Effects of chemical chaperones on partially retarded NaCl cotransporter mutants associated with Gitelman's syndrome in a mouse cortical collecting duct cell line
Joke C. de Jong1,
Peter H. G. M. Willems2,
Monique Goossens1,
Alain Vandewalle5,
Lambertus P. W. J. van den Heuvel3,
Nine V. A. M. Knoers4 and
René J. M. Bindels1
1Department of Physiology, 2Department of Biochemistry, 3Department of Pediatrics and 4Department of Human Genetics, University Medical Centre Nijmegen, Nijmegen, The Netherlands and 5INSERM U478, Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, Paris, France
Correspondence and offprint requests to: René J. M. Bindels, 160 Cell Physiology, University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Email: r.bindels{at}ncmls.kun.nl
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Abstract
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Background. Epithelial cells lining the distal convoluted tubule express the thiazide-sensitive NaCl cotransporter (NCC) that is responsible for the reabsorption of 510% of the filtered load of Na+ and Cl. Mutations in NCC cause the autosomal recessive renal disorder Gitelman's syndrome (GS). GS mutations give rise to mutant transporters that are either fully (class I) or partially (class II) retarded. Recent evidence indicates that class II mutations do not alter the intrinsic transport activity of NCC. These findings suggest that in GS caused by class II NCC mutations, pharmacological chaperones may be useful in treatment.
Methods. Initial attempts using 4-phenylbutyrate and glycerol to increase Na+ uptake in Xenopus laevis oocytes expressing the class II mutant L215P were unsuccessful. To study the effect of the chaperones in a more physiological setting, we next expressed hNCC in the polarized epithelial cell line of distal tubular origin, mpkCCD.
Results. mpkCCD cells readily expressed the class II mutant R955Q, but not the class I mutant G741R. Wild-type hNCC was predominantly present in the
1201403 kD complex glycosylated form. In contrast, the R955Q mutant was predominantly present in a lower molecular weight form of
100 kD. Pretreatment of R955Q expressing cells with 4-phenylbutyrate (5 mM, 16 h), but not thapsigargin (1 µM, 90 min), dimethyl sulfoxide (1%, 16 h) or glycerol (4%, 16 h), increased the expression of the complex glycosylated form and in parallel the number of hNCC positive cells.
Conclusions. Taken together, the data indicate that 4-phenylbutyrate is a promising candidate for rescuing partially retarded but otherwise functional class II GS mutants.
Keywords: mpk-CCD; NCC; thiazide
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Introduction
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The thiazide-sensitive NaCl cotransporter (NCC) is mainly expressed in the distal convoluted tubule (DCT), where it is responsible for the reabsorption of 510% of filtered Na+ and Cl [1]. NCC is the target of the thiazide diuretics, which are of particular therapeutic relevance for the treatment of hypertension [2,3]. These drugs exert their antihypertensive action primarily by inhibiting NCC transport activity, thus promoting the excretion of Na+ and Cl [4]. Loss-of-function mutations in NCC have been shown to cause Gitelman's syndrome (GS; OMIM 263800), an autosomal recessive disease characterized by salt wasting, hypokalaemic metabolic alkalosis, hypomagnesaemia and hypocalciuria [5]. At present, over 100 mutations in NCC have been identified in GS. These mutations are scattered throughout the SLC12A3 gene encoding hNCC and include missense, frameshift, nonsense and splice-site mutations. GS shows a considerable phenotypical variability but thus far no correlation between specific mutations and particular phenotypes has been reported.
In a recent study, we provided evidence for the existence of fully (class I: G439S, T649R, G741R) and partially (class II: L215P, F536L, R955Q, G980R, C985Y) retarded mutant transporters [6]. In a follow-up study, we provided evidence that the structural unit of the NCC is a homodimer [7]. In addition, we showed that a concatameric protein consisting of partially retarded G980R-hNCC and wild-type hNCC displayed normal Na+ transport following expression in Xenopus laevis oocytes. These findings demonstrate that the intrinsic transport activity of class II mutant transporters is unaffected by the mutation and opens the possibility for the development of pharmacological chaperones for therapeutic use. Due to the lack of a suitable mammalian cell system for overexpression of NCC, functional studies on the structural and regulatory requirements for membrane trafficking and ion-transporting activity of hNCC have been performed thus far in X.laevis oocytes [611]. However, a major disadvantage of this expression system is that it does not allow the study of the molecular mechanisms underlying the apical sorting process. Moreover, X.laevis oocytes predominantly express high mannose glycosylated hNCC, whereas we [6] and others [10] have recently shown that complex glycosylation is a prerequisite for functional targeting of this transporter to the plasma membrane. These findings suggest that X.laevis oocytes lack a chaperone type of protein necessary for proper processing of hNCC.
