human Na+-Cl- cotransporter (hNCC)-MDCK cells. MDCK cells stably expressing hNCC (Figure 1, clone 2) were cultured to subconfluence in 24-well plates. 22Na+ uptake was measured in the absence and presence of 100 µM hydrochlorothiazide (HCT), and uptake values were corrected for the number of cells present in the well. During Cl- depletion, cells were pretreated with aldosterone (1 µM, 15 min), phorbol 12-myriatate 13-acetate (PMA) (1 µM, 15 min), cyclic guanosine monophosphate (cGMP) (100 µM, 45 min), staurosporine (1 µM, 15 min), wortmannin (100 nM, 1 h), and forskolin (30 µM, 30 min). For each condition, the uptake value obtained in the presence of HCT (basal uptake) was set at 100%, to which the corresponding value obtained in the absence of inhibitor was related. Depicted is the percentage increase. Thiazide-sensitive 22Na+ uptake was significantly (P < 0.05) higher in cells pretreated with forskolin. Except for forskolin (six filters), the values presented are the mean ± SEM of three filters. *Significantly different from hNCC-MDCK (control).
Expression of hNCC Is Increased on Long-Term Treatment with PMA and Aldosterone
Long-term (16 h) treatment with 1 µM aldosterone markedlyincreased hNCC expression (Figure 6A, upper panel, lane 3).The figure shows that aldosterone preferentially upregulatedthe complex glycosylated species of hNCC (approximately 120to 140 kD). In contrast, long-term PMA treatment dramaticallyincreased the expression of both complex and high mannose glycosylatedNCC (data not shown). To visualize the effects of aldosteroneand PMA on the same blot, significantly less protein was loadedin lane 4 of the gel. Remarkably, this lane shows that PMA predominantlyincreased expression of the high mannose glycosylated form ofhNCC (approximately 110 kD). MDCK cells stably expressing aquaporin2 (AQP2) were used to investigate the specificity of the effectsof aldosterone and PMA. Figure 6A (bottom panel, lanes 3 and4) shows that both stimuli increased AQP2 expression to thesame extent. Note that in this case, equal amounts of proteinwere loaded onto the gel.
Figure 6. Long-term effects of aldosterone and PMA on human Na+-Cl- cotransporter (hNCC) expression and thiazide-sensitive Na+ uptake in hNCC-MDCK cells. hNCC-MDCK cells (Figure 1, clone 2) and aquahorin 2 (AQP2)–MDCK cells were transferred to 24-well plates and cultured in serum-free medium containing either 1 µM aldosterone or 1 µM PMA for 16 h. Control cells were cultured in serum-free medium without additions. At the end of the culturing period, cells were used for either Western blot testing (A) or 22Na+ uptake measurement (B). The Western blot results show that aldosterone increased the expression the approximately 120- to 140-kD and approximately 110-kD form of hNCC to the same extent (A, top). The blot furthermore shows that PMA predominantly increased the expression of the approximately 110-kD form of hNCC. It is of note that in the case of PMA, three times less protein was loaded onto the gel. The lower blot shows that aldosterone and PMA increased the expression of AQP2 to the same extent. 22Na+ uptake measurements were performed as described in Figure 5, and thiazide-sensitive uptake is expressed as percentage of basal uptake (B). The figure shows that 22Na+ uptake is significantly (P < 0.05) increased by aldosterone and virtually abolished by PMA. The values presented are the mean ± SEM of 12 filters. *Significantly different from hNCC-MDCK (control). nt indicates nontransfected cells; NCC, Na+-Cl- cotransporter.
Na+ Uptake in hNCC-MDCK Cells Is Increased on Long-Term Incubation with Aldosterone but Not PMA
To determine possible effects of aldosterone on thiazide-sensitiveNa+ uptake, MDCK cells stably expressing hNCC were incubatedin serum-free medium containing aldosterone for 16 h. Figure 6Bshows that this treatment resulted in a significant (P <0.05) increase in Na+ uptake. In contrast, long-term treatmentwith PMA dramatically reduced thiazide-sensitive Na+ uptake.
Here, we describe for the first time the generation of a high-resistanceMDCK cell line stably expressing the human thiazide-sensitiveNa+-Cl- cotransporter. NCC was largely present in the complexglycosylated form and localized to the apical domain of theMDCK cells. The cells exhibited a significant thiazide-sensitiveNa+ uptake, which was increased on short-term treatment withforskolin. Moreover, hNCC-MDCK cells cultured on permeable supportsdisplayed a thiazide-sensitive transepithelial Na+ transportfrom the apical to basolateral compartment. Together, thesedata show that hNCC-MDCK cells are a good model to study thestructural requirements and regulatory aspects of localizedtrafficking and ion-transporting activity of this cotransporter.
Thus far, studies on the molecular mechanisms underlying theapical sorting process and the regulation of the ion-transportactivity of hNCC have been hampered by the lack of an appropriatemammalian expression system. In the past years, many studieshave used polarized MDCK cells to functionally express a widevariety of transporter proteins (25,31–33). Here, we reportthe successful expression of hNCC in this polarized mammaliancell of renal origin. Both low- and high-expressing hNCC-MDCKcell lines exhibited normal growth rates, showing that overexpressionof the cotransporter did not affect cell viability. In agreementwith this, hNCC-MDCK cells were found to grow equally well inthe absence or presence of hydrochlorothiazide. Immunocytochemicalanalysis revealed that hNCC was present in the apical domainof the cell, with virtually no staining at the basolateral side.This is in agreement with the observation that in rat, NCC islocalized predominantly on apical microvilli of cells liningthe DCT (12). The observed increase in thiazide-inhibitableNa+ uptake and transepithelial transport indicates that at leastpart of the cotransporter is functional and therefore presentin the apical membrane. The same observation was reached witha number of other transporters with apical expression in theirnative cells, including the excitatory amino acid transporter3 (32), AQP2 (25), and glycine transporter 2 (33).
