CFTR disruption impairs cAMP-dependent Clminus secretion in primary cultures of mouse cortical collecting ducts

Marcelle Bens1, Jean-Paul Duong Van Huyen1, Françoise Cluzeaud1, Jacques Teulon2, and Alain Vandewalle1

Institut National de la Santé et de la Recherche Médicale, 1 Unité 478 et 2 Unité 426, Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, 75870 Paris, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-D-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(-/-)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of Na+ assessed by the short-circuit current (Isc) method in both CFTR(+/+) and CFTR(-/-) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in Isc induced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter Isc in BAm-treated CFTR(-/-) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of 36Cl- in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal 36Cl- flux in CFTR(-/-) CCDs but not the basal-to-apical 36Cl- flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted Cl- in mouse CCD.

cyclic adenosine monophosphate; kidney; epithelial sodium channel; cystic fibrosis transmembrane conductance regulator; cystic fibrosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ROLE OF THE CYSTIC FIBROSIS transmembrane conductance regulator (CFTR) in the kidney remains uncertain, because there is no major disruption of renal function in cystic fibrosis (CF) patients (29). The CFTR protein has been detected in most segments of the renal tubule, although its exact cellular and/or membranous localization remains to be identified. CFTR-like Cl- currents and/or CFTR mRNA have been reported in the amphibian A6 cells (19, 34) and in a variety of cultured cells from the distal convoluted tubule (22), cortical collecting duct (CCD) (8, 20), and inner medullary collecting duct (IMCD) (7, 14, 33). It is now generally agreed that CFTR is expressed mainly at the apical surface of distal and collecting duct cells, and there is increasing indirect evidence that renal Cl- secretion, a minor process under most physiological circumstances, is one function of CFTR in the terminal parts of the renal tubule, in particular in the IMCD (14, 33).

Because CFTR can act as a downregulator of the amiloride-sensitive epithelial sodium channel (ENaC) (3, 30, 31), which is normally expressed in principal collecting duct cells (6), it has been suggested that cAMP may stimulate CFTR-like Cl- channels and inhibit ENaC in cultured mouse M-1 CCD cells (17). More recently, Kibble et al. (15) have shown that Delta 508-CFTR transgenic mice exhibited normal urinary Na+ excretion under salt repletion and chronic salt depletion but that the fractional Na+ excretion induced by amiloride in mice subjected to salt restriction is greater in CF than in control animals. Na+ absorption mediated by ENaC may therefore be increased in CF CCDs, at least under particular physiological circumstances.

Nagy et al. (20) have previously shown that vasopressin (AVP), 8-bromo-adenosine 3',5'-cyclic monophosphate (8-bromo-cAMP), and isoproterenol (ISO) all stimulate a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-insensitive Cl- conductance in primary cultures of immunoselected rabbit CCD cells. These authors suggested that, in intercalated cells, CFTR may underlie at least part of this Cl- conductance. CFTR mRNA has been shown to be expressed mainly in intercalated cells and to a lesser extent in principal cells (32). CFTR and ENaC have also been shown to be expressed in the immortalized mouse M-1 CCD cells (17) and mouse principal mpkCCDcl4 cells (unpublished results). The questions therefore arise as to whether the stimulation of Na+ transport mediated by ENaC is accompanied by the secretion of Cl- into the CCD and to what extent CFTR may regulate ENaC activity in the CCD. To address these questions, we used cftrm1unc mice (28), which lack CFTR-mediated, cAMP-dependent Cl- secretion in the colon, airways, and exocrine pancreas cells (5). We used the short-circuit current (Isc) method and 36Cl- flux studies to carry out ion transport studies with the use of primary cultures of CCD cells microdissected from the kidneys of wild-type [CFTR(+/+)] and CFTR-knockout mice [CFTR(-/-)]. We investigated the effects of deamino-8-D-arginine vasopressin (dDAVP), an analog that binds to the V2 receptors and mimics the effects of vasopressin on Na+ absorption in principal cells (23), and of ISO, which is known to stimulate cAMP only in intercalated cells (9).