A preliminary study was conducted to look into the feasibility of using chemical chaperones including glycerol [12] and the transcriptional regulator 4-phenylbutyrate [13] to rescue class II mutant transporters. Unfortunately, neither chaperone was able to increase the Na+ transport activity of the partially retarded L215P mutant of hNCC in X.laevis oocytes. This negative result urged us to investigate the possibility of using immortalized epithelial cell lines of distinct tubular origin described by Vandewalle et al. [14]. The data presented demonstrate that the uncloned mpkCCD cells, derived from the cortical collecting duct (CCD) of a transgenic mouse originally described by Duong Van Huyen et al. [15], are a suitable expression system for studying the regulatory aspects of membrane trafficking of the human NCC and its mutants associated with GS.
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Subjects and methods
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Constructs
DNA encoding hNCC was obtained after digestion of the oocyte-expression vector pT7Ts-hNCC, pT7Ts-hNCC-G741R, pT7Ts-hNCC-R955Q and pT7Ts-hNCC-R904Q with BamHI and SpeI. The generation of these vectors has been described in detail previously [6]. Subsequently, the BamHI/SpeI fragment was ligated into the BglII/XbaI sites of the mammalian vector pCB6. Transcription of hNCC is driven by the cytomegalovirus promoter.
Culturing
The DCT cells (mpkDCT cells) were derived from isolated distal tubules microdissected from the kidney of an adult transgenic mouse (SV-PK/Tag) containing Tag under the control of the SV40 enhancer, located upstream from the 1000-BP fragment of the 5' rat L-L'-PK gene regulatory region [16]. Similarly, CCD cells (uncloned mpkCCD cells) were derived from a 1-month-old SV-PK/Tag transgenic mouse [15]. Cells were cultured in defined medium consisting of a 1:1 mixture of DMEM:Ham's F-12 (Invitrogen, Breda, The Netherlands), supplemented with 2% (v/v) heat-inactivated fetal calf serum, 5 mg/ml insulin (Sigma, St Louis, MO), 1 nM dexamethasone (Sigma), 90 nM sodium selenate (Sigma), 5 mg/ml transferrin (Sigma), 10 ng/ml EGF (Sigma), 4.5 mM glutamine (Gibco-BRL, Breda, The Netherlands), 0.2% (w/v) D-glucose (Sigma), 30 mg/ml gentamicin and 20 mM HEPES (Gibco-BRL), equilibrated with 5% CO295% air at 37°C.
Immunocytochemistry
For immunocytochemical analysis, cells were seeded at a density of 3 x 105 cells per cm2 on 0.33 cm2 filters and grown to confluence for 2 days. Cells were transfected with circular pCB6-hNCC using lipofectamine 2000 according the protocol of the manufacturer (Invitrogen). Confluent monolayers were fixed in PBS containing 3% (w/v) paraformaldehyde at room temperature (RT) and subsequently incubated in PBS containing 50 mM NH4Cl for 15 min. The filters were incubated overnight with 2530 ml of either rabbit anti-hNCC antibody A857 [17], affinity-purified rat anti-ECadherin antibody (Sigma), or affinity-purified mouse anti-Caveolin 1 antibody (Sigma), diluted 1:6000, 1:50 or 1:100 in PBS containing 0.05% (w/v) saponin, respectively. After three washes with PBS, the filters were incubated with 30 ml of secondary antibody for 30 min in the dark. The secondary antibodies, affinity-purified goat anti-rabbit IgG AlexaTM 488 conjugate, goat anti-rat IgG AlexaTM 594 conjugate and goat anti-mouse IgG AlexaTM 594 conjugate (Molecular Probes, Eugene, OR), were diluted 1:250 in PBS. Horizontal and vertical images were obtained with a BioRad MRC-100 laser scanning confocal imaging system. Immunocytochemical analysis of X.laevis oocytes was performed 2 days after injection as described previously [6].
