Vasopressin-stimulated chloride transport in transimmortalized mouse cell lines derived from the distal convoluted tubule and cortical and inner medullary collecting ducts
Jean-Paul Duong Van Huyen1,
Marcelle Bens1,
Jacques Teulon2 and
Alain Vandewalle,1
1 Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 478
2 Unité U426, Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, Paris, France
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Abstract
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Background. The fine control of NaCl absorption takes place in the distal parts of the renal tubule, but the regulation of Cl- transport in this region has not been fully elucidated. We have analysed the effects of dD-arginine vasopressin (dDAVP) on Cl- fluxes in cultured mouse distal convoluted tubule (mpkDCT), cortical collecting duct (mpkCCD) and inner medullary collecting duct (mpkIMCD) cell lines.
Methods. RT-PCR and Western blotting were used to detect the amiloride-sensitive sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNAs and protein in cultured mpkDCT, mpkCCD and mpkIMCD cells. Cl- fluxes were analysed by measuring the short-circuit current (Isc) and bidirectional 36Cl- fluxes on confluent cells grown on filters.
Results. All three cell lines expressed ENaC and CFTR and had Isc stimulated by dDAVP. The rise in Isc caused by dDAVP (10-8 M) was inhibited by amiloride, and to a lesser extent by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) in all three cell lines. The dDAVP-dependent Isc measured under apical Na+-free condition was reduced by Cl- channel blockers with a profile (NPPB>glibenclamide>DIDS), similar to that for rat CFTR. dDAVP stimulated the apical-to-basal 36Cl- flux and to a lesser extent the basal-to-apical 36Cl- flux under open-circuit condition in all three cultured cell lines. Adding NPPB to the apical side reduced the basal-to-apical 36Cl- flux but not the opposite 36Cl- flux from dDAVP-treated cells.
Conclusion. These results indicate that dDAVP stimulates the bi-directional flux of Cl-, resulting in net Cl-absorption, in these cultured mouse distal and collecting duct cells. Isc experiments also suggest the presence of a minor component of electrogenic Cl- secretion, possibly mediated by CFTR.
Keywords: arginine vasopressin; chloride flux; CFTR; ENaC; renal cell line; short-circuit current
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Introduction
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The fine control of Na+ and water reabsorption, K+ secretion, and acidbase balance takes place in the distal parts of the renal tubule and is regulated by hormones [1,2]. Distal and collecting duct cells form tight epithelia and exhibit apical amiloride-sensitive epithelial sodium channel (ENaC), which is the rate-limiting step on sodium entry, and a basolateral, Na+, K+ ATPase pump, which provides the driven force for sodium exit [3]. Cl- transport in the connecting tubule and the collecting duct system involves a net transepithelial Cl- absorption by mechanisms which are not totally understood. This has been most intensively studied in the microperfused rabbit cortical collecting duct (CCD), where there is a Cl- permeable paracellular pathway and transcellular transport through the ß-intercalated cells (IC) via an apically located Cl-/HCO3- exchange and a basolateral Cl- conductance [4]. This segment also has the capacity to secrete Cl-via a transcellular process, at least under conditions of K+ loading [5]. A number of studies using the short-circuit current (Isc) method have likewise demonstrated that amphibian A6 cells [6] and cultured CCD and inner medullary collecting duct (IMCD) cells can be stimulated by vasotocin or vasopressin to secrete Cl- [710]. However, under more physiological condition, i.e. open-circuit condition, vasopressin (or vasotocin) preferentially increases net Cl-absorption by the same cells [6,8,9]. Thus, net Cl-absorption probably results from a balance between absorption and secretion, as pointed out by Simmons [11]. It has also been suggested that the cystic fibrosis transmembrane conductance regulator (CFTR) mediates electrogenic secretion of Cl- in cultured mouse and rat IMCD cells [10,12]. Expression of CFTR has been also reported in rabbit DCT [13,14] and mouse M1 CCD cell lines [15]. Thus the question arises whether CFTR, like in IMCD cells, could also play a role in the secretion of Cl- in distal and cortical collecting duct cells.
