Unité Mixte de Recherche Centre National de la Recherche Scientifique 6548, Université de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France
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ABSTRACT |
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The role of cystic fibrosis transmembrane
conductance regulator (CFTR) in the control of Cl
currents was studied in mouse kidney. Whole cell clamp was used to
analyze Cl
currents in primary cultures of proximal and
distal convoluted and cortical collecting tubules from wild-type (WT)
and cftr knockout (KO) mice. In WT mice, forskolin activated
a linear Cl
current only in distal convoluted and
cortical collecting tubule cells. This current was not recorded in KO
mice. In both mice, Ca2+-dependent Cl
currents were recorded in all segments. In WT mice, volume-sensitive Cl
currents were implicated in regulatory volume decrease
during hypotonicity. In KO mice, regulatory volume decrease and
swelling-activated Cl
current were impaired but were
restored by adenosine perfusion. Extracellular ATP also restored
swelling-activated Cl
currents. The effect of ATP or
adenosine was blocked by 8-cyclopentyl-1,3-diproxylxanthine. The
ecto-ATPase inhibitor ARL-67156 inhibited the effect of hypotonicity and ATP. Finally, in KO mice, volume-sensitive Cl
currents are potentially functional, but the absence of CFTR precludes
their activation by extracellular nucleosides. This observation
strengthens the hypothesis that CFTR is a modulator of ATP release in epithelia.
kidney; cystic fibrosis; cell volume; regulatory volume decrease
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INTRODUCTION |
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THE CYSTIC FIBROSIS
TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) protein has been
detected by electrophysiological techniques in a variety of cultured
cells of the renal tubule, such as distal convoluted tubule (DCT)
(21), cortical collecting tubule (CCT) (2,
30), and inner medullary collecting duct (10). In
these segments, the presence of CFTR is correlated with activation of a
cAMP-activated Cl current. However, along the nephron,
CFTR is not always associated with these Cl
currents. For
instance, despite the presence of CFTR transcripts, CFTR expression,
along with forskolin-induced conductance, was not detected in rabbit
proximal tubule in primary culture (21). This observation
highlights the fact that CFTR could play an important role in the
control of different channels in kidney tissue. Such control is now
well established in secretory epithelia. In these structures, besides
the cAMP-sensitive Cl
secretion, CFTR controls the
epithelial Na+ channel (12, 14, 18, 28) and
the outwardly rectifying Cl
channel (24).
Moreover, CFTR is also needed for an effective volume regulation in
airway and intestinal epithelia (31, 32), suggesting that
it could modulate K+ and Cl
channels
implicated in regulatory volume decrease (RVD). Indeed, these multiple
functions of CFTR could explain the different phenotypes induced by
cystic fibrosis (CF) in secretory epithelia. In contrast, the role of
CFTR in the kidney remains uncertain, inasmuch as there is no major
disruption of renal function in CF patients (27).
Nevertheless, the reduced renal excretion of NaCl observed in CF
indicates that Cl
and Na+ channels could be
dependent on CFTR expression, suggesting that mutation of CFTR could
induce a primary defect in renal function. A better understanding of
the function of CFTR in the kidney, therefore, seems to be necessary.
For this reason, we chose to investigate the role of CFTR along the
nephron using primary cultures of proximal convoluted tubules (PCT),
DCT, and cortical collecting ducts microdissected from the kidney of
cftr
/
and cftr+/+ mice. The
cftr
/
mice lack cAMP-activated
Cl
currents in the colon, airways, and exocrine pancreas
cells (6) and represent a useful model for studying the
different ion channel defects due to CF. In the present study, using
patch-clamp methodology, we confirmed that cAMP-sensitive
Cl
conductances measured in primary cultures of DCT and
cortical collecting tubule (CCT) cells are linked to CFTR integrity.
Moreover, in contrast to the data reported in the literature on airways and endothelial cells (33), an increase in
Ca2+-dependent Cl
channels does not
compensate for the lack of CFTR Cl
channels in renal
tissue. The PCT, DCT, and CCT cells from
cftr
/
mice lost their capacity to regulate
their volume after a hypotonic shock because of the impairment of
swelling-activated Cl
channels. In
cftr
/
cells, the activity of these channels
could be restored by external application of adenosine. This suggests that CFTR controls the swelling-activated Cl
channels by
modulating adenosine autocrine production in renal cells.