Immunoblotting
Immunoblotting was performed as described previously [7]. Blots were incubated with rabbit anti-hNCC antibody A857 [17] diluted 1:10 000 in PBS buffer supplemented with 5% (w/v) non-fat dried milk. Subsequently, blots were incubated with sheep horseradish peroxidase conjugated to anti-rabbit IgG (Sigma) diluted 1:5000. NCC proteins were visualized using enhanced chemiluminescence (Pierce, Rockford, IL).
22Na+ uptake
For 22Na+ uptake experiments, 1.2 x106 CCD cells were transferred to a 2.0 cm2 well and allowed to attach to the bottom of the well. Next, the cells were washed three times with Optimem and incubated with 750 ml optimem containing 2.5 mg cDNA and 7.5 ml lipofectamine as described above. After 3 h, the transfection mixture was replaced by DCT medium and the cells were cultured overnight. The next day, the cells were trypsinized and divided over 11 wells of a 24 well plate. Cells were grown to subconfluence for another 2 days. Three days after transfection, the cells were washed and incubated in Cl-free KHB medium containing 5 mM HEPES/Tris (pH 7.4), 96 mM sodiumgluconate, 2 mM potassiumgluconate, 1.8 mM calciumgluconate, 1 mM Mg(NO3)2, 2.5 mM sodium pyruvate and 5 mg/dl gentamycin for 13 h at 37°C. Next, Cl-free medium was replaced by 500 ml 22Na+ uptake medium containing 20 mM 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 mM amiloride, 100 mM bumetanide and 1 mCi per ml 22Na+. 22Na+ uptake was measured in the absence and presence of 100 mM hydrochlorothiazide (HCT) for 15 min at RT. Ouabain was added to prevent Na+ exit via the Na+K+-ATPase, bumetanide to inhibit the Na+K+2Cl cotransporter, amiloride to block the Na+H+ antiporter and Na+ channels, and hydrochlorothiazide to inhibit NCC. The uptake reaction was stopped by washing the cells four times with ice-cold uptake medium. Next, cells were lysed in 500 µl of 0.1% (w/v) SDS and radioactivity was counted in a liquid scintillation counter. In each experiment, two wells were included for cell counting and one well for western blot analysis of NCC expression. The experimental conditions were tested in quadruplicate. 22Na+ uptake measurements in X.laevis oocytes were performed 2 days after injection as described previously [6].
Statistical analysis
Values are expressed as mean ± SEM. Statistical significance was determined by the Student's t-test. Differences in mean with P < 0.05 were considered statistically significant.
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Results
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Effect of chemical chaperones on mutant-hNCC activity in X.laevis oocytes
In a recent study we provided evidence that the intrinsic transport activity of partially retarded class II mutants is not affected by the mutation [7]. This finding urged us to test the effect of chemical chaperones on the transport activity of hNCC mutants associated with GS. Figure 1 shows, however, that neither glycerol (2 mM) nor 4-phenylbutyrate (2.5 mM) altered the transport activity of the partially retarded class II mutant L215P or the fully retarded class I mutant G439S in X.laevis oocytes.

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Fig. 1. Effect of glycerol and 4-phenylbutyrate on Na+ uptake in X.laevis oocytes expressing partially and fully retarded mutants of hNCC. Values are expressed as nmol Na+/oocyte/2 h. Oocytes were injected with 10 ng cRNA of wild-type, L215P- or G439S-hNCC. Prior to Na+ uptake, oocytes were incubated in the absence (black bars) or presence of either 2 mM glycerol (dotted bars) or 2.5 mM 4-phenylbutyrate (hatched bars) for 16 h at 18°C. The data presented are the mean ± SEM of 1520 oocytes.