The data from this study show that the component of secreted Cl- stimulated by dDAVP or ISO detected in wild-type CFTR(+/+) CCD cells was absent in CFTR(-/-) CCD cells. They also show that CFTR had almost no inhibitory action on dDAVP-stimulated Na+ absorption mediated by ENaC in this model of mouse CCD cells in primary culture.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
RESULTS
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Cell culture. Experiments were conducted on male cftrm1unc mice (28) bred at the Centre de Développement des Techniques Avancées pour l'Expérimentation Animale (Orleans, France). This strain of mice was originally derived from ES129/Sv cells injected into C57BL/6 embryos. Mice were back-crossed with C57BL/6 mice for three generations and then intercrossed. The 5- to 8-wk-old wild-type CFTR(+/+) mice (n = 21) and CFTR(-/-) mice homozygous for the disrupted CFTR gene (n = 16) were killed by cervical dislocation, and their kidneys were removed. CCDs were microdissected out under sterile conditons. Isolated CCDs (8-10 fragments, 0.2-0.5 mm long) were seeded on Transwell permeable filters (0.4-µm pore size, 0.33 cm2 insert growth area; Corning Costar, Cambridge, MA) and grown in a defined medium [DM (DMEM): Ham's F-12 (1:1 vol/vol), 60 nM sodium selenate, 5 µg/ml transferrin, 2 mM glutamine, 50 nM dexamethasone, 1 nM triiodothyronine, 10 ng/ml epidermal growth factor (EGF), 5 µg/ml insulin, 2% fetal calf serum (FCS), 20 mM HEPES, pH 7.4] at 37°C in a 5% C02-95% air atmosphere. After the first 5 days, the medium was changed every 2 days. Experiments were carried out 2 wk after seeding, by use of confluent cells that had developed high transepithelial electrical resistance (>700 Omega  · cm2). All experiments were performed in accordance with the guidelines of the French Agricultural Office and in compliance with the legislation governing animal studies.

Electronmicroscopy and immunohistochemical studies. Confluent cells grown on permeable filters were fixed for 2 h with 0.2% glutaraldehyde in 0.1 M cacodylate buffer, embedded in epon, and processed for electronmicroscopy. Cells were also fixed in ice-cold methanol and processed for immunofluorescence using biotinylated Dolichos biflorus agglutinin (DBA; Vector, Burlingame, CA), as previously described (1). Specimens were examined under a Zeiss microscope and photographed.

RNA extraction and RT-PCR. Total RNA was extracted from microdissected CFTR(+/+) and CFTR(-/-) CCDs by use of the RNA-PLUS extraction kit (Bioprobe Systems, Montreuil-sous-Bois, France) and reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Eragny, France) at 42°C for 45 min. cDNA and non-reverse-transcribed RNA were amplified for 28-38 cycles in a total volume of 100 µl of PCR buffer (50 mM KCl, 20 mM Tris · HCl, pH 8.4) containing 40 µM dNTP, 1.5 mM MgCl2, 1 µCi [alpha -32P]dCTP (NEN, Le Blanc Mesnil, France), 1 U Taq polymerase, 29 pmol of alpha -ENaC primers (13), and 1.5 pmol of beta -actin primers (8). The CFTR mRNA was also analyzed by RT-PCR with the use of 42 pmol of CFTR primers from the mouse CFTR exon 9 and exon 13, as described by Marvão et al. (18), and with 0.5 pmol of beta -actin primers. The thermal cycling program was as follows: 94°C for 30 s, 54°C (for alpha -ENaC) or 60°C (for CFTR) for 30 s, and 72°C for 60 s. Amplification products were run on 4% polyacrylamide gels and autoradiographed. The sets of CFTR and ENaC primers used yielded amplified products of the expected size, and sequence analysis of PCR products confirmed that they matched the expected cDNA analyses (data not shown).

Electrophysiological studies. Confluent CFTR(+/+) and (-/-) CCD cells were grown on 0.33-cm2 Transwell filters (Corning Costar) in DM medium until confluent (day 14), and then in DM containing no EGF, hormones, FCS, or HEPES [Ham's F-12 medium (HFM) containing 29 mM NaHCO3] for the final 2 h. The filters were mounted in a modified Ussing-type chamber (Diffusion Chamber System, Costar) connected to a voltage clamp apparatus via glass barrel Micro-Reference Ag/AgCl electrodes. Cell layers were bathed on both sides (0.2 ml for the apical side and 0.6 ml for the basal side) with HFM warmed to 37°C and continuously gassed with 95% 02-5% C02 to keep the pH at 7.4. Isc (µA/cm2) was measured by clamping the open-circuit transepithelial voltage (VT) to 0 mV for 1 s. By convention, a positive Isc value corresponded to a flow of positive charges from the apical to the basal solution.

