Original Contribution

CFTR mediates cadmium-induced apoptosis through modulation of ROS level in

mouse proximal tubule cells
Sebastien L'hoste
a
, Abderrahmen Chargui
a
, Radia Belfodil
a
, Christophe Duranton
a
, Isabelle Rubera
a
,
Baharia Mograbi
b
, Chantal Poujeol
a
, Michel Tauc
a
, Philippe Poujeol
a,

a
CNRS FRE 3093, Universit de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France
b
Inammation et Carcinogenese (INSERM ERI21) UFR Medecine 28, Avenue de Valombrose, 06107 Nice Cedex 2, France
a b s t r a c t a r t i c l e i n f o
Article history:
Received 19 June 2008
Revised 1 December 2008
Accepted 3 December 2008
Available online 24 December 2008
Keywords:
CFTR
Kidney
Patch clamp
Glutathione
Reactive oxygen species
The aim of this study was to characterize the role of CFTR during Cd
2+
-induced apoptosis. For this purpose
primary cultures and cell lines originated from proximal tubules (PCT) of wild-type cftr
+/+
and cftr
/
mice
were used. In cftr
+/+
cells, the application of Cd
2+
(5 M) stimulated within 8 min an ERK1/2-activated
CFTR-like Cl

conductance sensitive to CFTR

inh
-172. Thereafter Cd
2+
induced an apoptotic volume decrease
(AVD) within 6 h followed by caspase-3 activation and apoptosis. The early increase in CFTR conductance was
followed by the activation of volume-sensitive outwardly rectifying (VSOR) Cl

and TASK2 K
+
conductances.
By contrast, cftr
/
cells exposed to Cd
2+
were unable to develop VSOR currents, caspase-3 activity, and AVD
process and underwent necrosis. Moreover in cftr
+/+
cells, Cd
2+
enhanced reactive oxygen species (ROS)
production and induced a 50% decrease in total glutathione content (major ROS scavenger in PCT). ROS
generation and glutathione decrease depended on the presence of CFTR, since they did not occur in the
presence of CFTR
inh
-172 or in cftr
/
cells. Additionally, Cd
2+
exposure accelerates efuxes of uorescent
glutathione S-conjugate in cftr
+/+
cells. Our data suggest that CFTR could modulate ROS levels to ensure
apoptosis during Cd
2+
exposure by modulating the intracellular content of glutathione.
2008 Elsevier Inc. All rights reserved.
Introduction
The heavy metal cadmium is nephrotoxic, and chronic exposure
causes a Fanconi-like syndrome characterized by wasting of many ions,
small molecules, and proteins that are normally reabsorbed in the
proximal tubule ([1] and for review, see [2,3]). Several mechanisms
have been proposed to explain the toxic effects of Cd
2+
on renal cells
[4]. Of these mechanisms free Cd
2+
may generate reactive oxygen
species (ROS) [3,4]. This ROS increase probably results from the
inhibition of the mitochondrial oxidative phosphorylation [5,6]. More-
over, it seems that such ROS generation mediates Cd
2+
-induced
apoptosis in proximal tubule cells [7]. Nevertheless, the type of cell
death induced by Cd
2+
depends onthe heavy metal concentration since
it has been demonstrated that micromolar Cd
2+
concentration induces
more apoptosis than necrosis whereas higher Cd
2+
concentration
predominantly provokes necrosis in proximal cells [2,3].
It is well documented in the literature that cell volume decrease
(known as AVD apoptotic volume decrease) is an early prerequisite
event for apoptotic cell death [8]. This volume change is driven by a
loss of KCl via selective K
+
and Cl

channels (for review, see [9]).