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Transient expression of hNCC in mpk-DCT and -CCD cells
Subsequently, we investigated the possibility of expressing hNCC and its mutants in a polarized epithelial cell line of distal tubular origin. Here, we evaluated mpk-DCT [16] and -CCD cells [15] for functional expression of hNCC. Figure 2 reveals that both cell lines transiently express wild-type hNCC. In mpkDCT cells, the predominant form of the transporter had an apparent molecular size of
100 kD (Figure 2A). In addition, DCT cells expressed relatively low amounts of hNCC with molecular weights ranging from
120 to
140 kD. We have previously shown that these high molecular weight transporters are complex glycosylated [6,7]. In contrast, CCD cells expressed relatively more protein of high molecular size. Relatively little straining was observed at a weight of
100 kD. CCD cells endogenously produced an immunoreactive protein with a size of
100 kD, which was not present in the untransfected DCT4 cells. For immunolocalization studies, cells were grown on permeable supports until they formed a confluent monolayer. In DCT cells, hNCC was present throughout the cell, whereas in CCD cells the transporter showed a predominantly apical distribution (Figure 2B). However, minor amounts of transporter protein appeared to be present basolaterally. Because the glycosylation (relatively more complex glycosylated hNCC) and localization (predominantly apical localization) pattern of hNCC in mpkCCD cells resembled the in vivo situation best, it was decided to continue with this cell line.

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Fig. 2. Localization of hNCC expressed in mpkDCT and mpkCCD cells. (A) Total cell lysates of hNCC-DCT and hNCC-CCD cells were subjected to western blotting using rabbit anti-hNCC antibody A857. (B) MpkDCT cells and mpkCCD cells transiently expressing hNCC were grown to confluence on permeable supports. Immunocytochemistry was performed using rabbit anti-hNCC antibody A857 as a primary antibody and goat anti-rabbit IgG Alexa 488 conjugate as the secondary antibody. XY and XZ images were obtained by confocal laser scanning microscopy.
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Plasma membrane localization of hNCC in mpkCCD cells
To determine the subcellular localization of hNCC following transient expression in mpkCCD cells, we performed co-localization studies with the basolateral marker E-cadherin (Figure 3A, left panel) and the apical marker caveolin-I (Figure 3B, left panel). The Z-scans confirm the polarized expressions of these two markers in mpkCCD cells. In addition, the scans show that the apical staining of caveolin-I was rather diffuse as compared with the lateral staining of E-cadherin. In this respect, the apical staining pattern of caveolin-I resembled that of hNCC (Figure 3, right panels). Subsequent merging of the confocal images revealed that apical hNCC perfectly co-localized with caveolin-I, whereas basolateral hNCC was confined to the cytosolic compartment underneath the plasma membrane.

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Fig. 3. Subcellular localization of hNCC in mpkCCD cells. hNCC-CCD cells were grown to confluence on permeable supports. Immunocytochemistry was performed using rabbit anti-hNCC antibody A857, rat anti-E-cadherin and mouse anti-caveolin-1. Secondary antibodies were IgG Alexa 488 conjugated goat anti-rabbit, IgG Alexa 594 conjugated goat anti-rat and goat anti-mouse. XY and XZ images were obtained by confocal laser scanning microscopy.
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Thiazide-sensitive Na+ uptake in mpkCCD cells transiently expressing hNCC
Thiazide-sensitive 22Na+ uptake was determined to assess the functionality of the transporter following transient expression in mpkCCD cells. Figure 4 shows that Na+ uptake in cells expressing hNCC was significantly increased as compared to untransfected cells (P < 0.05). Although HCT (1 mM) significantly reduced Na+ uptake in hNCC expressing cells, complete inhibition was not observed.

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Fig. 4. Thiazide-sensitive Na+ uptake in hNCC-CCD cells. MpkCCD cells transiently expressing hNCC were cultured to subconfluence in 24-well plates. 22Na+ uptake was measured in the absence and presence of 100 mM hydrochlorothiazide (hct). The uptake values were corrected for cell number. The values presented are the mean ± SEM of 12 filters. *Significantly (P < 0.05) different from non-transfected (nt). #Significantly (P < 0.05) different from corresponding control.
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Transient expression of GS mutants in mpkCCD cells
Western blot analysis revealed that mpkCCD cells readily expressed the class II mutant transporter R955Q (Figure 5). Similarly to wild-type hNCC, this mutant transporter was mainly present in the complex glycosylated form as indicated by the heavily stained band of
120140 kD. Relatively little transporter was present in the high mannose glycosylated form (
110 kD). Conversely, the class I mutant transporter G741R was only scarcely expressed. The putative gain of function mutant transporter R904Q was expressed in roughly the same quantity and with virtually the same ratio between the complex and high mannose glycosylated forms as the wild-type transporter.