Apical Na+ substitution experiments. Isc was measured on CFTR(+/+) and (-/-) CCD cells in which the apical NaHCO3-HFM medium was replaced by a Na+-free solution (in mM: 156 N-methyl-D-glucamine, 4 KCl, 0.7 MgCl2, 0.4 MgSO4, 1.05 CaCl2, 20 glucose, 8 HEPES, pH 7.4) (8). The basal side of the filter was bathed with the HFM warmed to 37°C and continuously gassed with 95% 02-5% CO2, and Isc was measured after a 1-h equilibration period. For all experiments, dDAVP (Ferring Pharmaceutical, Malmo, Sweden) or ISO (Sigma, St. Louis, MO) was added to the basal side of the filters. Benzamyl amiloride (BAm; Sigma), 5-nitro-2-(3-phenylpropamino)benzoate (NPPB; Research Biochemicals, Natick, MA), glibenclamide (Glb; Sigma) or DIDS (Sigma) was added to the apical side of the filters. These agents were dissolved at 0.1 M in DMSO, and diluted 1:1,000 (10-4 M final concentration for NPPB, Glb, and DIDS) or 1:100,000 (10-6 M final concentration for BAm) in appropriate medium.

36Cl- flux studies. Transepithelial fluxes of 36Cl- (Amersham, Les Ulis, France) were measured on confluent cells grown on Transwell filters (0.33-cm2 insert growth area). The inside of the filters was filled with 150 µl, and the outside was immersed in 600 µl of NaHCO3-HFM. 36Cl- (400 nCi/ml) was added to the apical or basal medium bathing the cells. Cell layers were incubated at 37°C, and 50-µl samples of apical or basal medium were collected from opposite sides of the filters 60 min after the radioactive tracers were added. The radioactivity was measured in a liquid scintillation counter (LKB; Pharmacia). In parallel, VT and transepithelial electrical resistance (RT) were monitored using the Millicell electrical resistance clamp apparatus (Precision Instrument Design, Tahoe City, CA) as previously described (1, 8).

Statistical analysis. Results are expressed as means ± SE from (n) experiments. Significant differences between groups were analyzed by unpaired or paired Student's t-test. A P value <0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENaC and CFTR mRNAs in isolated CCDs and the morphology of primary cultured CCD cells. ENaC was detected by RT-PCR using alpha -ENaC specific primers (13) in both CFTR(+/+) and CFTR(-/-) microdissected CCDs (Fig. 1). CFTR transcripts, analyzed by RT-PCR using primers from the mouse CFTR exon 9 and exon 13 (18), were detected in microdissected CFTR(+/+) CCDs, but not in CFTR(-/-) CCDs (Fig. 1). CFTR(+/+) and CFTR(-/-) CCDs grown to confluence on permeable filters formed monolayers of cuboid cells (Fig. 2A). Both CFTR(+/+) and CFTR(-/-) CCD cells in primary culture exhibited a similar heterogeneous pattern of lectin DBA binding (Fig. 2B), suggesting that, as intact CCDs, they were composed of both intercalated and principal cells (12). The electrophysiological parameters also showed that CCDs seeded on permeable filters had the typical electrophysiological features of tight epithelia. Confluent CFTR(+/+) and CFTR(-/-) CCD cells seeded and grown on 0.33-cm2 permeable filters (day 14 after seeding) exhibited almost identical high RT, negative VT, and positive Isc under basal conditions (Table 1). Taken together, these results indicate that cultured CCDs microdissected from wild-type and CFTR-knockout mice and grown on filters formed confluent layers of tight epithelial cells suitable for electrophysiological studies.


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Fig. 1.   Cystic fibrosis transmembrane conductance regulator (CFTR; A) and epithelial Na channel (ENaC; B) expression in microdissected CFTR(+/+) and CFTR(-/-) cortical collecting ducts (CCDs). Autoradiograms of 32P-labeled fragments obtained by RT-PCR from microdissected CFTR(+/+) and CFTR(-/-) CCD cDNAs. alpha -ENaC-amplified products (28 cycles) of expected size (564 bp) were detected in both isolated CFTR(+/+) and CFTR(-/-) CCDs, whereas amplified products (38 cycles) of expected size (636 bp) obtained with CFTR primers were detected only in CFTR(+/+), but not in CFTR(-/-), CCDs or by omitting cDNA [control (C)]. In all cases, beta -actin-amplified products (241 bp) were used as the internal standard.



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Fig. 2.   Morphology of primary cultures of CFTR(+/+) and CFTR(-/-) CCD cells. Confluent CFTR(+/+) and CFTR(-/-) CCD cells grown on filters (A) formed monolayers of closely apposed cells. Both CFTR(+/+) and CFTR(-/-) CCD cells exhibited a similar heterogeneous pattern of lectin Dolichos biflorus agglutinin (DBA)- positive and DBA-negative cells (B). Bars: 2 (A) and 20 µm (B).