The Cl

channel activated during AVD exhibits similar biophysical

properties to the volume-sensitive outward rectifying (VSOR) Cl

channels and the literature data converge toward the conclusion

that this Cl

channel type is probably ubiquitously expressed in

animal cells [10,11]. Concerning the K
+
conductance, different classes
of channels could be involved in apoptosis [12] but in mouse proximal
tubule cells staurosporine-induced AVD was mainly associated with
the activity of TASK2 K
+
channel [13]. Interestingly, it has been
shown that ROS mediate the staurosporine-induced activation of the
VSOR channels [11] and TASK2 K
+
channels [13]. In previous
studies, we have also demonstrated that CFTR is involved in the
control of apoptosis [14] and in the control of RVD during hypotonic
shock [15]. This RVD control could be due to a CFTR-dependent
autocrine mechanism, which controls both VSOR Cl

and TASK2 K
+
conductances [15]. However, until now, the interplay among ROS
production, K
+
and Cl

channels activation, and regulation by CFTR

of AVD phenomenon during Cd
2+
-induced apoptosis had not yet
been elucidated. Thus the rst aim of this study is to address the
question as to whether such interplay exists.
After that, it is interesting to further elucidate the mechanisms
implicated in this control. Based on the putative sensibility of Cl

Free Radical Biology & Medicine 46 (2009) 10171031

Abbreviations: AEBSF, 4-(2-aminoethyl)benzenesulfonyl uoride; AVD, apoptotic
volume decrease; BSO, buthionine sulfoximine; carboxy-H
2
DCFDA, (5-and-6)-carboxy-
2,7-dichlorodihydrouorescein diacetate; CMFDA, 5-chloromethyluorescein diace-
tate; DMSO, dimethyl sulfoxide; DTNB, 5,5-dithiobis(2-nitrobenzoic) acid; NAC, N-
acetylcysteine; NMDGCl, N-methyl-D-glucamine chloride; PCT, proximal tubules; ROS,
reactive oxygen species; TBS, Tris-buffered saline; TNB 5-thiobis(2-nitrobenzoic) acid,
VSOR, volume-sensitive outwardly rectifying chloride channel.
Corresponding author. Fax: +04 92 07 68 50.
E-mail address: Philippe.POUJEOL@unice.frf (P. Poujeol).
0891-5849/$ see front matter 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2008.12.009
Contents lists available at ScienceDirect
Free Radical Biology & Medicine
j our nal homepage: www. el sevi er. com/ l ocat e/ f r eer adbi omed
and K
+
channels to ROS, it becomes apparent that CFTR could
modulate the intracellular level of ROS produced by Cd
2+
intoxication.
It is an important point as it has been proposed that subtoxic
concentration of ROS may act as a second messenger in several
biological process including apoptosis [16].
It is well known that reduced glutathione (GSH) is the most
abundant intracellular ROS scavenger. Moreover nephrotoxic agents
(such as Cd
2+
) may formconjugates with GSH(GS conjugates) and can
be exported out of the cells [17]. Therefore, protection against the
actions of Cd
2+
can be achieved through this antioxidant system. In
proximal tubule cells the content of GSHis remarkably regulatedvia the
coexistence of pathways for its synthesis, degradation, efux, and
uptake[18]. Interestinglyit has beenproposedthat multidrugresistance
proteins (MRP2/4) participate in the efux of GSH and GS conjugates
through the brush-border membrane of the proximal cells [18]. These
transporters are probably induced by long-term Cd
2+
intoxication and
could represent a detoxication mechanismfor the proximal cells [19].
