Vasopressin-stimulated chloride transport in transimmortalized mouse ...

Nephrol Dial Transplant (2001) 16: 238±245

Original Article

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 Vandewalle1 Institut National de la SanteÂ et de la Recherche MeÂdicale (INSERM), 1 UniteÂ 478 and 2 UniteÂ U426, Institut FeÂdeÂratif de Recherche 02, FaculteÂ de MeÂdecine Xavier Bichat, Paris, France

Abstract Background. The ®ne 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 ¯uxes 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 ®brosis transmembrane conductance regulator (CFTR) mRNAs and protein in cultured mpkDCT, mpkCCD and mpkIMCD cells. Cl ¯uxes were analysed by measuring the short-circuit current (Isc) and bidirectional 36Cl ¯uxes on con¯uent cells grown on ®lters. 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-nitro2-(3-phenylpropylamino)-benzoic acid (NPPB) in all three cell lines. The dDAVP-dependent Isc measured under apical Naq-free condition was reduced by Cl channel blockers with a pro®le (NPPB)glibenclamide)DIDS), similar to that for rat CFTR. dDAVP stimulated the apical-to-basal 36Cl ¯ux and to a lesser extent the basal-to-apical 36Cl ¯ux under open-circuit condition in all three cultured cell lines. Adding NPPB to the apical side reduced the basalto-apical 36Cl ¯ux but not the opposite 36Cl ¯ux from dDAVP-treated cells. Conclusion. These results indicate that dDAVP stimulates the bi-directional ¯ux of Cl , resulting in net Cl absorption, in these cultured mouse distal and collecting duct cells. Isc experiments also suggest the Correspondence and offprint requests to: Dr Alain Vandewalle, INSERM U478, FaculteÂ de MeÂdecine Xavier Bichat, BP 416, F-75870 Paris CeÂdex 18, France. #

presence of a minor component of electrogenic Cl secretion, possibly mediated by CFTR. Keywords: arginine vasopressin; chloride ¯ux; CFTR; ENaC; renal cell line; short-circuit current

Introduction The ®ne control of Naq and water reabsorption, Kq secretion, and acid±base balance takes place in the distal parts of the renal tubule and is regulated by hormones w1,2x. Distal and collecting duct cells form tight epithelia and exhibit apical amiloride-sensitive epithelial sodium channel (ENaC), which is the ratelimiting step on sodium entry, and a basolateral, Naq, Kq ATPase pump, which provides the driven force for sodium exit w3x. 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 b-intercalated cells (IC) via an apically located Cl uHCO3 exchange and a basolateral Cl conductance w4x. This segment also has the capacity to secrete Cl via a transcellular process, at least under conditions of Kq loading w5x. A number of studies using the shortcircuit current (Isc) method have likewise demonstrated that amphibian A6 cells w6x and cultured CCD and inner medullary collecting duct (IMCD) cells can be stimulated by vasotocin or vasopressin to secrete Cl w7±10x. However, under more physiological condition, i.e. open-circuit condition, vasopressin (or vasotocin) preferentially increases net Cl absorption by the same cells w6,8,9x. Thus, net Cl absorption probably results from a balance between absorption and secretion, as pointed out by Simmons w11x. It has also been suggested that the cystic ®brosis transmembrane

2001 European Renal Association±European Dialysis and Transplant Association

Chloride transport by cultured mouse distal and collecting duct cells

conductance regulator (CFTR) mediates electrogenic secretion of Cl in cultured mouse and rat IMCD cells w10,12x. Expression of CFTR has been also reported in rabbit DCT w13,14x and mouse M1 CCD cell lines w15x. 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 ¯ux in three immortalized mouse cell lines derived from microdissected DCT, CCD, and IMCD. We used the mpkDCT w16x, and mpkCCD w9x cell lines, which were derived from the kidneys of the same SV-PKuTag 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 w17x, and a new mpkIMCD cell line derived from IMCDs microdissected from the same strain of SV-PKuTag mice.

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(DMEM : Ham's F12, 1 : 1 voluvol; 60 nM sodium selenate; 5 mguml transferrin; 2 mM glutamine; 50 nM dexamethasone; 1 nM triiodothyronine; 10 nguml epidermal growth factor (EGF); 5 mguml insulin; 20 mM D-glucose; 2% fetal calf serum (FCS); and 20 mM HEPES, pH 7.4) w9x at 378C in 5% CO2±95% air atmosphere. After several passages, the cells grew faster and were cultured in 25cm2 culture ¯askes (Corning Costar Corp., Cambridge, MA). All mpkDCT, mpkCCD, and mpkIMCD cells were grown in the same modi®ed DM medium described above. The medium was changed every 2 days and all studies described in this report were performed on con¯uent cells (day 7±10 after seeding) taken between the 10th and 25th passages.