                              
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Table 1.   Electrophysiological properties of confluent CFTR(+/+) and CFTR(-/-) CCD cells grown on filters

Effect of Na+ and Cl- channel inhibitors on dDAVP-stimulated Isc in primary cultures of CFTR(+/+) and CFTR(-/-) CCDs. The effect of dDAVP, which acts on ion transport via a cAMP-dependent pathway, was tested on confluent cultured CFTR(+/+) and CFTR(-/-) CCD cells grown on filters. All subsequent experiments were performed using 10-8 M dDAVP, a concentration shown to give greatest rise in Isc in cultured mouse CCD cells (8). As shown in Fig. 3, the rise in Isc caused by dDAVP after 20 min was slightly, but not significantly, lower in the CFTR(+/+) CCD cells (50.6 ± 10 µA/cm2, n = 17) than in the CFTR(-/-) CCD cells (61.8 ± 12.1 µA/cm2, n = 16). In both cases, the apical addition of BAm (10-6 M), a potent ENaC channel blocking agent (16), almost completely inhibited the dDAVP-stimulated Isc after 10 min (Fig. 3). As a result, the BAm-sensitive component of Isc was slightly, but not significantly, greater in CFTR(-/-) CCD than in CFTR(+/+) CCD cells (Fig. 3). Thus, under our experimental conditions and by using symetrical high Na+ solutions (i.e., HFM), ENaC-mediated Na+ absorption was not statistically significantly increased in primary cultured mouse CFTR(-/-) CCDs expressing ENaC but lacking functional CFTR compared with cultured wild-type CCDs coexpressing CFTR and ENaC.


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Fig. 3.   Effects of deamino-D-arginine vasopressin (dDAVP) and benzamyl amiloride (BAm) on short-circuit current (Isc). Isc was measured on confluent CFTR(+/+) and CFTR(-/-) CCD cells grown on filters and sequentially incubated without (open circle ) and with 10-8 M dDAVP () and 10-6 M BAm (triangle ). dDAVP was applied to the basal medium, and the sodium channel blocker was added to the apical medium. Bars represent the BAm-sensitive (BAms) component of dDAVP-stimulated Isc (BAms Isc) measured in CFTR(+/+) and CFTR(-/-) CCD cells. Values are means ± SE from 17 (+/+) and 16 (-/-) separate filters.

We next analyzed the effects of dDAVP on Isc in BAm-pretreated CCD cells to test whether this vasopressin analog stimulated Cl- secretion (Fig. 4). Apical addition of 10-6 M BAm almost completely inhibited Isc in both CFTR(+/+) and CFTR(-/-) CCD cells (Fig. 4A). The subsequent addition of 10-8 M dDAVP to the basal side of the CFTR(+/+) CCD cells caused a discrete, but significant, rise in Isc (untreated cells: 1.4 ± 0.2 µA/cm2; +dDAVP for 20 min: 8.4 ± 2.7 µA/cm2, n = 7, P < 0.0001; Fig. 4, A and B). Addition of 10-4 M NPPB, a commonly used Cl- channel blocker (35), to the apical side of the dDAVP-treated CFTR(+/+) CCD cells significantly (P < 0.05) reduced the dDAVP-stimulated Isc by 33% (Fig. 4, A and B). In sharp contrast, dDAVP failed to increase the Isc from BAm-treated CFTR(-/-) CCD cells, and the subsequent addition of NPPB had no impact on the Isc in these cells (Fig. 4, A and B). Thus blockade of Na+ absorption by BAm unmasked a dDAVP component of Cl- secretion in cultured wild-type CCDs, but not in CFTR(-/-) CCDs. These data also demonstrated that CFTR is involved in this process.


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Fig. 4.   Effect of 5-nitro-2-(3-phenylpropamino)benzoate (NPPB) on dDAVP-stimulated Isc. A: Isc was measured on confluent cultures of CFTR(+/+) (n = 7) and CFTR(-/-) (n = 5) CCD cells grown on filters and sequentially incubated without (open circle ) and with 10-6 M BAm (triangle ), 10-8 M dDAVP (), and 10-4 M NPPB (). dDAVP was applied to the basal medium, and the ion channel blockers were added to the apical medium. B: bars represent mean Isc values ± SE measured 10 or 20 min after addition of the agents tested on CFTR(+/+) (open) and CFTR(-/-) (filled) CCD cells. *P < 0.05 and ***P < 0.001 represent significant differences (unpaired t-test) between groups.