Since CFTRbelongs to the same ABCtransporter family, the last purpose
of this study is to investigate the role of CFTR inthe modulationof GSH/
GSSG or GS conjugate efuxes in the proximal tubule.
Materials and methods
Animals
Knockout CFTR mice (cftr
/
) were generated with the gene
targeting methodology previously described [20] in the Centre de
Dveloppement des Techniques Avances pour l'Exprimentation
Animale (Orlans, France). Four-to 6-week-old wild-type cftr
+/+
mice and cftr
/
mice homozygous for the disrupted cftr gene were
killed by cervical dislocation, and the kidneys were removed. All
experiments were performed in accordance with the guidelines of the
French Agricultural Ofce and the legislation governing animal studies.
Cell cultures
Primary cultures of PCT tubules from cftr
+/+
and cftr
/
mice were
obtained as previously described [21,22]. PCT tubules corresponding to
the 1-to 1.5-mm segment of tissue located immediately following the
glomerulus were microdissected under sterile conditions.
Immortalized cell lines of proximal tubule from wild-type cftr
+/+
and cftr
/
were obtained after transfection of 10-day-old primary
cultures with pSV3 neo plasmid and G418 selection [23].
Cultures were seeded on collagen-coated petri dishes with a
culture medium composed of equal quantities of DMEM and Ham's
F-12 (GIBCO BRL). The medium was supplemented with 15 mM
NaHCO
3
, 20 mMHepes at pH 7.4, 1% fetal calf serum, 2 mMglutamine,
5 mg/L insulin, 50 nM dexamethasone, 10 g/L epidermal growth
factor-1, 5 mg/L transferrin, 30 nM sodium selenite, and 10 nM
triiodothyronine. Cultures were maintained at 37 C in a 5% CO
2
95%
airwater-saturated atmosphere.
Electrophysiological studies
Whole-cell currents were recorded from cultured cells grown on
collagen-coated supports (35 mm petri dishes) maintained at 37 C
for the duration of the experiments. The ruptured patch whole-cell
conguration was used. Patch pipettes (2-to 4-M resistance) were
made fromborosilicate capillary tubes (1.5 mmo.d., 1.1 mmi.d.; Fisher
Manufacturing) using a two-stage vertical puller (Model PP 830,
Narishige, Tokyo, Japan). Cells were observed using an inverted
microscope; the stage of the microscope was equipped with a water
robot micromanipulator (Model WR 89, Narishige). The patch pipette
was connected via an AgAgCl wire to the head stage of a patch
amplier (Model VP 500, Biologic). After formation of a gigaseal, the
fast-compensation system of the amplier was used to compensate
for the intrinsic input capacitance of the head stage and the pipette
capacitance. The membrane was ruptured by additional suction to
achieve the conventional whole-cell conguration. Settings available
on the amplier were used to compensate for cell capacitance. No
series resistance compensationwas applied, but experiments inwhich
the series resistance was N20 M were discarded. Solutions were
perfused in the extracellular bath using a four-channel glass pipette,
with the tip placed as close as possible to the clamped cell. Voltage-
clamp commands, data acquisition, and data analysis were controlled
via a VP 500 patch clamp amplier connected to a computer. The
membrane currents resulting from voltage stimuli ltered at 1 kHz,
sampled at 2.5 kHz, were stored directly on the computer hard
disk. Cells were held at -50 mV, and 400 ms pulses from 100 to
+100 or +120 mV were applied in 20 mV increments.
For Cl