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MATERIALS AND METHODS |
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Animals
Knockout CFTR mice were generated with the gene-targeting methodology previously described (26) at Centre de Développement des Techniques Avancées pour l'Expérimentation Animale (Orléans, France). This strain of mice was originally derived from ES129/Sv cells injected into C57BL/6 embryos. They were backcrossed with C57BL/6 mice for three generations and then intercrossed. Mice were allowed free access to food and water in a facility at 25 ± 1°C with a 12:12-h light-dark cycle. The 4- to 6-wk-old wild-type cftr+/+ mice and cftrPrimary Cell Cultures
PCT, DCT, and collecting tubules were microdissected under sterile conditions. Kidneys were perfused with Hanks' solution (GIBCO) containing 700 kU/l collagenase (Worthington), cut into small pyramids that were incubated for 1 h at room temperature in perfusion buffer (160 kU/l collagenase, 1% Nuserum, and 1 mM CaCl2), and continuously aerated. The pyramids were then rinsed thoroughly in the same buffer devoid of collagenase. The individual nephrons were dissected by hand in this buffer under binoculars using stainless steel needles mounted on Pasteur pipettes. The criteria used to identify the nephron segments have been described elsewhere (4). Briefly, PCT corresponded to the 1- to 1.5-mm segment of tissue located immediately following the glomerulus. The DCT portion was the segment between the macula densa and the first branching with another tubule [i.e., connecting tubule (CNT)]. The CNT segment was discarded. The CCT was identified as the straight, poorly branched portion that followed the CNT segment. After they were rinsed in dissecting medium, tubules were transferred to collagen-coated 35-mm petri dishes filled with culture medium composed of equal quantities of DMEM and Ham's F-12 (GIBCO) containing 15 mM NaHCO3, 20 mM HEPES, pH 7.4, 1% serum, 2 mM glutamine, 5 mg/l insulin, 50 nM dexamethasone, 10 µg/l epidermal growth factor, 5 mg/l transferrin, 30 nM sodium selenite, and 10 nM triiodothyronine. Cultures were maintained at 37°C in a 5% CO2-95% air water-saturated atmosphere. The medium was removed 4 days after seeding and then every 2 days.Electrophysiological Studies
Whole cell currents were recorded from 6- to 20-day-old cultured cells grown on collagen-coated supports maintained at 33°C for the duration of the experiments. The ruptured-patch whole cell configuration of the patch-clamp technique was used. Patch pipettes (2- to 3-MData acquisition and analysis.
Voltage-clamp commands, data acquisition, and data analysis were
controlled via a computer equipped with a Digidata 1200 interface (Axon
Instruments). pCLAMP software (versions 5.51 and 6.0, Axon Instruments)
was used to generate whole cell current-voltage (I-V) relations, with the membrane currents resulting from voltage stimuli filtered at 1 kHz, sampled at 2.5 kHz, and stored directly on the
computer hard disk. Cells were held at 50 mV, and 400-ms pulses from
100 to +120 mV were applied in 20-mV increments every 2 s.