Immunohistochemical studies Con¯uent cells were ®xed with ice-cold methanol and processed for immuno¯uorescence using anti-large T antigen, anti-ZO-1, and anti-a-Naq, Kq-ATPase antibodies as previously described w9x. Specimens were examined under a Zeiss photomicroscope or by confocal laser scanning microscopy (CLSM, Leica).

Subjects and methods Materials Culture media (DMEM, Ham's F12) were from Life Technologies (Eragny, France). wa-32PxdCTP 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-a-Naq, Kq-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 ®lters (0.4 mm 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) w18x. We used the mpkDCT and mpkCCD cells previously established in our laboratory w9,16x. The mpkDCT and mpkCCD cells were both derived from isolated DCTs and CCDs microdissected from the kidneys of SV-PKuTag transgenic mice w17x. The protocol used to establish the mpkIMCD cells was similar to that described for mpkCCD and mpkDCT cells. Brie¯y, the kidneys of 1-month-old SV-PKuTag transgenic mouse were removed under sterile conditions, sliced, and incubated in medium (DMEM : Ham's F12, 1 : 1 voluvol) containing 0.1% (wtuvol) collagenase fo 1 h at 378C. The slices were rinsed in medium, and IMCD fragments were microdissected. Pools of 5±10 isolated IMCD were rinsed, transferred to collagen-coated 24-well trays, and cultured in a modi®ed DM medium

Western blotting ENaC and CFTR were detected by Western blotting using an immuno-puri®ed anti-rabbit a-ENaC antibody w19x and the antibody 169 directed against the R domain of CFTR w20x. Con¯uent cells were scraped off, collected in phosphatebuffered saline (PBS), centrifuged (150 g for 5 min) and homogenized in 50 ml lysis buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM Tris±HCl, pH 7.4, containing 0.5% (wuv) SDS, 0.1 mM PMSF and 100 mguml leupeptin). Protein content was determined by the Bradford method w21x using bovine serum albumin as standard. Cell homogenates (50 mg) were subfractionated by SDS±PAGE on 10% (for ENaC) or 6% (for CFTR) acrylamide gels. The proteins were transferred to polyvinylidene di¯uoride membranes (NEN, Le Blanc Mesnil, France), probed (4 h at 48C) with the antia-ENaC or the anti-CFTR (1 : 500) antibody and then incubated with alkaline phosphatase-conjugated goat antirabbit 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 con¯uent cells using the RNA-PLUS extraction kit. RNA (2 mg) was reversetranscribed with M-MLV reverse transcriptase at 428C for 45 min, and 100 ng cDNA and non-reverse-transcribed RNA were ampli®ed for 28 cycles in 100 ml total volume containing 50 mM KCl, 20 mM Tris±HCl (pH 8.4), 40 mM dNTP, 1.5 mM MgCl2, 1mCi wa-32PxdCTP, 1 unit Taq polymerase, 29, 31 or 29 pmol of a , b , c-rENaC primers w22x respectively, 31 pmol of CFTR primers w9x and 16.5 pmol of hGAPDH w22x or 0.3 pmol of b-actin w9x primers. The thermal cycling programmes were as follows: 948C for 30 s, 558C (CFTR), 548C (a rENaC) or 538C (b- and c-rENaC) for 30 s, and 728C for 60 s.

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cAMP assay The in¯uence of deamino-8-D-arginine vasopressin (dDAVP) on the cell cAMP content of cultured cells was assayed as described earlier w9x.

Electrophysiological studies The three mpk cell lines were grown on Snapwell ®lters in DM medium until con¯uent (day 6), and then in DM without EGF, hormones or FCS (HFM containing 29 mM NaHCO3) for a ®nal 18 h. The ®lters were mounted in a diffusion chamber (Costar Corp.) connected to a voltage clamp apparatus via glass barrel Micro-Reference AguAgCl electrodes ®lled with 3M KCl. The cell layers were bathed on each side with 8 ml HFM medium warmed to 378C and continuously gassed with 95% O2±5% CO2 to keep the pH at 7.4. Isc (mAucm2) 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 Naq-free solution w9x. By convention, a positive Isc corresponded to a ¯ow 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. 36

Cl ¯ux studies

The transepithelial transport of 36Cl was measured on cells grown on Transwell ®lters w9x. The inside of the ®lters was ®lled with 600 ml and the outside with 1.2 ml HFM medium. For apical-to-basal ¯ux measurements 36Cl (150 nCiuml) was added to the apical medium bathing the cells (inside of the ®lter). 36Cl (150 nCiuml) was added to the basal medium bathing the cells (outside of the ®lter) for basal-to-apical ¯ux measurements. Cell layers were incubated at 378C, and 50 ml samples of apical or basal medium were collected from opposite sides of the ®lters, 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 ®lter area.