whole-cell experiments, pipettes were lled with a

solution containing (in mM) 140 N-methyl-D-glucamine chloride
(NMDGCl), 5 EGTA, 5 MgATP and 10 Hepes (pH 7.4, HCl). The bath
solution contained (in mM) 140 NMDGCl, 1 CaCl
2
, 1 MgCl
2
, 10 Hepes
(pH7.4, HCl), and 60 mannitol. Osmotic pressure (P
osm
) was adjusted
to 340350 mosmol kg
1
H
2
O.
For K
+
whole-cell experiments, pipettes were lled with a solu-
tion containing (in mM) 145 K-gluconate, 10 Hepes, 10 EGTA, 1 CaCl
2
(10 nM free Ca
2+
concentration), and 5 MgATP while the bath
solution contained 140 Na-gluconate, 10 Hepes, 1 CaCl
2
, 1 MgCl
2
, and
50 mannitol (P
osm
adjusted to 330340 mosmol kg
1
H
2
O).
Western blot analysis
Cells lysates were prepared in ice-cold lysis buffer containing in
mM: 50 Hepes, pH 7.4, 150 NaCl, 100 NaF, 10 EDTA, 10 Na
4
P
2
O
7
, and
2 Na
3
VO
4
with 1% Triton X-100 and supplemented with protease
inhibitors: aprotinin (2 g/ml), leupeptin (10 M), and 4-(2-
aminoethyl)benzenesulfonyl uoride (AEBSF; 1 mM). Equivalent
amounts of protein (20 g) were separated by SDS-PAGE on 10%
acrylamide gels. Proteins were electrically transferred to Hybond-C
Extra membrane (Amersham) and stained with amidoblack to
verify an even transfer, and then the blots were incubated in blocking
buffer [1Tris-buffered saline (TBS), 0.1% Tween 20, 5% nonfat dry
milk] for 1 h at room temperature. The membranes were washed in
washing buffer (1 TBS, 0.1% Tween 20) three times, for 5 min per
wash, and probed with the primary antibody (anti-phosphospecic
ERK1/2, dilution 1:10,000; Sigma, Saint-Quentin Fallavier France)
and then horseradish peroxidase-conjugated secondary antibody for
1 h in 1X TBS, 0.1% Tween 20, 1% nonfat dry milk. After each
incubation, membranes were washed three times for 10 min in
washing buffer. Proteins were then visualized by the Amersham ECL
system. After stripping, equal loadings of proteins were veried by
reprobing the blots with total anti-ERK1/2 (1:10,000; Sigma). Band
intensity was quantied using PCBas software.
Quantication of apoptosis and/or necrosis induced by Cd
2+
exposure
Cd
2+
-induced apoptosis and/or necrosis was studied in cftr
+/+
and cftr
/
cell lines. After incubation for 6 h with Cd
2+
, the cell
preparations were carefully washed with fresh culture medium and
incubated for 10 min in the presence of both annexin-5-Fluos (Roche,
dilution 1/50) and propidium iodide (Roche, 1 g/ml) in a buffer
solution containing (in mM): 140 NaCl, 10 Hepes (adjusted to pH 7.4
with NaOH), and 5 CaCl
2
. Digital micrographs were successively taken
at 550 nm for annexin-V-Fluos and N600 nm for propidium iodide
labeling. Apoptotic cells were counted by comparing the two
stainings. A cell was considered apoptotic only if the plasma
membrane was stained by annexin-V-uos and the nucleus was not
stained by propidium iodide. The numbers of cells positive for
annexin-V-uos or propidium iodide staining were expressed as the
percentage of total cells.
1018 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
Fig. 1. Cd
2+
-induced whole-cell Cl

currents in primary culture cells from proximal convoluted tubule (PCT) of cftr
+/+
and cftr
/
mice. (A,B) Whole-cell currents recorded under
control conditions and 8 min after an extracellular perfusion of Cd
2+
(5 M CdCl
2
). Experiments were performed in primary culture (1015 days old) fromcftr
+/+
(A) and cftr
/
mice
(B). Currents were recorded in symmetrical 140 mMNMDGCl bath and pipette solutions. The membrane potential was held at-50 mV and currents were elicited by a train of 12 voltage
steps (400 ms duration) between-100 and +120 mV in increments of +20 mV. (C) Mean current-voltage (I/V) relationships measured at 350 ms after the onset pulse corresponding to
experiments performed with cftr
+/+
PCT cells under control condition (squares, n=16) and after a 8-min Cd
2+
exposure (circles, n=13) or after substitution of the Cl

from the
bath by I

(triangles, n=8). (D) Mean current-voltage (I/V) relationships corresponding to the experiments performed with cftr
/
PCT cells under control condition (squares,
n=15) and after an 8-min Cd
2+
exposure (circles, n=15). (E) Histograms of whole-cell Cl

current amplitudes (measured at +100 mV) in PCT cells from cftr

+/+
and cftr
/
mice
recorded in the absence (control) or presence of Cd
2+
(5 M). Whole-cell Cl

current amplitudes were also measured in PCTcells fromcftr

+/+
mice after Cd
2+
exposure in the presence
of NAC (10 mM), DIDS (1 mM), glibenclamide (glib.100 M), CFTR
inh
-172 (5 M), or when replacing the extracellular Cl