Cell Volume Measurement
The relative cell volume was monitored by image analysis with fura 2 as fluorescent volume indicator, as previously reported (22). Six- to 20-day-old cell monolayers grown on petri dishes were loaded with a solution of 2 µM fura 2 containing 0.01% pluronic acid for 20 min at 37°C and then washed with an NaCl solution. The fluorescence was monitored with 360-nm excitation wavelength. At 360 nm, the variations in the signal emitted by the probe are directly proportional to the variations in cell volume. In a typical experiment, the cells were first perfused with an isotonic NaCl solution containing (in mM) 110 NaCl, 5 KCl, 1 CaCl2, 90 mannitol, and 10 HEPES, pH 7.4 [osmotic pressure (Posm) = 320 mosmol/kgH2O] at 30 ml/min, and images were averaged eight times and recorded every 5 s for 15 min. Once the fluorescence was stabilized, a hypotonic shock was induced by perfusing the NaCl solution without mannitol (Posm = 200 mosmol/kgH2O). The relative change in cell volume was estimated from the fluorescent signal by assuming that a 30% decrease in osmolarity caused a decrease in the fluorescent signal corresponding to a maximum swelling of 30% compared with the initial volume. The means of relative volume changes were obtained by analysis of 10-20 zones in each culture (n) chosen with the software. Each zone delimited a cytoplasmic area chosen in individual cells.Image analysis. The optical system was composed of a Zeiss ICM-405 inverted microscope and a Zeiss ×40 objective, which was used for epifluorescent measurement with a 75-W xenon lamp. The excitation beam was filtered through a narrow-band filter centered at 360 nm, mounted in a motorized wheel (model Lambda 10-2, Sutter Instrument), and equipped with a shutter to control the exposure times. The incident and the emitted fluorescence radiation were separated through a Zeiss chromatic beam splitter. Fluorescence emission was selected through a 510-nm narrow-band filter (Oriel). The transmitted light images were viewed by an intensified camera (Extended ISIS, Photonic Science, Sussex, UK). The eight-bit Extended ISIS camera was equipped with an integration module to maximize signal-to-noise ratio. The video signal from the camera proceeded to an image processor integrated in a DT2867 image card (Data Translation) installed in a Pentium 100 personal computer. The processor converts the video signal to 512 lines by 768 square pixels per line by 8 bits per pixel. The 8-bit information for each pixel represents one of the 256 possible gray levels, ranging from 0 (for black) to 255 (for white). Image acquisition and analysis were performed with the AIW software (version 2.0, Axon Instruments). The final calculations were made using Excel software (Microsoft).
Calibration. We used the methods described by Tauc et al. (29) using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and improved more recently by Raat et al. (17) using fura 2. After cells were loaded with the fluorescent probe in the culture medium, they were perfused with a solution adjusted to various osmolarities (150-400 mosmol/kgH2O) by omitting mannitol. For each osmolarity, two images were stored, averaged, and subsequently corrected for fading after background subtraction. The mean fluorescence (360 nm) of five areas was plotted against the inverse of Posm (in mosmol/kgH2O). Data showed that when the cells were exposed to a hyposmotic solution, fluorescence decreased linearly with Posm according to Boyle's law. To verify that cells in culture behave as osmometers in a reversible manner, we performed experiments in which the cultures were perfused successively and randomly with 200-300 mosmol/kgH2O solutions. The fluorescent signal was related to Posm in a reversible way. In all calibration experiments, images were recorded 1-2 min after the beginning of perfusion, at which time the swelling in hypotonic solutions reached the maximum value. These methods measure variations in the relative volume as a function of Posm of the perfusion medium (29).
Intracellular Ca2+ Measurements
Intracellular Ca2+ concentration ([Ca2+]i) was measured in cells grown in petri dishes and loaded for 45 min at room temperature with a solution of 2 µM fura 2-AM containing 0.01% pluronic acid. The cells were washed with NaCl solution containing (in mM) 140 NaCl, 5 KCl, 1 MgSO4, 5 glucose, 20 HEPES, pH 7.40, and 1 Tris. Cells were successively excited at 350 and 380 nm, with images digitized and stored on the computer hard disk for later analysis. Each raw image was the result of an integration of four to five frames averaged four times. The acquisition rate was one image every 10 s. For each monolayer, [Ca2+]i was monitored in 18-20 random cells. The equation of Grynkiewicz et al. (9) was used to calculate [Ca2+]i from the dual wavelength-to-fluorescence ratio.Expression in Cultured Cells
The cDNA encoding CFTR was introduced into a polycistronic expression vector derived from the pIRESneo plasmid (cytomegalovirus promoter; Clontech) in which the neomycin resistance gene had been replaced by cDNA encoding the chain of the human CD8 cell surface antigen. Cells were transfected using the DAC-30 method according to the manufacturer's instructions (Eurogentec, Herstal, Belgium). Six-day-old cultured cells grown on 35-mm-diameter petri dishes were serum starved for 24 h before transfection. Transfected cells with 2 µg of CD8-CFTR coexpress CFTR and CD8 at their plasma membrane and can be visualized using anti-CD8 antibody-coated beads (Dynabeads M-450, Dynal, Oslo, Norway) (11a). Cells were electrophysiologically tested 48 h after transfection.Chemicals
5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; Calbiochem) was prepared at 100 mM in DMSO and used at 0.1 mM in final solutions. 4-4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was directly dissolved at a final concentration of 1 mM. Forskolin and ionomycin were prepared at 10 and 2 mM, respectively, in ethanol and used at 10 and 2 µM, respectively, in bath medium. DIDS, forskolin, ARL-67156 (6-N,N-diethyl- ![]() |
RESULTS |
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Cl Currents Activated by Forskolin
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The forskolin-sensitive Cl currents measured at +100 mV
are compared in primary cultures of PCT, DCT, and CCT from
cftr+/+ and cftr
/
mice in Fig.