As shown in Fig. 5, NPPB (10-4 M) was more efficient in inhibiting the dDAVP-stimulated Isc in BAm-treated CFTR(+/+) CCD cells than Glb (10-4M), an ATP-sensitive K+ channel blocker that partially inhibits the CFTR Cl- channel (26). Consistent with an inhibitory effect of NPPB and Glb on CFTR, these two channels blockers had no effect on Isc stimulated by dDAVP in BAm-treated CFTR(-/-) CCD cells (Fig. 5). DIDS, which has no inhibitory action on CFTR, did not alter Isc in either dDAVP-treated CFTR(+/+) or CFTR(-/-) CCD cells (Fig. 5).


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Fig. 5.   Effects of ion channel inhibitors on dDAVP-stimulated Isc. Confluent CFTR(+/+) (open circle , n = 6) and CFTR(-/-) (, n = 4) CCD cells grown on filters were incubated with 10-6 M BAm and 10-8 M dDAVP, as described in the legend of Fig. 4. Thereafter, Isc was measured before and after apical additions of 10-4 M DIDS, glibenclamide (Glb), or NPPB. The fractional change of Isc (Delta Isc) was calculated using the Isc value measured before and 10 min after addition of the inhibitor to be tested. Results are means ± SE in each group.

ISO-stimulated Isc in primary cultures of CFTR(+/+) CCDs. In the absence of reliable anti-CFTR antibodies suitable for immunofluorescence studies on mouse kidney sections or cultured CCD cells, the exact intrarenal and cellular distribution of the CFTR protein in the mouse kidney still remains to be determined. Our results strongly suggest that the concomitant increase in Na+ absorption mediated by ENaC and the electrogenic secretion of Cl- mediated by CFTR induced by dDAVP both occur in principal cells. However, it has been shown that CFTR mRNA is also expressed in intercalated cells (32). We therefore tried to find out whether ISO (10-5 M), a beta -adrenergic agonist known to stimulate cAMP in intercalated cells (9), would also increase the secretion of Cl- without affecting Na+ absorption in cultured mouse CCD cells. Isc recordings were first performed with symetrical, high Na+ levels (i.e., HFM) to find out whether ISO affected Isc in the absence of BAm. Basal addition of ISO increased Isc in CFTR(+/+) CCD cells (Fig. 6A). Results from individual monolayers showed that the Isc was always significantly greater in ISO-treated than in untreated CFTR(+/+) CCD cells (Fig. 6B). Furthermore, the BAm-sensitive Isc, which reflected ENaC-mediated Na+ absorption, remained unchanged in untreated and ISO-treated CFTR(+/+) CCD cells (Fig. 6C).


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Fig. 6.   Effect of isoproterenol (ISO) on Isc. A: Isc was measured on confluent cultures of CFTR(+/+) (n = 6) CCD cells grown on filters before (open circle ) and after adding 10-5 M ISO () to the basal medium. B: representation of the Isc from individual monolayers (n = 6) and the mean Isc (open circle ) values measured before and 10 and 20 min after the addition of ISO. C: bars represent BAms Isc measured in untreated (control, open bars) and ISO-treated (filled bars) CFTR(+/+) CCD cells. Values are means ± SE from 6 individual monolayers in each group. *P < 0.05 and ***P < 0.001 represent significant differences (paired t-test) vs. untreated Isc values (open circle ).

dDAVP- and ISO-stimulated Isc in primary cultures of CFTR(+/+) and CFTR(-/-) CCDs measured in the absence of apical Na+. Apical Na+ replacement experiments were undertaken to clarify the contribution of CFTR in the cAMP-dependent secretion of Cl- in cultured mouse CCD cells incubated either with dDAVP or ISO. Because the symmetrical (apical plus basal) replacement of Na+ by N-methyl-D-glucamine-supplemented solutions did not permit sufficiently stable and reproducible Isc recordings, experiments were performed on primary-cultured CCD cells that were bathed on the apical side with the Na+-free solution, while the basal side of the filter was bathed with the HFM, as described in MATERIALS AND METHODS. Under these experimental conditions, Isc significantly fell by 74% in the untreated cultured CFTR(+/+) CCDs and by 63% in the CFTR(-/-) CCD cells. The possibility could not still be excluded that some HFM containing Na+ remained in the apical compartment after washing and replacement of the apical (200 µl) HFM medium by the Na+-free solution. Therefore, BAm (10-6 M) was once again added to the apical Na+-free solution. BAm further reduced the Isc (by ~4-5 µA/cm2) in both CFTR(+/+)and CFTR(-/-) CCD cells. Under these experimental conditions, the subsequent addition of 10-8 M dDAVP still induced a rise in Isc in CFTR(+/+) CCDs that was inhibited by 41% after the apical addition of NPPB (Fig. 7, top). This confirmed that dDAVP does stimulate the secretion of Cl- in wild-type cultured mouse CCDs. In contrast, dDAVP did not induce any significant change in the Isc recorded under apical Na+-free condition in cultured CFTR(-/-) CCDs (Fig. 7, top). Taken together, these results provide further evidence that CFTR mediates the dDAVP-stimulated secretion of Cl- in this model of cultured mouse CCDs.