ions of the bath solution by I

. Values are meansSE; n,

number of monolayers from 4 different mice. (F) Histograms of whole-cell Cl

current amplitudes (measured at +100 mV) in PCT cftr

+/+
cells recorded after NAC preincubation
(3 h) and 10 min after Cd
2+
exposure. Values are means SE; n, number of monolayers from 2 different mice.
1019 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
Measurement of caspase-3 activity
Caspase-3 activity was measured using commercial colorimetric
assays (CaspACE assay system, Promega). The activity was assayed in
triplicate or quadruplicate on protein extracts obtained after lysis of
proximal cftr
+/+
and cftr
/
cells. As indicated by the supplier, the
involvement of other related proteases was excluded by observing the
difference between color intensity in the absence and presence of a
specic caspase-3 inhibitor (Z-VAD). The absorbance was measured at
405 nm using an ELX-800 automated microplate reader (Bio-Tek
instruments, Inc.).
Cell volume measurements
Cell volume was measured by an electronic sizing technique using
a CASY 1 cell counter (Schrfe System) according to L'Hoste et al. [23].
Briey, proximal cftr
+/+
and cftr
/
cell lines exposed to the different
experimental conditions were rapidly trypsinized (1X, 45 s) and cell
volume measurement was performed just after suspending the cells in
Casyton solution (NaCl isotonic solution).
Measurement of reactive oxygen species
The level of cellular oxidative stress was measured using the
uorescent probe (5-and-6)-carboxy-2,7-dichlorodihydrouores-
cein diacetate (carboxy-H
2
DCFDA). Carboxy-H
2
DCFDA is a cell-
permeant indicator for ROS that becomes spontaneously uorescent
when the acetate groups are removed by intracellular esterase activity
and cell oxidation. This probe is trapped mainly in the cytoplasm and
is oxidized by several ROS, most notably hydrogen peroxide. Briey,
proximal cftr
+/+
and cftr
/
cell lines were incubated in petri dishes
at 37 C for 30 min in the presence of carboxy-H
2
DCFDA (10 M) and
gently washed in serum-free culture medium. Cells were then rapidly
trypsinized (10X, 45 s) and incubated in the absence or presence of
either CdCl
2
(5 M) or NAC (N-acetylcysteine, 10 mM) or both
substances. Variations of uorescence of the cell suspension were
measured every 2 min using a Genius Spectrouorimeter (SAFAS,
Monaco) at 538 nm.
Measurements of intracellular GSH-GSSG content and intracellular
uorescent glutathione S-conjugate
Variations of intracellular GSH-GSSG content in cftr
+/+
and cftr
/
cell lines were quantied using an enzymatic kit (glutathione assay
kit, Sigma) according to Grifth [24]. Cells were incubated with
CdCl
2
(5 M) for 10, 20, or 30 min at 37 C and rapidly lysed (liquid
nitrogen treatment). The cell lysate was analyzed for glutathione
content (GSH+GSSG) by quantifying the formation of TNB (5-thiobis
(2-nitrobenzoic) acid) amount obtained by reduction of DTNB (5,5-
dithiobis(2-nitrobenzoic) acid) in the presence of glutathione reduc-
tase. The quantity of TNB was measured at 412 nm using the ELX-800
automated microplate reader and reected the total amount of GSH/
GSSG content of the sample.
Intracellular glutathione S-conjugate was measured with the
uorescent probe CMFDA (5-chloromethyluorescein diacetate,
Molecular Probes, USA). CMFDA is a cell-permeant indicator for
glutathione that becomes spontaneously uorescent when the acetate
groups are removed by intracellular esterase activity [25]. This probe
is trapped mainly in the cytoplasm and is rapidly bound by
glutathione. Fifteen-day-old PCT primary cultures grown on 35-mm
petri dishes were loaded with 1 M CMFDA for 150 s in culture
mediumat roomtemperature. After rinsing in culture mediumdevoid
of phenol red, cells were installed on the stage of an inverted