1B. Only DCT and CCT from wild-type mice exhibited
forskolin-activated Cl
currents that were blocked by 0.1 mM NPPB and insensitive to 1 mM DIDS in the extracellular bath.
Moreover, in these segments, replacing external Cl
with
I
strongly inhibited the Cl
currents
activated by forskolin and caused Erev to shift
toward positive values: Erev for
I
= 37.5 ± 1.4 and 17.0 ± 6.8 mV for DCT
and CCT, respectively (n = 4 monolayers from 4 different mice).
Ca2+-Induced
Cl Currents
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Cl Currents Induced by a Hypotonic
Shock
The monolayers were then perfused with a 290 mosmol/kgH2O
solution. Figure 3A gives the
currents recorded in PCT, DCT, and CCT cells. In >95% of the
cftr+/+ cells, an increase in the whole cell current was
observed within 1 min. In all epithelial cell types, the currents
reached a maximum after 4-5 min. Under these conditions, the
initial currents recorded at +100 mV were ~2.5 times the amplitude of
the currents recorded at 100 mV. These large, outwardly rectifying
currents showed a small time-dependent inactivation at depolarizing
potentials
60 mV in cultured PCT and CCT cells and
40 mV in
cultured DCT cells. In most cases, the time course of this inactivation
could be well fitted with a single exponential irrespective of the
recording time. When the cells were reexposed to the hyperosmotic
solution, the currents returned to the control level within 2-3
min (Fig. 3B). In the three cultured segments, the
currents induced by hypotonicity were strongly blocked by 1 mM DIDS
(Fig. 3B).
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In cftr/
mice, hypotonic shock was completely
inefficient for increasing Cl
conductance in the
three different cultured segments (Fig. 3, A and
B). In all nephron segments studied, an absence of response to hypotonic shock was observed in 100% of the recorded cells. This
result implicates CFTR in the control of the swelling-activated Cl
conductance in renal epithelium.
The results reported above clearly show that Cl
conductances developed in the presence of forskolin, Ca2+,
or hypotonic shock in DCT cells were roughly similar to those recorded
in CCT cells under the same experimental conditions. Therefore, in the
following experimental series, no distinction was made between DCT and
CCT cells.
Cl Currents in Cultured PCT and DCT
Cells From cftr
/
Mice Transfected With CFTR cDNA
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In another experimental series, the effect of a hypotonic shock was
studied in cftr/
PCT and DCT cells
transfected with the cftr plasmid. In both cell types, after
the hypotonic shock, the coated cells developed Cl
currents within 3 min (Figs. 4B and 5B). The
initial currents measured 20 ms after the onset of the voltage pulse
rectified in the outward direction (Figs. 4Bd and
5Bd). For PCT cells, they reversed at +0.6 ± 0.4 mV
(n = 4 cells), and the total current at +100 mV was 3.8 times that at
100 mV: 1,555 ± 150 vs.
405 ± 18 pA
(n = 4 cells). For DCT cells, they reversed at
+0.9 ± 0.3 mV (n = 4 cells), and the total
current at +100 mV was 2.2 times that at
100 mV: 1,123 ± 155 vs.
508 ± 86 pA (n = 4 cells). These large
outwardly rectifying currents showed time-dependent inactivation at
depolarizing step potential >40 mV. Finally, replacement of the
hypotonic bath solution by a hypertonic solution inhibited the
Cl
currents by 74 ± 3 and 80 ± 4% for PCT
and DCT cells, respectively (n = 4). As expected, the
uncoated cells remained insensitive to the hypotonic shock (Fig.
5B). Therefore, transfection of CFTR also restores the
swelling-activated Cl
conductance in
cftr
/
PCT and DCT cells.