The present study analyses the effect of dD-arginine vasopressin (dDAVP) and anion channel inhibitors on the short-circuit curent (Isc) and bi-directional 36Cl- flux in three immortalized mouse cell lines derived from microdissected DCT, CCD, and IMCD. We used the mpkDCT [16], and mpkCCD [9] cell lines, which were derived from the kidneys of the same SV-PK/Tag transgenic mice carrying the SV40 large T antigen (Tag) placed under the control of the L-type pyruvate kinase (L-PK) promoter fused to the SV40 enhancer [17], and a new mpkIMCD cell line derived from IMCDs microdissected from the same strain of SV-PK/Tag mice.
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Subjects and methods
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Materials
Culture media (DMEM, Ham's F12) were from Life Technologies (Eragny, France). [
-32P]dCTP was from New England Nuclear (Le Blanc Mesnil, France). 36Cl-was purchased from Amersham (Les Ulis, France). NPPB was from Research Biochemicals Incorp. (Natrick, MA). All hormones and reagents were from Sigma (St Louis, MO). The anti-large T antigen antibody was a gift from D. Hanahan (University of San Francisco, CA). The anti-ZO-1 antibody was from Chemicon International Inc. (Temecula, CA). The anti-
-Na+, K+-ATPase antibody was a gift from M. Caplan (Yale University, New Haven, CT). The RNA-PLUS extraction kit was purchased from Bioprobe Systems (Montreuil-sous-Bois, France). The Moloney murine leukaemia virus (M-MLV) reverse transcriptase was from Life Technologies. Tissue Culture Treated Snapwell or Transwell filters (0.4 µm pore size, 1 cm2 insert growth area) were from Corning Costar Corp. (Cambridge, MA).
Cell lines
The strategy of targeted oncogenesis in transgenic mice has proved to be a powerful method to derive immortalized renal cells from the tissues of transgenic mice expressing the simian virus 40 (SV40) [18]. We used the mpkDCT and mpkCCD cells previously established in our laboratory [9,16]. The mpkDCT and mpkCCD cells were both derived from isolated DCTs and CCDs microdissected from the kidneys of SV-PK/Tag transgenic mice [17]. The protocol used to establish the mpkIMCD cells was similar to that described for mpkCCD and mpkDCT cells. Briefly, the kidneys of 1-month-old SV-PK/Tag transgenic mouse were removed under sterile conditions, sliced, and incubated in medium (DMEM : Ham's F12, 1 : 1 vol/vol) containing 0.1% (wt/vol) collagenase fo 1 h at 37°C. The slices were rinsed in medium, and IMCD fragments were microdissected. Pools of 510 isolated IMCD were rinsed, transferred to collagen-coated 24-well trays, and cultured in a modified DM medium (DMEM:Ham's F12, 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; 20 mM D-glucose; 2% fetal calf serum (FCS); and 20 mM HEPES, pH 7.4) [9] at 37°C in 5% CO295% air atmosphere. After several passages, the cells grew faster and were cultured in 25cm2 culture flaskes (Corning Costar Corp., Cambridge, MA). All mpkDCT, mpkCCD, and mpkIMCD cells were grown in the same modified DM medium described above. The medium was changed every 2 days and all studies described in this report were performed on confluent cells (day 710 after seeding) taken between the 10th and 25th passages.
Immunohistochemical studies
Confluent cells were fixed with ice-cold methanol and processed for immunofluorescence using anti-large T antigen, anti-ZO-1, and anti-
-Na+, K+-ATPase antibodies as previously described [9]. Specimens were examined under a Zeiss photomicroscope or by confocal laser scanning microscopy (CLSM, Leica).
Western blotting
ENaC and CFTR were detected by Western blotting using an immuno-purified anti-rabbit
-ENaC antibody [19] and the antibody 169 directed against the R domain of CFTR [20]. Confluent cells were scraped off, collected in phosphate-buffered saline (PBS), centrifuged (150 g for 5 min) and homogenized in 50 µl lysis buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM TrisHCl, pH 7.4, containing 0.5% (w/v) SDS, 0.1 mM PMSF and 100 µg/ml leupeptin). Protein content was determined by the Bradford method [21] using bovine serum albumin as standard. Cell homogenates (50 µg) were subfractionated by SDSPAGE on 10% (for ENaC) or 6% (for CFTR) acrylamide gels. The proteins were transferred to polyvinylidene difluoride membranes (NEN, Le Blanc Mesnil, France), probed (4 h at 4°C) with the anti-
-ENaC or the anti-CFTR (1:500) antibody and then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Dako, Trappes, France) for 60 min at room temperature. Negative controls were performed by omitting the primary antibody. The antigen-antibody complexes were detected using the NBT-BCIP alkaline phosphatase substrate (Sigma).