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Fig. 5. Immunoblot of partially and fully retarded mutants of hNCC expressed in mpkCCD cells. Total lysates of mpkCCD cells transiently expressing wild-type, G741R- (class I), R955Q- (class II), or the putative gain of function mutant R904Q-hNCC were subjected to immunoblot analysis using rabbit anti-hNCC antibody A857 (nt, non-transfected cells).
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Effect of chemical chaperones on the glycosylation state of mutant-hNCC in mpkCCD cells
In a second series of experiments, mpkCCD cells were transfected with either wild-type hNCC or the partially retarded GS mutant R955Q. To investigate the effect of chemical chaperones, transfected cells were divided over six permeable filters (0.33 cm2; immunocytochemistry) and six wells (2 cm2; western blotting) and cultured in the absence (control) and presence of either thapsigargin (1 µM), a specific inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase, 4-phenylbutyrate (5 mM), dimethyl sulfoxide (DMSO; 1%, v/v), or glycerol (4%, v/v) for 16 h. Immunocytochemical analysis revealed that 4-phenylbutyrate increased the number of hNCC-positive cells both after transfection with wild-type and mutant hNCC. In contrast, the number of immunopositive cells was not altered by thapsigargin and virtually annulated by DMSO and glycerol (data not shown). Figure 6 shows that, irrespective of the culturing conditions, non-transfected mpkCCD cells expressed a doublet of immunoreactive proteins. In addition to the
100 kD protein described above we now observed a second immunoreactive protein of slightly higher molecular weight. Cells transfected with wild-type hNCC showed the expected immunopositive band of
120140 kD. Thapsigargin and 4-phenylbutyrate increased the expression of wild-type hNCC, without changing the staining pattern. In contrast, both DMSO and glycerol (data not shown) reduced the staining intensity of the
120140 kD band. Cells transfected with R955Q-hNCC showed a relatively strong staining of the
100 kD band. Thapsigargin did not alter this staining pattern, whereas 4-phenylbutyrate increased the intensity of the
120140 kD band and caused the disappearance of the
100 kD band.

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Fig. 6. Effect of chemical chaperones on the glycosylation state of wild-type and partially retarded R955Q-hNCC expressed in mpkCCD cells. Total lysates of mpkCCD cells transiently expressing wild-type or R955Q- (class II) hNCC were subjected to immunoblot analysis using rabbit anti-hNCC antibody A857. (nt, non-transfected cells). Cells were cultured in the absence (control) or presence of 1 µM thapsigargin (90 min), 5 mM 4-phenylbutyrate (16 h) and 1% (v/v) DMSO (16 h).
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Discussion
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The data presented in this study demonstrate the potential of the immortalized mpkCCD cell line originally described by Duong Van Huyen et al. [15] for studying the regulatory aspects of polarized trafficking of the human NCC and its mutants associated with GS.
Western blot analysis of total cell lysates revealed that mpkCCD cells expressed significant amounts of hNCC following transfection by lipofectamin. Importantly, hNCC was predominantly present in the complex glycosylated (
120140 kD) form. We and others have previously shown that complex glycosylation is a prerequisite for functional expression on the plasma membrane [6,10]. In addition to hNCC-related bands, blots of transfected mpkCCD cells showed a positive band of
100 kD. This band was also present on blots of untransfected mpkCCD cells, but not on blots of mpkDCT cells. Because untransfected mpkCCD cells showed no signal in immunofluorescence studies we conclude that the signal on the blot reflects an epitope that is specific for the mpkCCD cell but that is not exposed under normal physiological conditions.
Immunofluorescence studies revealed that the greater part of hNCC co-localized with caveolin-I in the apical part of the mpkCCD cell, whereas a minor part was confined to the basolateral part of the cell. The apical staining of hNCC was diffuse, which suggests, in addition to the apical membrane, localization of hNCC in subapical structures like (recycling) endosomes. A similar apical distribution of hNCC has been reported for the rat distal nephron beginning in the initial distal convoluted tubule and terminating within the connecting tubule [18]. The observed co-localization with caveolin-1 provides evidence that hNCC is partially located in caveolae. Caveolae are non-clathrin membrane invaginations on the inner surface of the plasma membrane that fuse to endosomes. These endosomes either recycle proteins or direct them to lysosomes for degradation. Our finding of a partial caveolar localization of hNCC suggests that internalization may play a role in the regulation of hNCC activity. A minor part of hNCC was observed in the basolateral part of the mpkCCD cell. No co-localization with the basolateral membrane marker E-cadherin was observed, indicating that the transporter was present in the cytosolic compartment.