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Fig. 7.   Effect of apical substitution of Na+ on Isc elicited by dDAVP and ISO. Isc was measured on confluent CFTR(+/+) and CFTR(-/-) CCD cells with apical Na+ replaced by N-methyl-D-glucamine. After a 1-h equilibration period, BAm (10-6 M) was added to the apical side of the filters, and Isc was then measured after sequential addition of basal 10-8 M dDAVP () or 10-5 M ISO () and apical 10-4M NPPB (). Values are means ± SE from 4-7 individual recordings performed in each group.

We then tested the effects of ISO on Isc under the same apical Na+-free conditions as had been used to test the effect of dDAVP. As also shown in Fig. 7, the basal addition of ISO caused a rapid rise in Isc in CFTR(+/+) CCD cells. As we had observed in dDAVP-treated cells, the subsequent addition of apical NPPB decreased the ISO-stimulated Isc by 32% (Fig. 7, bottom). However, the concomitant addition of ISO and dDAVP induced a greater rise (P < 0.05) in Isc than that of dDAVP alone (+dDAVP for 4 min: 2.9 ± 0.4 µA/cm2, n = 5; +ISO: 3.4 ± 0.5 µA/cm2, n = 6, +dDAVP +ISO: 5.0 ± 0.7 µA/cm2, n = 5). These findings suggest that the CFTR-mediated secretion of Cl- also occurs in intercalated cells. Once again, neither ISO nor NPPB altered the Isc in CFTR(-/-) CCD cells under apical Na+-free conditions (Fig. 7, bottom).

Effect of dDAVP on Cl- flux in primary cultures of CFTR(+/+) and CFTR(-/-) CCDs. Unidirectional ion flux studies using 36Cl- were then performed on confluent CCD cells grown on 0.33-cm2 Transwell filters. The internal diameter of the filters used to grow the CCD cells and the small volume of apical medium required made it impossible to collect the serial 50-µl samples necessary for the isotopic flux measurements under short-circuit current conditions. The Cl- flux studies were therefore performed under the more physiological open-circuit condition (8, 34). Continuous monitoring of RT and VT with the Millicel electrical resistance clamp apparatus showed that 10-8 M dDAVP induced a slight decrease in RT and a sustained increase in VT that was more marked in CFTR(-/-) than in CFTR(+/+) CCD cells (data not shown). As a consequence, the equivalent short-circuit current (Ieq) calculated from RT and VT was significantly greater (P < 0.05) after dDAVP-treatment in CFTR(-/-) CCD cells (44.5 ± 5.5 µA/cm2, n = 19) than in CFTR(+/+) CCD cells (27.9 ± 3.1 µA/cm2, n = 21). The open-circuit conditions therefore seemed to be suitable for ion flux studies. In untreated CFTR(+/+) and CFTR(-/-) CCD cells, 36Cl- flux was slightly greater in the apical-to-basal direction than the basal-to-apical direction (Fig. 8), indicating a net absorption of Cl-. dDAVP doubled the basal-to-apical flux of 36Cl- in CFTR(+/+) CCD cells, but, in sharp contrast, it had no impact on the basal-to-apical 36Cl- flux in CFTR(-/-) CCD cells (Fig. 8). dDAVP also increased the apical-to-basal 36Cl- flux to a similar, significant extent in both CFTR(+/+) CCD cells (2.5-fold) and CFTR(-/-) CCD cells (2.9-fold) (Fig. 8). As a consequence, the net absorption of Cl- measured under open-circuit condition in CFTR(-/-) CCD cells was about twofold greater than in CFTR(+/+) CCD cells.


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Fig. 8.   Effects of dDAVP on Cl- fluxes in primary cultures of CFTR(+/+) and CFTR(-/-) CCD cells. The apical-to-basal (Aright-arrowB) and basal-to-apical (Bright-arrowA) 36Cl- fluxes were measured on confluent cultured CFTR(+/+) (n = 27), and CFTR(-/-) (n = 22) CCD cells grown on filters and incubated without (open bars) or with (filled bars) 10-8 M dDAVP added to the basal side of the filters. Bars are means ± SE in each group. *P < 0.05 and **P < 0.01 represent significant differences (unpaired t-test) between groups.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

Using Isc experiments and 36Cl- flux studies using primary cultures of isolated CCDs microdissected from the kidneys of wild-type and cftrm1unc mice, we have shown that Cl- is secreted in response to cAMP stimulation and have demonstrated the role of CFTR in this process.