RNA extraction and reverse transcription-PCR
Total RNA was extracted from confluent cells using the RNA-PLUS extraction kit. RNA (2 µg) was reverse-transcribed with M-MLV reverse transcriptase at 42°C for 45 min, and 100 ng cDNA and non-reverse-transcribed RNA were amplified for 28 cycles in 100 µl total volume containing 50 mM KCl, 20 mM TrisHCl (pH 8.4), 40 µM dNTP, 1.5 mM MgCl2, 1µCi [
-32P]dCTP, 1 unit Taq polymerase, 29, 31 or 29 pmol of
-, ß-,
-rENaC primers [22] respectively, 31 pmol of CFTR primers [9] and 16.5 pmol of hGAPDH [22] or 0.3 pmol of ß-actin [9] primers. The thermal cycling programmes were as follows: 94°C for 30 s, 55°C (CFTR), 54°C (
-rENaC) or 53°C (ß- and
-rENaC) for 30 s, and 72°C for 60 s.
cAMP assay
The influence of deamino-8-D-arginine vasopressin (dDAVP) on the cell cAMP content of cultured cells was assayed as described earlier [9].
Electrophysiological studies
The three mpk cell lines were grown on Snapwell filters in DM medium until confluent (day 6), and then in DM without EGF, hormones or FCS (HFM containing 29 mM NaHCO3) for a final 18 h. The filters were mounted in a diffusion chamber (Costar Corp.) connected to a voltage clamp apparatus via glass barrel Micro-Reference Ag/AgCl electrodes filled with 3M KCl. The cell layers were bathed on each side with 8 ml HFM medium warmed to 37°C and continuously gassed with 95% O25% CO2 to keep the pH at 7.4. Isc (µA/cm2) was measured by clamping the open-circuit VT to 0 mV for 1 s. Isc was also measured on cells in which the apical NaHCO3-HFM medium was replaced by a Na+-free solution [9]. By convention, a positive Isc corresponded to a flow of positive charges from the apical to the basal solution. The transepithelial electrical resistance (RT) was calculated with Ohm's law from transepithelial voltage (VT) and Isc.
36Cl- flux studies
The transepithelial transport of 36Cl-was measured on cells grown on Transwell filters [9]. The inside of the filters was filled with 600 µl and the outside with 1.2 ml HFM medium. For apical-to-basal flux measurements 36Cl- (150 nCi/ml) was added to the apical medium bathing the cells (inside of the filter). 36Cl- (150 nCi/ml) was added to the basal medium bathing the cells (outside of the filter) for basal-to-apical flux measurements. Cell layers were incubated at 37°C, and 50 µl samples of apical or basal medium were collected from opposite sides of the filters, 10, 30 or 60 min after adding the radioactive tracers. The radioactivity was then measured in a liquid scintillation counter (LKB, Pharmacia). The results are expressed as nEq per cm2 filter area.
Statistical analysis
Results are expressed as means±SE from (n) experiments. Significant differences were analysed by Student's t test. P value <0.05 was considered significant.
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Results
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Morphological features of mpkDCT, mpkCCD and mpkIMCD cells and ENaC and CFTR mRNA and protein expressions
Confluent cells grown on Petri dishes formed monolayers of cuboid cells and formed domes (Figure 1A
, B,C). All cells were positive for nuclear large T antigen (Figure 1D
,E
,F
). They possessed the tight junction-associated protein ZO-1 (Figure 1G
,H
,I
) and their basolateral membranes were positively immunostained with the anti-
-Na+, K+-ATPase antibody (Figure 1J
,K
,L
).

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Fig. 1. Morphology of immortalized late DCT, CCD and IMCD cells.Confluent mpkDCT (A), mpkCCD (B) and mpkIMCD (C) cells have a uniform shape and form domes. All the cells had nuclei that were positive for large T antigen (DF), ZO-1 immunostaining delineated the cell borders (GI), and the basolateral membranes were labelled with the anti- -Na+, K+-ATPase antibody (JL). Bars: AC, 50 µm; DL, 10 µm.