Regulation of the Cl Conductance
Induced by Hypotonic Shock in
cftr+/+ and
cftr
/
DCT and CCT Cells
Role of extracellular Ca2+ in the
presence of high EGTA concentration in the pipette solution.
In cftr+/+ cells, to eliminate the implication of cytosolic
Ca2+ in the development of hypotonicity-induced
Cl currents, experiments were generally performed using
pipette solutions containing 5 mM EGTA without additional
Ca2+. The effects of extracellular Ca2+ on the
development of hypotonicity-induced Cl
currents were also
tested in cftr+/+ DCT and CCT cells. When the hypotonic
shock was carried out in the absence of bath Ca2+,
development of the Cl
current was significantly impaired
(Fig. 6A). As previously
reported in rabbit distal bright convoluted tubule (DCTb) in primary
culture (20), these experiments confirm that extracellular
Ca2+ was required to activate the swelling-activated
Cl
conductance in DCT and CCT cells cultured from
cftr+/+ mice. Using this information, we therefore decided
to study the effect of an influx of Ca2+ on
swelling-activated Cl
conductance in
cftr
/
DCT and CCT cells. For this purpose,
the effects of ionomycin were tested on whole cell Cl
currents recorded in the absence of intracellular free
Ca2+. Whole cell currents were recorded in the presence of
20 mM EGTA in the pipette solution and 1 mM free Ca2+ in
the bath (Fig. 6B). In the absence of ionomycin in the bath solution, the hypotonic shock remained inefficient for triggering Cl
currents in cftr
/
cells
(Fig. 6Ba). In contrast, when the hypotonic shock was
performed in the presence of 2 µM ionomycin, Cl
currents were activated within 5 min (Fig. 6Bb). These
currents showed time-dependent inactivation at depolarizing step
potentials >60 mV and displayed an outwardly rectified instantaneous
I-V plot (Fig. 6Be) with an
Erev of + 1.1 ± 0.3 mV
(n = 7). When the cells were reexposed to the
hyperosmotic solution, the currents returned toward control level
within 2-3 min (Fig. 6, Bc and Be). Alternatively, addition of DIDS rapidly reduced the Cl
currents (89.7 ± 4% inhibition at +100 mV, n = 5; Fig. 6Bd). Overall, the ionomycin-induced
Cl
currents developed during hypotonicity in DCT and CCT
cells from cftr
/
mice were quite similar to
the swelling-activated Cl
currents measured in
cftr+/+ mice.
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Role of extracellular Ca2+ in the
absence of EGTA in the pipette solution.
The experiments described above indicate that Ca2+ influx
induced by ionomycin could restore the swelling-activated
Cl currents in cftr
/
cells. To
further analyze this phenomenon, the effect of ionomycin was tested in
the absence of EGTA in the pipette solution. Two successive, increased
external Ca2+ concentrations were applied to the same
cftr
/
DCT cells. The results are reported in
Fig. 7A. Control currents were
recorded, and the cells were perfused with a Ca2+-free
solution containing 2 µM ionomycin. After 2 min, raising the
Ca2+ concentration to 0.1 µM induced Cl
currents that were identical to the swelling-activated Cl
currents (Fig. 7Ab). A further new increase in
Ca2+ concentration to 1 µM enhanced the currents (Fig.
7Ac). These currents showed virtually no inactivation during
the 400-ms voltage pulse. Currents obtained by subtracting the current
recorded at 0.1 µM external Ca2+ from that recorded at 1 µM Ca2+ are shown in Fig. 7Ad. The resulting
currents exhibited the characteristic profile of the
Ca2+-sensitive Cl
currents.
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Role of extracellular adenosine.
We previously demonstrated that stimulation of A1 adenosine
receptors could be implicated in the control of swelling-induced Cl currents in rabbit DCT (20), and
experiments were therefore performed to determine the role of adenosine
in Cl
permeability of PCT and DCT cells from
cftr+/+ and cftr
/
mice. Results of
whole cell experiments performed in cftr
/
PCT
and DCT cells are illustrated in Fig. 8.