Total blockade of ENaC by BAm and the use of Na+-free solutions applied to the apical side of the cells grown on filters almost completely inhibited the basal transport of Na+ but did not impair the small rise in Isc (corresponding, in this case, to a flow of negative charges from the basal to the apical medium) induced by dDAVP or ISO in cultured wild-type CCD cells. This small component of Isc can therefore be attributed to Cl- secretion, because it occurs in the presence of BAm and the absence of Na+ and is inhibited, at least to some extent, by agents known to block Cl- channels. The fact that NPPB, the most potent, but nonselective, Cl- channel blocker (35) tested, partially inhibited both dDAVP- and ISO-stimulated Isc in amiloride-treated CFTR(+/+) CCD cells provides a further indication that CFTR is implicated in this process. Previous studies have shown that cAMP agonists stimulate Cl- secretion in amphibian A6 cells (4, 19, 34) and mammalian CCD and IMCD cells (8, 20) and that the electrogenic anion secretion by cultured IMCD cells can be inhibited by inhibitors of the CFTR Cl- channel (14, 33). The detection of CFTR mRNA and CFTR-like Cl- currents in several cultures of DCT, CCD, and IMCD cells of different origins has also provided further indirect evidence of the involvement of CFTR in the Cl--secreting pathway (7, 14, 17, 22). Furthermore, Morris et al. (19) have shown that vasotocin, which stimulates Na+ absorption and secretory Cl- currents in amphibian A6 cells, concomitantly induced an apparent microtubule-dependent recruitment of cytoplasmic CFTR to the apical cell surface. In this study, we have shown that the component of Cl- secretion elicited by dDAVP in mouse wild-type CCD cells is partially inhibited by NPPB and, to a lesser extent, Glb, but not by DIDS. This pharmacological inhibition profile closely resembles previously reported observations that were attributed to CFTR, in murine kidney cultured cells (14, 22). Thus the results obtained in mouse CFTR(+/+) CCD cells in primary culture agree well with those of similar studies of other cultured renal cells.

The implication of CFTR in the cAMP-stimulated Cl- secretory pathway in CCD was directly demonstrated by showing that dDAVP and ISO failed to stimulate Isc in BAm-treated CFTR (-/-) CCD cells and that the anion channel blockers NPPB and Glb had no effects. Our results therefore demonstrate that the Cl- diffusion pathway stimulated by cAMP agonists in the apical membrane of cultured mouse CCD cells is formed by CFTR. The CFTR protein is expressed in most, if not all, parts of the renal tubule, including the distal collecting duct system. Results from RT-PCR experiments confirmed that CCDs microdissected from wild-type mouse kidneys express CFTR mRNA. However, the precise cellular localization of the CFTR protein in the CCD (principal and/or intercalated cells) has not yet been unambiguously identified. Not all the anti-CFTR antibodies that we tested were suitable for performing a proper immunofluorescence study to determine the precise localization of the protein in frozen mouse kidney sections and cultured mouse CCD cells. CFTR mRNA has been found in both principal and intercalated cells from immunodissected rabbit CCD cells, with the highest level of expression found in the beta -intercalated cells (32). Letz and Korbmacher (17) also showed that principal cells in culture coexpress CFTR and ENaC. Here, we have shown that dDAVP stimulates a predominent component of amiloride-sensitive Na+ absorption mediated by ENaC in both CFTR(+/+) and CFTR(-/-) CCD cells, as well as a minor component of Cl- secretion, which is inhibited by NPPB in CFTR(+/+) but not CFTR(-/-) CCD cells. These results strongly suggest that CFTR is coexpressed with ENaC in principal cells. The fact that ISO, which does not stimulate Na+ transport, also increases the secretion of Cl- in CFTR(+/+) cells but not in CFTR(-/-) CCD cells, indicates that functional CFTR is also expressed in intercalated cells. dDAVP plus ISO also has a greater impact on NPPB-sensitive Isc than dDAVP alone, and this once again is consistent with the presence of CFTR in both principal and intercalated cells.