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RT-PCR was used to detect ENaC and CFTR mRNAs. The three mouse renal cell lines all contained ENaC and CFTR transcripts (Figure 2A
). There was always more
-ENaC mRNA than ß and
mRNAs, as compared to the amount of GAPDH mRNA used as internal standard (Figure 2A
). Western blotting analyses using the anti-
-ENaC and anti-CFTR antibodies showed that the three cultured cell lines contained both ENaC and CFTR proteins (Figure 2B
). The single ENaC (
100 kDa) and CFTR (
170 kDa) protein bands identified appeared to be specific, since they were not detected when the primary antibodies were omitted (Figure 2B
). These results indicated thus that these three mouse distal and collecting duct cell lines have conserved the typical features of polarized epithelial cells and have conserved ENaC and CFTR expression.
Electrophysiological properties of mpkDCT, mpkCCD, and mpkIMCD cells
Confluent cells grown on filters developed high RT (mpkDCT, 2041±146; mpkCCD, 3166±337; mpkIMCD, 3363±262
/cm2, n=23-32), negative VT (mpkDCT, -41±4; mpkCCD, -54±4; mpkIMCD, -50±5 mV) and positive Isc (mpkDCT, +24±3; mpkCCD, +22±3; mpkIMCD, +16±2 µA/cm2). As previously reported for mpkDCT cells [16] and mpkCCD cells [9], dDAVP (10-6 M) significantly increased (P<0.001) the cAMP content of IMCD cells (basal: 23±4; +dDAVP: 734±136 pmol/7 min/mg protein, n=8). dDAVP added to the basal side of mpkDCT, mpkCCD, and mpkIMCD cells also increased Isc in a dose-dependent manner (Figure 3
). The maximal Isc values were always obtained with 10-9 to 10-8 M dDAVP. Accordingly, all subsequent experiments were performed with 10-8 M dDAVP. dDAVP added to the basal side of cultured DCT, CCD and IMCD cells caused a rapid rise in Isc (1 min) which plateaued after 10 min (Figure 4
). Adding NPPB (10-4 M), the most potent Cl- channel blocker available [23], to the apical side rapidly decreased (by 1530%) the dDAVP-stimulated Isc. The subsequent addition of apical amiloride (10-6 M) almost completely inhibited Isc (Figure 4
). Thus these short-circuit current experiments indicated that all mouse cultured DCT, CCD and IMCD cells had a predominantly amiloride-sensitive Na+ conductance (ENaC), and more discrete NPPB-sensitive Cl- conductance(s).

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Fig. 3. Effects of dDAVP on short-circuit current. Isc was measured on confluent mpkDCT, mpkCCD and mpkIMCD cells in the absence ( ) or presence () of increasing concentrations of dDAVP applied to the basal medium. Values are means±SE from 610 separate experiments.
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Effects of anion transporter blockers on Cl- secretion
To test whether electrogenic secretion of Cl- mediated by CFTR occurred in cultured mouse DCT, CCD, and IMCD cells, Isc was measured under apical Na+-free condition in the presence of various inhibitors of anion transporters. Replacing Na+ by N-methyl-D-glucamine on the apical side of all cultured DCT, CCD, and IMCD cells resulted in a dramatic decrease in Isc under basal conditions (Figure 5
). This decrease in Isc was more marked in DCT cells, which developed a high negative Isc, than in CCD and IMCD cells. Adding NPPB to the apical side further decreased the positive Isc measured in basal conditions and fully prevented the discrete rise in Isc (corresponding to Cl- secretion) caused by dDAVP (Figure 5
). Apical µM glibenclamide, which partially inhibits CFTR [24], also decreased the Isc of cultured DCT, CCD and IMCD cells and prevented the rise in Isc caused by dDAVP (Figure 5
). However, the inhibition caused by glibenclamide was less marked than that of NPPB. In contrast, apical 100 µM 4,4'-diisothiocyanatostilbene-2,2'-di-sulphonic acid (DIDS) neither altered the Isc of untreated cells nor impaired the dDAVP-stimulated Isc (Figure 5
). Thus, these mouse distal and collecting duct cells all had similar profiles of Isc inhibition caused by the anion transporter blocking agents (NPPB>glibenclamide>DIDS), although the inhibitory actions of these agents appeared to be more marked in DCT cells than in CCD and IMCD cells.