These results were strictly identical to those obtained with
cftr+/+ PCT and DCT cells. In both types of primary
cultures, 10 µM adenosine activated an outwardly rectifying
Cl
conductance with a time-dependent inactivation at
depolarizing potentials and with a maximal effect at 3-4 min (Fig.
8A). Erev of the stimulated current
were 3.8 ± 3.7 mV (n = 5 monolayers) and 0.3 ± 2.9 mV (n = 4) for PCT and DCT cells, respectively. In the presence of adenosine, the maximal slope conductances reached 19 ± 9 nS (n = 5) and 11 ± 4 nS
(n = 4) in PCT and DCT cells, respectively. These
adenosine-sensitive Cl
currents were decreased in the
presence of 1 mM DIDS by 90 and 78% in PCT and DCT cells,
respectively. To determine whether the response to adenosine occurred
via receptor-mediated mechanisms, we examined the effect of a
P1-selective receptor antagonist, 8-cyclopentyl-1,3-diproxylzanthine (DPCPX). Treatment of DCT cells with
10 µM DPCPX completely inhibited the development of outward Cl
currents first induced by 10 µM adenosine in PCT and
DCT cells (Fig. 8).
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Role of extracellular ATP.
In addition to adenosine, it has been postulated that ATP could
activate a volume-sensitive-like Cl conductance in
immortalized rabbit distal cells (20). To check this
possibility in PCT and DCT cells from cftr+/+ and
cftr
/
mice, we studied the role of ATP in the
control of whole cell Cl
currents in the presence of 5 mM
EGTA in the pipette solution. In PCT and DCT monolayers, addition of 10 µM ATP to the bath solution induced activation of Cl
currents within 4-5 min. This ATP-activated Cl
current showed time-dependent inactivation at depolarizing step potentials >60 mV (Fig. 10,
A and B) and displayed an outwardly rectified
instantaneous I-V plot (data not given) with
Erev close to 0 mV. DIDS (1 mM) strongly
decreased ATP-activated currents in both types of monolayers. Overall,
these currents were quite similar to those induced by adenosine.
Moreover, the effect of ATP was completely blocked by 10 µM DPCPX,
indicating that the action was triggered via P1, rather
than P2, receptors. Such results suggested that stimulation
of Cl
currents in the presence of ATP was most probably
due to an action of adenosine generated by degradation of ATP.
Experiments were therefore carried out to check this hypothesis. For
this purpose, DCT cells from cftr+/+ mice were subjected to
a hypotonic shock in the presence of the selective ecto-ATPase
inhibitor ARL-67156. ARL-67156 (100 µM) completely blocked the
swelling-activated Cl
currents (Fig. 10C).
Adenosine (10 µM) restored a swelling-activated Cl
conductance, which displayed an outwardly rectified instantaneous I-V plot with Erev close to 0 mV
(Fig. 10Cd) and was strongly inhibited by DIDS (Fig.
10Cb).
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RVD in PCT and DCT Cells From
cftr+/+ and
cftr/
Mice
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DISCUSSION |
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The aim of the present study was to investigate the putative role
of CFTR in the control of Cl conductances along the
different segments of the mouse nephron. Using the patch-clamp
technique to measure whole cell conductance, we analyzed three distinct
types of Cl
currents in primary cultures of PCT, DCT, and
CCT segments obtained by microdissection of kidney cortex from
wild-type cftr+/+ and cftr
/
mice. These
Cl
conductances consisted of forskolin-activated,
volume-sensitive, and Ca2+-activated Cl
currents.
In the first series of experiments, the effect of forskolin on
Cl conductance was tested in PCT, DCT, and CCT cells. For
this purpose, swelling-activated currents were blocked by exposing the
cells to a hyperosmotic solution, and Ca2+-activated
conductances were impaired by the use of high EGTA concentrations in
the pipette solution. In cftr+/+ mice, external application
of forskolin activated a linear Cl
current in DCT and
CCT, but not PCT, cells. The halide selectivity was consistent with low
relative I
permeability and with an inhibitory effect of
I
. Moreover, this forskolin-stimulated conductance was
blocked by NPPB but was quite insensitive to DIDS. These
characteristics are very similar to those reported previously in rabbit
distal bright convoluted tubule (DCTb) in primary culture
(21). In contrast, in cultured DCT and CCT cells from
cftr
/
mice, addition of forskolin remained completely
inefficient for increasing Cl
conductances. Taken
together, the results obtained in primary cultures from
cftr+/+ and cftr
/
mice clearly demonstrate
that, at least in DCT and CCT cells, the activity of
forskolin-activated Cl
channels is consistent with CFTR.