Functional analysis of mpkCCD cells transiently expressing hNCC revealed a 4-fold increase in Na+ uptake, which is consistent with functional targeting of the transporter to the plasma membrane. However, only part of the hNCC-mediated Na+ uptake appeared to be thiazide-sensitive. At present, we do not have a clear explanation for this result. Taken together, these data show that hNCC has an apical membrane localization, is properly glycosylated, and is functionally active following transient expression in mpkCCD cells.
In this study we show that the class II mutant R955Q, but not the class I mutant G741R, is expressed in mpkCCD cells following transfection by lipofectamin. This finding suggests that class I mutants, in contrast to class II mutants, are rapidly degraded. In addition to the GS mutants we also expressed the R904Q mutant, which was recently suggested to give an increased risk of development of primary hypertension [19]. It was speculated that this mutation results in increased Na+ uptake and therefore represents a gain-of-function mutation. The data presented in this study suggest that this mutant is not expressed to a higher extent than wild-type hNCC.
Using the X.laevis oocyte expression system we previously identified two classes of mutant GS transporters [6]. Class II in particular, consisting of partially retarded GS mutant transporters, might be of therapeutic interest because in a follow-up study using concatemeric proteins of wild-type and mutant hNCC we provided evidence that the intrinsic activity of these mutant transporters is not affected by the mutation. This indicates that these mutant transporters have a major routing problem and may be subject to rescue by pharmacological chaperones [7]. In a first attempt to test this idea, we evaluated the effects of glycerol and 4-phenylbutyrate, two widely used chemicals with a chaperone type of function, on Na+ uptake by X.laevis oocytes expressing either the partially retarded class II mutant L215P or the fully retarded class I mutant G439S. The data presented show that neither of the two chemicals was able to restore Na+ uptake in oocytes expressing mutant hNCC. The ability of chaperones to improve NCC activity in the oocyte expression system was recently reported by Wyse et al. [9]. They showed that the glucose-regulated protein 58 (grp58), a chaperone with thiol-dependent reductase activity, associated with NCC and increased NCC-mediated Na+-uptake.
Next, we used mpkCCD cells to investigate the effect of pharmacological chaperones on the expression and glycosylation state of the partially retarded GS mutant R955Q in a more physiological background. Thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase, was recently shown to allow DF508-CFTR to reach the cell surface, where it functioned effectively as a Cl channel [20]. The present study shows that thapsigargin increased the expression of wild-type hNCC, suggesting a role of Ca2+-dependent chaperones. Remarkably, thapsigargin did not change the expression of the partially retarded GS mutant. In contrast to wild-type hNCC, the GS mutant R955Q was mainly present at a low molecular weight (
100 kD) form. Most promising, this low molecular weight form was virtually absent in cells cultured in the presence of 4-phenylbutyrate, whereas these cells showed an increased expression of the complex glycosylated (
120140 kD) form of the mutant transporter. This effect of 4-phenylbutyrate on the amount of complex glycosylated hNCC was paralleled by an increase in the number of hNCC positive cells. Unfortunately, the transfection efficiency in this experiment was too low to perform Na+ uptake measurements. Finally, both glycerol and DMSO virtually abolished expression of wild-type and mutant hNCC. Taken together, the data presented indicate that 4-phenylbutyrate is a promising candidate for rescuing partially retarded but otherwise functional class II GS mutants.
Conflict of interest statement. None declared.