J-P. Duong Van Huyen et al.

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 a-ENaC mRNA than b and c mRNAs, as compared to the amount of GAPDH mRNA used as internal standard (Figure 2A). Western blotting analyses using the anti-a-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 identi®ed appeared to be speci®c, 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 Con¯uent cells grown on ®lters developed high RT (mpkDCT, 2041"146; mpkCCD, 3166"337; mpkIMCD, 3363"262 Vucm2, ns23 32), negative VT (mpkDCT, 41"4; mpkCCD, 54"4; mpkIMCD, 50"5 mV) and positive Isc (mpkDCT, q24"3; mpkCCD, q22"3; mpkIMCD, q16"2 mAucm2). As previously reported for mpkDCT cells w16x and mpkCCD cells w9x, dDAVP (10 6 M) signi®cantly

Statistical analysis Results are expressed as means"SE from (n) experiments. Signi®cant differences were analysed by Student's t test. P value -0.05 was considered signi®cant.

Results Morphological features of mpkDCT, mpkCCD and mpkIMCD cells and ENaC and CFTR mRNA and protein expressions Con¯uent 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 junctionassociated protein ZO-1 (Figure 1G,H,I) and their basolateral membranes were positively immunostained with the anti-a-Naq, Kq-ATPase antibody (Figure 1J,K,L).

Fig. 1. Morphology of immortalized late DCT, CCD and IMCD cells.Con¯uent 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 (D±F), ZO-1 immunostaining delineated the cell borders (G±I), and the basolateral membranes were labelled with the anti-a-Naq, Kq-ATPase antibody (J±L). Bars: A±C, 50 mm; D±L, 10 mm.


increased (P-0.001) the cAMP content of IMCD cells (basal: 23"4; qdDAVP: 734"136 pmolu7 minumg protein, ns8). dDAVP added to the basal side of mpkDCT, mpkCCD, and mpkIMCD cells also

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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 w23x, to the apical side rapidly decreased (by 15±30%) 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 Naq conductance (ENaC), and more discrete NPPB-sensitive Cl conductance(s).

Effects of anion transporter blockers on Cl secretion

Fig. 2. ENaC and CFTR mRNAs and proteins in cultured late distal and collecting duct cells. (A) cDNA from mpkDCT, mpkCCD and mpkIMCD cells were ampli®ed by RT-PCR (lanes 1) with primers speci®c for a-, b-, and c-rENaC and CFTR (arrows) and with sets of hGAPDH or b-actin primers (arrowheads). No ampli®ed product was detected with non-reverse-transcribed RNA (lanes 2). (B) mpkDCT (lanes 1), mpkCCD (lanes 2) and mpkIMCD (lanes 3) cell homogenates were immunoblotted using the anti-a-ENaC (left panels) and anti-CFTR (right panels) antibodies. No band was detected when the primary antibodies were omitted (lanes 4). Molecular weight markers are indicated on the left-hand side of the ®gures.

To test whether electrogenic secretion of Cl mediated by CFTR occurred in cultured mouse DCT, CCD, and IMCD cells, Isc was measured under apical Naq-free condition in the presence of various inhibitors of anion transporters. Replacing Naq by N-methyl-Dglucamine 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 mM glibenclamide, which partially inhibits CFTR w24x, 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 mM 4,49-diisothiocyanatostilbene-2,29-di-sulphonic acid (DIDS) neither altered

Fig. 3. Effects of dDAVP on short-circuit current. Isc was measured on con¯uent mpkDCT, mpkCCD and mpkIMCD cells in the absence (k) or presence (m) of increasing concentrations of dDAVP applied to the basal medium. Values are means"SE from 6±10 separate experiments.

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Fig. 4. Effects of dDAVP and ion channel blockers on short-circuit current. Representative traces of Isc measured on cells incubated ®rst without (k) and then with 10 8 M dDAVP (m), 10 4 M NPPB (^) and 10 6 M amiloride (Am, (I). dDAVP was applied to the basal medium and the channel blockers were added to the apical medium.