In other words, as we concluded in a previous study (21),
the channel involved in the Cl
currents activated by
forskolin in DCT and CCT is the small-conductance CFTR Cl
channel.
Interestingly, application of forskolin did not stimulate any
Cl current in primary cultures of PCT cells from
cftr+/+ mice. Such an observation was reported in primary
culture of rabbit PCT, in which no CFTR expression and no
forskolin-activated Cl
currents were detected in the
apical membrane, despite the presence of CFTR mRNA (21).
The presence of CFTR in the mammalian kidney is now well documented
(2, 15, 16), but the absence of detectable renal disease
in CF patients led several authors to postulate that an increase of
another type of Cl channel might compensate for the lack
of cAMP-activated Cl
channels in renal tissue (6,
11). On the other hand, the cftr
/
mice
used in the present study did not present significant pulmonary
disease. Moreover, in these mice, it has been shown that the
Ca2+-activated Cl
channels could be
candidates for compensation of the missing CFTR Cl
channels (6). To determine whether this possibility could arise in the renal epithelium, we studied the
Ca2+-activated conductance in PCT, DCT, and CCT cells. As
expected, in cftr+/+ mice, extracellular application of
ionomycin rapidly activated currents in all types of monolayers. This
Ca2+-sensitive conductance was similar to that previously
described in rabbit PCT and DCTb cells under identical experimental
conditions (21, 22). In cftr
/
mice, the
increase of Cl
conductance triggered by ionomycin was
strikingly identical to that observed in wild-type mice, eliminating
the hypothesis that Ca2+-activated conductance could
substitute for CFTR Cl
conductance in renal epithelium.
In cftr+/+ mice, cultured PCT, DCT, and CCT cells developed
a volume-sensitive Cl current when exposed to a hypotonic
shock. The biophysical and pharmacological characteristics of this
Cl
conductance show strong similarities to the properties
of swelling-activated Cl
currents described in many other
epithelial cells, including rabbit PCT and DCTb in primary culture
(7, 25). Null mutation of the cftr gene
strongly impaired the swelling-activated Cl
currents in
the three different nephron segments. Moreover, cftr
/
DCT or CCT cells transfected with cftr cDNA displayed
complete restoration of cAMP-dependent and swelling-activated
Cl
currents. Transfection of PCT cells with cftr
cDNA also restored both conductances. These observations indicate
that PCT cells have maintained their ability to insert exogenous CFTR
into the apical membrane. Therefore, the lack of forskolin-induced
Cl
conductance in wild-type PCT cells is probably due to
a difference in the protein function, rather than a modification of the
intracellular trafficking leading to protein retention in intracellular membranes.
It is well established that the swelling-activated Cl
channels participate in the RVD phenomenon, which is induced by
exposure of cells to hyposmotic solutions. In the present study, to
determine whether cultured PCT, DCT, and CCT cells develop RVD after a
hypotonic shock, we used a simple fluorescence method for studying
relative cell volume variations (29). The findings
indicate that cultured cells from cftr+/+ mice are sensitive
to osmolarity changes in the bathing medium and that they are capable
of RVD after hypotonic shock. RVD was also examined in cultured cells
from cftr
/
mice. These cells exhibited a defective
volume regulation after a hyposmotic shock. This observation confirms
the results in the literature (31) and indicates that CFTR
could play a role in the RVD of epithelia. Obviously, this defective
RVD is due to the fact that the hypotonic shock is completely
inefficient for increasing Cl
conductances. The
intervention of CFTR in the control of swelling-activated Cl
conductances has been proposed by Chan et al.