Effects of dDAVP on Cl- fluxes
We then measured the bidirectional transport of 36Cl- in untreated and dDAVP-treated cells under the more physiological open-circuit condition (Figure 6
). In all cases, both the apical-to-basal and basal-to-apical 36Cl- fluxes increased linearly over 60 min (Figure 6
, left panels). The apical-to-basal transport of 36Cl- was slightly higher than the basal-to-apical transport of 36Cl- in all untreated cell lines (Figure 6
, left panels). The apical-to-basal Cl- flux was significantly greater in cells treated with dDAVP for 60 min (Figure 6
, left panels). The increase in apical-to-basal Cl- flux caused by dDAVP was also greater in cultured DCT than in cultured CCD and IMCD cells (Figure 6
, right panels). dDAVP also stimulated the basal-to-apical component of Cl- flux, but to a lesser extent (Figure 6
, right panels). As a consequence, dDAVP caused a significant increase (p<0.05) in the net Cl- absorbed by cultured DCT (241 nEq/60 min/cm2), CCD (148 nEq/60 min/cm2) and IMCD (94 nEq/60 min/cm2) cells. Adding NPPB (10-4 M) to the apical side of the cells did not alter the apical-to-basal transport of 36Cl- from dDAVP-treated cells, but did prevent the rise in the basal-to-apical 36Cl- flux caused by dDAVP (Table 1
). However, the inhibition of Cl-secretion by NPPB was more marked in the cultured DCT and CCD cells than in IMCD cells. These results therefore indicate that dDAVP stimulated both apical-to-basal and basal-to-apical Cl- fluxes and that NPPB inhibited, at least partially, the secreted component of Cl- flux in these mouse distal and collecting duct cell lines.
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Discussion
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The results of this study indicate that cultured mouse DCT, CCD, and IMCD cells derived from the same strain of transgenic mice are all sensitive to dDAVP, and express ENaC mRNAs and proteins. The short-circuit current experiments show that dDAVP stimulates mainly Na+ absorption mediated by amiloride-sensitive ENaC channels, in all three mouse cultured DCT, CCD, and IMCD cells. It is clear, however, that a minor electrogenic Cl- secretion is also present in the same cell lines. A similar Cl- secretion has been reported for other cultured CCD cells [89] and IMCD cells [10,12,25], but not for DCT cells, where the rise in Cl- secretion was the greatest.
We designed specific experiments to determine the pharmacological profile of Cl- secretion. The fraction of Isc due to Cl- secretion is inhibited by the Cl- channel blockers NPPB and glibenclamide, and the basal-to-apical 36Cl- flux measured under open-circuit condition that is stimulated by dDAVP, although to a lesser extent than the opposite Cl- flux, is partially inhibited by NPPB. DIDS has no effect. This pattern of inhibition compares well with that obtained in previous studies on CCD and IMCD cells. In rabbit CCD cells in primary culture, the fraction of Isc that is dependent on Cl- is inhibited by NPPB but not by DIDS [8]. Similar results have been obtained in rat IMCD cells in primary culture [12] and this last study showed that glibenclamide, a blocker of the CFTR Cl- channel, is also effective. Thus there is a Cl- conductive pathway at the apical membrane of DCT, CCD and IMCD cells, with an inhibition profile all along the distal tubule that is similar in the mouse, rat and rabbit.
The presence of Cl- channels at the apical membrane, as a basis for Cl- secretion into the lumen, has been documented in a variety of cultured cells from DCT through IMCD, which contain several types of Cl- channels [26]. There are multiple indirect indications that the CFTR protein takes part in this phenomenon. CFTR mRNA has been detected by RT-PCR all along the nephron, in renal tissues and in cultured cells [26]. Specifically in this study we have shown that CFTR mRNA and protein are present in the three cell types. The profile of inhibition of the Cl--dependent fraction of Isc demonstrated in the present and previous studies is reminiscent but not strictly identical to that of CFTR [27]. Patch-clamp studies have also shown that there are CFTR-like Cl- currents in cultured DCT [13,14], CCD [15,28] and IMCD [12,25] cells. However, electrophysiological information alone is insufficient to demonstrate the presence of CFTR, since, for example, thick ascending limb cells also contain a distinct Cl- channel that has some properties of a CFTR Cl- channel [29]. Finally, a study on microsomes from the rat kidney inner medulla has shown partial inhibition of Cl--dependent cell swelling using a purified antibody against CFTR [30]. Thus there is a growing body of evidence that CFTR takes part in a Cl-secreting pathway in the apical membrane of the distal nephron, but final proof will only be obtained by comparing Cl-fluxes in renal cells from normal and CFTR knock-out mice.