Besides the fact that an electrogenic secretion of Cl- can be demonstrated by the Isc method (4, 8, 14, 20, and the present study), vasopressin (or vasotocin) has also been shown preferentially to increase net Cl- absorption by cultured A6 or CCD cells under more physiological, open-circuit conditions (7, 20, 34). We also found that dDAVP stimulates the basal-to-apical component of Cl- flux measured in CFTR(+/+) cells but has no effect on CFTR(-/-) CCD cells. However, dDAVP also produces a similar increase in the predominant apical-to-basal component of Cl- flux in both CFTR(+/+) and CFTR(-/-) cells. Because only Cl- secretion can be detected under short-circuit conditions, the results obtained under the more physiological, open-circuit condition indirectly suggest that the apical-to-basal flux of Cl- stimulated by dDAVP occurs via the paracellular pathway (25). All together, the net cAMP-stimulated Cl- absorption observed in cultured cells under open-circuit conditions (8, 20, 33, and the present study) is in agreement with in vivo data demonstrating net Cl- absorption in isolated, microperfused CCD tubules (25). Although the presence of apical Cl- conductance has been documented in cultured CCD cells as well as in isolated, perfused CCD tubules (24), the evidence that Cl- secretion is part of Cl- transport across CCD in vivo is poorly documented (27) and relies almost entirely on the fact that microperfused CCD tubules dissected from rabbits fed a K+-rich diet exhibit active Cl- secretion (36).

It is now clear that CFTR, besides acting as a Cl- channel, can modulate the activity of several ion channels, including ENaC (3, 30, 31). The question arises whether CFTR can modulate Na+ absorption mediated by ENaC in principal CCD cells. We found that the amiloride-sensitive component of dDAVP-stimulated Isc, which reflects ENaC-mediated Na+ absorption, although slightly higher, was not significantly increased in CFTR(-/-) compared with CFTR(+/+) CCD cells. Thus CFTR has no effect on the basal Na+ transport and only slightly decreases the stimulatory effect of cAMP on Na+ transport in CCD. In contrast to what is normally observed in CCD, Boucher et al. (2) found that cAMP inhibits rather than stimulates Na+ absorption in normal nasal epithelial cells. These authors reported that basal Na+ absorption is higher in airway cells from CF patients than in normal cells and that it could be stimulated by cAMP. Enhanced basal Na+ absorption also occurs in the nasal epithelium and colon from the cftrm1unc mice (10, 11). In sharp contrast with the above studies, Reddy et al. (21) recently reported that ENaC activity is dependent on, and increases with, CFTR in freshly isolated normal sweat glands and that Na+ conductance is lower in CF sweat glands. The possibility cannot be excluded that there may be different regulatory pathways in different cell systems with differing vectorialized ion transport properties such as CCD, where CFTR mediates Cl- secretion, and sweat glands, where CFTR mediates Cl- absorption. Hence, disrupting CFTR in the CCD seems to have a marginal effect on Na+ absorption compared with that reported for most CFTR target tissues. The cellular heterogeneity from intact CCDs and primary cultured CCD cells may explain the lack of apparent interaction between CFTR and ENaC, just because intercalated cells (lacking ENaC) contain much more CFTR than principal cells and that, under most circumstances, there will be no or very poor interactions between ENaC and CFTR. Another possible explanation could be that the level of expression of CFTR in principal CCD cells remains very low and is therefore insufficient to regulate the predominant, more highly expressed ENaC in these cells. This would explain why the rate of Na+ absorption (which accounts for >80% of the total Isc measured) stimulated by dDAVP in CFTR(+/+) CCD cells does not change much when CFTR is disrupted and why an increase in fractional Na+ excretion can be demonstrated only under particular circumtances in CF mice (15).

In conclusion, this study provides direct evidence that CFTR mediates Cl- secretion, a minor component of overall Cl- transport, in primary-cultured mouse CCD cells and suggests that the increase in NaCl absorption elicited by vasopressin in vivo is not greatly different in CCD cells in which functional CFTR is lacking.


    ACKNOWLEDGEMENTS

We thank M. F. Bertrand (Centre de Développement des Techniques Avancées pour l'Expérimentation Animale, Orleans, France) and the Association Française de Lutte contre la Mucoviscidose, who kindly provided us the cftrm1Unc mice. We thank M. Ghosh for editing assistance.


    FOOTNOTES

This study was supported by INSERM and in part by a grant from the "Mucoviscidose:ABCF Proteins" and the Association pour l'aide à la recherche contre la mucoviscidose et l'assistance aux malades.

Present address of J.-P. Duong Van Huyen: Service d'Anatomie Pathologique, Hôpital Georges Pompidou, 75015 Paris, France.

Address for reprint requests and other correspondence: A. Vandewalle, INSERM U478, Faculté de Médecine Xavier Bichat, BP 416, 75870 Paris, Cédex 18, France (E-mail: vandewal{at}bichat.inserm.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 February 2001; accepted in final form 27 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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