Fig. 5. Apical Naq-substituted experiments. Isc was measured on mpkDCT, mpkCCD and mpkIMCD cells with apical Naq replaced by N-methyl-D-glucamine. (Left panels) Representative traces of Isc measured before (k) and after adding 10 4 M NPPB (I), 10 4 M glibenclamide (Glb,^) or 10 4 M DIDS (e) and 10 8 M dDAVP (m). (Right panels) Bars are the mean Isc"SE values (ns6) performed on cells incubated without (open bars), or with dDAVP (black bars) in the presence of anion inhibitors. The C bars represent the mean control Isc"SE values (ns6) from cells incubated without (open bars) or with dDAVP (black bars) in absence of any anion inhibitors. *P-0.05, **P-0.01, ***P-0.001 represent signi®cant differences between untreated and dDAVP-treated cell values.

the Isc of untreated cells nor impaired the dDAVPstimulated Isc (Figure 5). Thus, these mouse distal and collecting duct cells all had similar pro®les 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

¯uxes 36

We then measured the bidirectional transport of Cl 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 36 Cl ¯uxes 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 36 Cl in all untreated cell lines (Figure 6, left panels). The apical-to-basal Cl ¯ux was signi®cantly greater in cells treated with dDAVP for 60 min (Figure 6, left panels). The increase in apical-to-basal Cl ¯ux 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 ¯ux, but to a lesser extent (Figure 6, right panels). As a consequence, dDAVP caused a signi®cant increase (p-0.05) in the net Cl absorbed by cultured DCT (241 nEqu60 minucm2), CCD (148 nEqu60 minucm2) and IMCD (94 nEqu60 minucm2) 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 ¯ux caused by dDAVP (Table 1). However, the inhibition of Cl secretion by

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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 ¯uxes and that NPPB inhibited, at least partially, the secreted component of Cl ¯ux in these mouse distal and collecting duct cell lines.

Table 1. Effect of apical side NPPB on dDAVP-stimulated bi-directional 36Cl ¯uxes 36

Cl ¯ux (nEqu60 minucm2)

Apical-to-basal

mpkDCT mpkCCD mpkIMCD

Basal-to-apical

NPPB

qNPPB

NPPB

qNPPB

695"34 349"30 246"61

674"25 313"28 263"86

391"55 236"54 190"41

227"30* 113"22* 92"29

The apical-to-basal and basal-to-apical ¯uxes of 36Cl were measured on con¯uent cells incubated with 10 8 M dDAVP and without ( NPPB) or with (qNPPB) 10 4 M NPPB. Values are means"SE from 7±12 separate experiments. *P-0.05 vs NPPB basal-to-apical values.

Fig. 6. Effects of dDAVP on 36Cl ¯uxes. The apical-to-basal (circles, Ap £Bl) and basal-to-apical (squares, Bl £Ap) ¯ux of 36Cl were measured on con¯uent mpkDCT, mpkCCD and mpkIMCD cells incubated without (open circles and squares and open bars) or with 10 8 M dDAVP (black circles and squares and black bars). Bars are means"SE from 5 to 10 experiments. *P-0.05, **P-0.01 represent signi®cant differences between untreated and dDAVP-treated cell values.

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Discussion 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 shortcircuit current experiments show that dDAVP stimulates mainly Naq 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 w8±9x and IMCD cells w10,12,25x, but not for DCT cells, where the rise in Cl secretion was the greatest. We designed speci®c experiments to determine the pharmacological pro®le 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 ¯ux measured under open-circuit condition that is stimulated by dDAVP, although to a lesser extent than the opposite Cl ¯ux, 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 w8x. Similar results have been obtained in rat IMCD cells in primary culture w12x 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 pro®le 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 w26x. 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 w26x. Speci®cally in this study we have shown that CFTR mRNA and protein are present in the three cell types. The pro®le 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 w27x. Patch-clamp studies have also shown that there are CFTR-like Cl currents in cultured DCT w13,14x, CCD w15,28x and IMCD w12,25x cells. However, electrophysiological information alone is insuf®cient 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 w29x. Finally, a study on microsomes from the rat kidney inner medulla has shown partial inhibition of Cl -dependent cell swelling using a puri®ed antibody against CFTR w30x. 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 ®nal proof will only be obtained by comparing Cl ¯uxes 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 ¯ux 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 w6x and in rabbit CCD cells in primary culture w8x. 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 speci®c 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 ¯uxes 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 Naq and Cl transport stimulated by vasopressin. Isc experiments using anion channel blockers and Cl ¯uxes studies also evidenced the presence of a Cl secretory pathway, not only in IMCD cells w12,25x, but also in DCT and CCD cells. Acknowledgements. 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 proteÂines and the Association pour l'aide aÁ 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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Received for publication: 9.6.00 Accepted in revised form: 14.9.00