Western blot analysis revealed that hNCC was present in boththe high mannose (approximately 110 kD) and complex glycosylated(approximately 120 to 140 kD) form, whereas the unglycosylatedprotein, obtained after PGNaseF or EndoH treatment, had an apparentmolecular size of approximately 100 kD. By use of the Xenopuslaevis expression system, we (24) and others (29) have previouslyshown that complex glycosylation is a prerequisite for functionaltargeting to the plasma membrane. Here, we show that MDCK cellspredominantly express the approximately 120- to 140-kD formof the hNCC, with only very little expression of the 110-kDprotein. Importantly, the same profile has been reported formouse kidney cortical membranes (28). The glycosylation patternwas not altered in cells in which the expression of hNCC wasmoderately increased after long-term stimulation with aldosterone.On the other hand, it was completely reversed when hNCC expressionwas dramatically increased after chronic treatment with PMA.This suggests that the cellular sorting machinery becomes obstructedat high protein production rates. Alternatively, long-term PMAtreatment may lead to inhibition at some step of the sortingprocess. In contrast to hNCC-MDCK cells, Xenopus laevis oocytespredominantly express the approximately 110-kD protein (24,28).This glycosylation pattern is not likely to be due to excessivehNCC production because AQP2, when expressed at similar levels,was hardly detected in the high mannose glycosylated form (34).Therefore, this finding rather suggests that Xenopus laevisoocytes lack a chaperone type of protein necessary for properprocessing of hNCC. In agreement with this, recent coexpressionstudies that used Xenopus laevis oocytes showed that the glucose-regulatedprotein 58 (grp58), a chaperone with thiol-dependent reductaseactivity, associates with NCC and increases NCC-mediated Na+uptake (35). The present demonstration of a "native" glycosylationpattern in hNCC-MDCK cells suggests that these cells of distal/collectingtubule origin do possess such a chaperone. Therefore, this modelmay be of use in developing strategies for improvement of apicalsorting of (partially) retained Gitelman syndrome mutants.
To date, little has been learned about the regulation of hNCC.Here, we report that hNCC-mediated Na+ uptake is stimulatedby forskolin, a drug that promotes the adenylyl cyclase–catalyzedproduction of cAMP. In view of the rapidity of the response,the effect of cAMP, acting through PKA, is not likely to bedue to increased hNCC expression. Rather, it reflects activationof cotransporters already present on the cell surface and/ortheir increased shuttling between intracellular vesicles andthe apical plasma membrane. In this context, forskolin-inducedredistribution from intracellular vesicles to the apical membranehas been demonstrated in MDCK-HRS cells stably expressing AQP2water channels (25). Of all species studied thus far, only hNCCcontains a potential phosphorylation site for PKA (36). Thisexplains the lack of effect of cAMP on thiazide-sensitive Na+uptake in Xenopus laevis oocytes expressing flounder NCC (20)and suggests that this site is important in cAMP-dependent activationof hNCC-mediated Na+ transport. At present, it is unclear whetherand which hormones or paracrine factors act through cAMP toregulate hNCC in DCT. In rat, DCT expresses prostaglandin EP4(37) and vasopressin V2 (38) receptors. Moreover, immortalizedmouse DCT cells accumulate Na+ in response to -adrenergic receptoragonists (39). Finally, microdissected distal tubules from rabbitdisplay cAMP rises in response to calcitonin and vasoactiveintestinal peptide in DCT and to parathyroid hormone and isoprenalinein connecting tubule (CNT) (40).
In the long term, aldosterone caused a significant increasein thiazide-sensitive Na+ uptake. This was accompanied by increasedexpression of both the high mannose and complex glycosylatedform of the transporter, but without changing their relativeabundances. Because expression of hNCC is driven by a cytomegaloviruspromoter/enhancer, this effect of aldosterone is not likelyto be specific. This was confirmed by the observation that aldosteronesignificantly increased AQP2 expression driven by the same cytomegaloviruspromoter/enhancer in MDCK-HRS cells stably expressing this waterchannel.
Short-term treatment with either PMA or staurosporine did notalter thiazide-sensitive Na+ uptake in hNCC-MDCK cells. Thisshows that PKC does not act directly or indirectly on hNCC activityand/or cell surface expression in these cells. In contrast,PMA was found to markedly decrease Na+ uptake in Xenopus laevisoocytes expressing either flounder (20) or rat (21) NCC. Thissuggests that PKC affects NCC activity and/or trafficking differentiallyin polarized MDCK cells and oocytes. It should be noted thatall NCC sequenced thus far contain putative PKC consensus sites(36). The lack of effect of cGMP and wortmannin indicates thatneither PKG nor PI3K play a role in hNCC-mediated Na+ uptake.Recently, the serine-threonine kinase WNK4 was shown to associatewith NCC and to reduce NCC membrane expression and NCC-mediatedNa+ uptake after coexpression in Xenopus laevis oocytes (41).Taken together, these findings are compatible with a model inwhich PKA directly or indirectly activates hNCC and/or increasesits membrane expression, whereas WNK4 works in the oppositedirection.
Acknowledgments
We thank Dr. Mark Knepper for providing hNCC antibody A857 andIrene Konings for technical assistance with the transfectionof the MDCK cells.
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Received for publication April 29, 2003.
Accepted for publication July 19, 2003.
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Abstract
Introduction
-adrenergic receptor agonists (39). Finally, microdissected distal tubules from rabbit display cAMP rises in response to calcitonin and vasoactive intestinal peptide in DCT and to parathyroid hormone and isoprenaline in connecting tubule (CNT) (40). 