The net transepithelial transport of Cl-results in Cl- absorption in DCT as well as in CCD and IMCD cells. Cl- flux studies performed under open-circuit conditions show that dDAVP preferentially stimulates net Cl- absorption. These results are similar to those obtained in the amphibian A6 cell line [6] and in rabbit CCD cells in primary culture [8]. We also found that the net Cl- absorption is greater in cultured mouse DCT cells than in cultured mouse CCD and IMCD cells counterparts. It cannot be excluded that the balance between absorption and secretion is altered under specific conditions, so that the overall Cl- transport may shift from absorption to secretion, especially in IMCD cells where the difference between apical-to-basal and basal-to-apical Cl- fluxes is small.
In conclusion, these transimmortalized mouse renal cells derived from microdissected distal and collecting duct cells expressed both ENaC and CFTR and have retained Na+ and Cl- transport stimulated by vasopressin. Isc experiments using anion channel blockers and Cl- fluxes studies also evidenced the presence of a Cl- secretory pathway, not only in IMCD cells [12,25], but also in DCT and CCD cells.
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Acknowledgments
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This study was supported by INSERM and in parts by grants from INSERM (Contrat Prisme to J. Teulon) and by grants from the Muscoviscidose: ABC protéines and the Association pour l'aide à la recherche contre la mucoviscidose et l'assistance aux malades. We thank L. Pascual Letallec for technical assistance, F. Cluzeaud for her help in confocal microscopy, S. Roger and P. Disdier for photographic work. We thank M. Caplan, B. C. Rossier, and W. B. Guggino for generously providing us with valuable antibodies and O. Parkes for editorial assistance.
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Notes
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Correspondence and offprint requests to: Dr Alain Vandewalle, INSERM U478, Faculté de Médecine Xavier Bichat, BP 416, F-75870 Paris Cédex 18, France. 
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References
|
---|
-
Kirk KL, Schafer JA. Water transport and osmoregulation by antidiuretic hormone in terminal nephron segments. In: Seldin DW, Giebisch G, eds. The Kidney: Physiology and Physiopathology. Raven Press, New York, 1992; 16931725
-
Koeppen BM, Stanton BA. Sodium chloride transport: distal nephron. In: Seldin DW, Giebisch G, eds. The Kidney: Physiology and Physiopathology. Raven Press, New York, 1992; 20032039
-
Garty H, Palmer LG. Epithelial sodium channels: function, structure and regulation. Physiol Rev1997; 77: 359396[Abstract/Free Full Text]
-
Schuster VL, Stokes JB. Chloride transport by the cortical and outer medullary collecting duct. Am J Physiol1987; 253: F202212[ISI]
-
Wingo BA. Active and passive chloride transport by the rabbit collecting duct. Am J Physiol1990; 258: F13881393[Abstract/Free Full Text]
-
Verrey F. Antidiuretic hormone action in A6 cells: Effect of apical Cl and Na conductances and synergism with aldosterone for NaCl reabsorption. J Membr Biol1994; 138: 6576[ISI][Medline]
-
Chalfant ML, Coupaye-Gerard B, Kleyman TR. Distinct regulation of Na+ reabsorption and Cl- secretion by arginine vasopressin in the amphibian cell line A6. Am J Physiol1993; 264: C14801488[Abstract/Free Full Text]
-
Nagy E, Náray-Fejes-Tóth A, Fejes-Tóth G. Vasopressin activates a chloride conductance in cultured cortical collecting duct cells. Am J Physiol1994; 267: F831838[Abstract/Free Full Text]
-
Duong Van Huyen JP, Bens M, Vandewalle A. Differential effects of aldosterone and vasopressin on chloride fluxes in transimmortalized mouse cortical collecting duct cells. J Membr Biol1998; 164: 7990[ISI][Medline]
-