(5), who demonstrated, in the human colonic cell line T84,
that an antibody against CFTR inhibited the cAMP- as well as the
swelling-induced whole cell Cl
conductances but did not
affect the Ca2+-activated Cl
channel. In
previous studies, we found that development of Cl
conductance after a hypotonic shock in rabbit DCT cells was related to
an influx of external Ca2+ through Ca2+
channels (21, 22). Such a hypothesis could also apply to the data obtained in DCT cells from cftr+/+ mice, because
removal of external Ca2+ just before the hypotonic shock
completely impaired the increase in Cl
current,
suggesting that Ca2+ influx could participate in activation
of the Cl
channels in mouse kidney. It is now proposed
that CFTR could regulate other ion channel proteins (23)
and could also be implicated in different cell functions such as
apoptosis (1, 8, 13) or cytosolic Ca2+
regulation (19). Moreover, a recent study by Braunstein et al. (3) clearly demonstrated that CFTR participates in
cell volume regulation via control of ATP release. Taken together, these observations led us to propose the hypothesis that the absence of
swelling-activated Cl
conductance in DCT cells from
cftr
/
could be related to an alteration of the
Ca2+ entry. This hypothesis is strengthened by two main
results: 1) The hypotonic shock induced an increase of
[Ca2]i in cftr+/+ DCT cells, but
not in cftr
/
cells. 2) In
cftr
/
cells, addition of ionomycin in the presence of
high intracellular EGTA concentration restored the ability of the cells
to respond to the hypotonic shock by increasing swelling-sensitive
Cl
conductance. It remains to be shown how intracellular
Ca2+ can increase in the presence of a high EGTA
concentration. The observations of Evans and Marty (6a) shed light on
this problem by indicating that, with EGTA as a buffer, a whole region
of the cell could escape control by the Ca2+ buffer.
Because this region could extend to a large part of the plasma membrane
(10), a local transient increase of Ca2+ could
arise in the presence of EGTA. In accordance with the model proposed by
Braunstein et al. (3), the defect in cell volume regulation that we observed in cftr
/
renal cells could
be due to a defect in the ATP release pathway. We have proposed
(20) that hypotonic shock stimulates ATP release from
rabbit DCT cells. A1 receptors are then activated by
adenosine generated by the degradation of ATP by membrane ectoenzymes,
and this stimulation of A1 receptors induces an influx of
extracellular Ca2+. Finally, this Ca2+ influx
activates the Cl
channel. The observation that adenosine
restores swelling-activated Cl
conductance and RVD in
cftr
/
cells confirms that adenosine is a mediator of
RVD, at least in renal epithelium. Therefore, in cftr
/
cells, there is no volume-sensitive ATP release, and the cascade of
events that triggers the final increase in Cl
conductance
no longer occurs.
The present results confirm that autocrine ATP release is probably an
essential step in the cell volume regulation phenomenon. However, it
could be questioned why adenosine or ATP did not activate a current
consistent with a Ca2+-activated Cl current
during a hypotonic shock. We have demonstrated that swelling-activated and Ca2+-sensitive Cl
currents could be
additive but also that their thresholds of activation by
Ca2+ were quite different. Thus the former was activated at
0.1 µM Ca2+, whereas the latter was activated at 1 µM
Ca2+. The cytosolic Ca2+ concentration induced
by hypotonic shock never exceeded 0.15 µM. This small increase is
consistent with the fact that CFTR control of ATP release during
swelling involved probably low ATP concentration and, consequently, low
adenosine production. In the present study, the effect of adenosine in
increasing swelling Cl
currents was concentration
dependent, with a half-maximal effect at 5.0 × 10
7
M. As previously reported in rabbit DCT cells, this adenosine concentration raised cell Ca2+ to 0.11 nM, which was
sufficient to trigger swelling-activated Cl
currents but
too low to induce Ca2+-dependent Cl
currents.
The RVD process involves Cl and K+ efflux.
Previous data indicate that impairment of RVD in jejunal crypts of
cftr
/
mice was due to a defective K+ channel
(31, 32). However, the nature of the K+
channels stimulated during hypotonic shock remains very uncertain and
appears to depend on the tissue under investigation (32). Therefore, in the companion article (1a), we investigate the
K+ conductances along the different nephron segments of
cftr
/
and cftr+/+ mice.
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FOOTNOTES |
---|
Address for reprint requests and other correspondence: P. Poujeol, UMR CNRS 6548, Bâtiment Sciences Naturelles Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France (E-mail: poujeol{at}unice.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.
First published December 10, 2002;10.1152/ajprenal.00237.2002
Received 26 June 2002; accepted in final form 3 December 2002.
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