[See related article by Gross and Schöneberg (this issue, pp. 10291032)]
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References
|
---|
- Obermuller N, Bernstein P, Velazquez H, Reilly R, Moser D, Ellison DH, Bachmann S. Expression of the thiazide-sensitive Na-Cl cotransporter in rat and human kidney. Am J Physiol 1995; 269: F900F910[ISI][Medline]
- Ellison DH, Velazquez H, Wright FS. Thiazide-sensitive sodium chloride cotransport in early distal tubule. Am J Physiol 1987; 253: F546F554[ISI][Medline]
- Appel LJ. The verdict from ALLHAT thiazide diuretics are the preferred initial therapy for hypertension. JAMA 2002; 288: 30393042[Free Full Text]
- Rossier BC. Negative regulators of sodium transport in the kidney: key factors in understanding salt-sensitive hypertension? J Clin Invest 2003; 111: 947950[Free Full Text]
- Gitelman HJ, Graham JB, Welt LG. A familial disorder characterized by hypokalemia and hypomagnesemia. Ann NY Acad Sci 1969; 162: 856864[ISI][Medline]
- De Jong JC, Van Der Vliet WA, Van Den Heuvel LP, Willems PH, Knoers NV, Bindels RJ. Functional expression of mutations in the human NaCl cotransporter: evidence for impaired routing mechanisms in Gitelman's syndrome. J Am Soc Nephrol 2002; 13: 14421448[Abstract/Free Full Text]
- De Jong JC, Willems PH, Mooren FJ, Van Den Heuvel LP, Knoers NV, Bindels RJ. The structural unit of the thiazide-sensitive NaCl cotransporter (NCC) is a homodimer. J Biol Chem 2003; 278: 2430224307[Abstract/Free Full Text]
- Kunchaparty S, Palcso M, Berkman J et al. Defective processing and expression of thiazide-sensitive Na-Cl cotransporter as a cause of Gitelman's syndrome. Am J Physiol 1999; 277: F643F649[ISI][Medline]
- Wyse B, Ali N, Ellison DH. Interaction with grp58 increases activity of the thiazide-sensitive Na-Cl cotransporter. Am J Physiol Renal Physiol 2002; 282: F424F430[Abstract/Free Full Text]
- Hoover RS, Poch E, Monroy A, Vazquez N, Nishio T, Gamba G, Hebert SC. N-glycosylation at two sites critically alters thiazide binding and activity of the rat thiazide-sensitive Na+:Cl cotransporter. J Am Soc Nephrol 2003; 14: 271282[Abstract/Free Full Text]
- Wilson FH, Kahle KT, Sabath E et al. Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4. Proc Natl Acad Sci USA 2003; 100: 680684[Abstract/Free Full Text]
- Sato S, Ward CL, Krouse ME, Wine JJ, Kopito RR. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J Biol Chem 1996; 271: 635638[Abstract/Free Full Text]
- Rubenstein RC, Egan ME, Zeitlin PL. In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR. J Clin Invest 1997; 100: 24572465[Abstract/Free Full Text]
- Vandewalle A, Bens M, Duong Van Huyen JP. Immortalized kidney epithelial cells as tools for hormonally regulated ion transport studies. Curr Opin Nephrol Hypertens 1999; 8: 581587[CrossRef][ISI][Medline]
- Duong Van Huyen J, Bens M, Vandewalle A. Differential effects of aldosterone and vasopressin on chloride fluxes in transimmortalized mouse cortical collecting duct cells. J Membr Biol 1998; 164: 7990[CrossRef][ISI][Medline]
- Peng KC, Cluzeaud F, Bens M, Van Huyen JP, Wioland MA, Lacave R, Vandewalle A. Tissue and cell distribution of the multidrug resistance-associated protein (MRP) in mouse intestine and kidney. J Histochem Cytochem 1999; 47: 757768[Abstract/Free Full Text]
- Biner HL, Arpin-Bott MP, Loffing J et al. Human cortical distal nephron: distribution of electrolyte and water transport pathways. J Am Soc Nephrol 2002; 13: 836847[Abstract/Free Full Text]
- Plotkin MD, Kaplan MR, Verlander JW et al. Localization of the thiazide sensitive Na-Cl cotransporter, rTSC1 in the rat kidney. Kidney Int 1996; 50: 174183[ISI][Medline]
- Melander O, Orho-Melander M, Bengtsson K et al. Genetic variants of thiazide-sensitive NaCl-cotransporter in Gitelman's syndrome and primary hypertension. Hypertension 2000; 36: 389394[Abstract/Free Full Text]
- Egan ME, Glockner-Pagel J, Ambrose C et al. Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells. Nat Med 2002; 8: 485492[CrossRef][ISI][Medline]
Received for publication: 21. 6.03
Accepted in revised form: 25. 7.03