Kizer NL, Lewis B, Stanton B. Electrogenic sodium reabsorption and chloride secretion by an inner medullary collecting duct cell line (mIMCD-K2). Am J Physiol1995; 268: F347355[Abstract/Free Full Text]
-
Simmons NL. Renal epithelial Cl- secretion. Exp Physiol1993; 78: 117137[ISI][Medline]
-
Husted RF, Volk KA, Sigmund RD, Stokes JB. Anion secretion by the inner medullary collecting duct: evidence for involvement of the cystic fibrosis transmembrane conductance regulator. J Clin Invest1995; 95: 644650[ISI][Medline]
-
Poncet V, Tauc M, Bidet M, Poujeol P. Chloride channels in apical membranes of primary cultures of rabbit distal bright convoluted tubule. Am J Physiol1994; 266: F543553[Abstract/Free Full Text]
-
Rubera I, Tauc M, Verheecke-Mauze C et al. Regulation of cAMP-dependent chloride channels in DCT1 immortalized rabbit distal tubule cells in culture. Am J Physiol1999; 276: F104121[Abstract/Free Full Text]
-
Letz B, Korbmacher LB. cAMP stimulates CFTR-like Cl- channels and inhibits amiloride-sensitive Na+ channels in mouse CCD cells. Am J Physiol1997; 272: C657666[Abstract/Free Full Text]
-
Peng K-C, Cluzeaud F, Bens M et al. Tissue and cell distribution of the multidrug resistance-associated protein (MRP) in mouse intestine and kidney. J Histochem Cytochem1999; 47: 757768[Abstract/Free Full Text]
-
Miquerol L, Cluzeaud F, Porteu A, Alexandre Y, Vandewalle A, Kahn A. Tissue specificity of L-Pyruvate kinase transgenes results from combinatorial effects of proximal promoter and distal activator regions. Gene Express1996; 5: 315330[ISI]
-
Briand P, Kahn A, Vandewalle A. Targeted oncogenesis: A powerful method to derive renal cell lines. Kidney Int1995; 47: 388394[ISI][Medline]
-
Duc C, Farman N, Canessa CM, Bonvalet JP, Rossier BC. Cell-specific expression of epithelial sodium channel
, ß,
subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol1994; 127: 19071921[Abstract]
-
Zeitlin PL, Crawford I, Lu L et al. CFTR protein expression in primary and cultured epithelia. Proc Natl Acad Sci USA1992; 89: 344347[Abstract]
-
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem1976; 72: 248254[ISI][Medline]
-
Hummler E, Barker P, Gatzy J et al. Early death due to defective neonatal lung liquid clearance in
ENaC-deficient mice. Nature Genet1996; 12: 325328[ISI][Medline]
-
Wangemann P, Wittner M, Di Stephano A et al. Cl--channel blockers in the thick ascending limb of the loop of Henle. Structure activity relationship. Pflügers Arch1986; 407: S128141[ISI][Medline]
-
Sheppard DN, Welsh MJ. Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane regulator chloride currents. J Gen Physiol1992; 100: 573591[Abstract]
-
Vandorpe D, Kizer N, Ciampollilo F et al. CFTR mediates electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells. Am J Physiol1995; 269: C683689[Abstract]
-
Schwiebert EM, Lopes AM, Guggino WB. Chloride channels along the nephron. In: Guggino WB, ed. Current Topics in Membranes, vol. 42. Academic Press Inc, San Diego, 1994; 265315[ISI]
-
Schultz BD, Singh AAK, Devor DC, Bridges RJ. Pharmacology of CFTR chloride channel activity. Physiol Rev1999; A [Suppl]: S109212
-
Ling BN, Kokko KE, Eaton DC. Prostaglandin E2 activates clusters of apical Cl- channels in principal cells via a cyclic adenosine monophosphate-dependent pathway. J Clin Invest1994; 93: 829837[ISI][Medline]
-
Marvão P, De Jesus Ferreira M-C, Bailly C et al. Cl- absorption across the thick ascending limb is not altered in cystic fibrosis mice: a role for a pseudo-CFTR Cl- channel. J Clin Invest1998; 102: 19861993[Abstract/Free Full Text]
-
Benharouga M, Lipecka J, Fanen P et al. Properties of a Cl--conductive pathway(s) in microsomes from rat kidney inner medulla. Involvement of cystic fibrosis transmembrane conductance regulator protein. Eur J Biochem1996; 240: 268273[Abstract]
Received for publication: 9. 6.00
Revision received 14. 9.00.