microscope maintained at 37 C and illuminated with a xenon lamp
through a dichroic lter (excitation 490 nm and emission over 510
nm). Digital images were recorded every 30 s with a low light level
camera and processed for gray level analysis. Values were expressed
as the percentage of the gray level recorded at t-0.
To follow simultaneously the variations of intracellular and
extracellular CMFDA glutathione contents, PCT cftr
+/+
immortalized
cells were seeded on 24 well coated collagen plates. After 6 days cells
were loaded with 10 M CMFDA (1 h at 10 C). After rinsing in phenol
red-free culture medium, cells were incubated at 37 C in culture
medium containing CdCl
2
(5 M) in the presence or the absence of
CFTR
inh
-172 (5 M). Total culture medium from each well (1 ml) was
collectedalong time and analyzed for GS-CMFDAuorescence content.
The remaining intracellular GS-CMFDA uorescence was determined
on the adherent cells from the corresponding culture well after cell
solubilization in 1 ml of culture medium containing 1% Triton X-100.
Efux of Cd
2+
Cd
2+
efux was performed in cftr
+/+
and cftr
/
PCT cell lines.
Briey, 2-day-old conuent PCT monolayers were cultured in serum-
free medium for 24 h and incubated for 10 min in the presence of
CdCl
2
(5 M). The monolayers were washed one time with calcium-
free PBS solution and one time with calcium-free PBS solution
containing EGTA (1 mM). At the end of the washing period, 1 ml of
PBS medium supplemented or not with CFTR
inh
172 (5 M) or the
inhibitor of glutathione biosynthesis BSO (buthionine sulfoximine,
0.5 mM) was added to each monolayer. The entire bathing medium
was collected every 10 min and immediately replaced by fresh
medium for 1 h. Cd
2+
contents of all collected samples were
determined using atomic absorption spectrometry with a Zeeman
furnace system(Solaar 969, Thermo Optek). Experiments performed
with BSO required a 24-h preincubation of the cells with serum-free
culture medium containing BSO (0.5 mM). Each point represented
the cumulative amounts of Cd
2+
determined at 10, 20, 30, 40, or 60
min. Under each experimental condition, rates of Cd
2+
efuxes were
also calculated between 10 and 20 min.
Chemical compounds
CdCl
2
was prepared from a stock solution of 5 mM in distilled
water. Stock solutions of CFTR
inh
-172 (5 mM), CMFDA (5-chloro-
methyluorescein diacetate, 10 mM), forskolin (10 mM), and -
tocopherol (100 mM) were prepared in DMSO and diluted accord-
ingly. DIDS (1 mM), NAC (10 mM) H
2
O
2
, and BSO (0.5 mM) were
prepared directly at nal concentrations every day.
Results
Cd
2+
application induced CFTR like Cl
-
currents in cultured PCT cells
Whole-cell currents were recorded in primary cultures of cftr
+/+
or
cftr
/
PCT cells in the absence or presence of Cd
2+
(5 M, CdCl
2
).
Experiments were performed with symmetrical 140 mM NMDG-
chloride in pipette and bath solutions. The external osmolarity was
maintained at 350 mosmol/kgH
2
O (by addition of mannitol) to avoid
spontaneous development of volume-activated Cl

currents [15,26].
Under control conditions, the voltage-step protocol elicited small
currents that changed linearly with the membrane potential in PCTcells
from cftr
+/+
mice (Fig. 1A). The corresponding I/V curve (Fig. 1C)
indicated a reversal potential (Erev
v
) of -2.9 1.8 mV and slope
conductance of 2.9 0.5 nS (n=16 monolayers from 4 mice).
Exposing the cftr
+/+
cells to Cd
2+
(5 M) induced a large increase in
the linear currents within 4 min after the onset of the Cd
2+
application
(Fig. 1A). These Cl

currents were maximal after 10 min and remained

constant for 45 min with a slope conductance of 9.5 1.5 nS and a
reversal potential of 0.660.75 mV (Fig. 1C, n=16 monolayers from 4
mice). Replacing external Cl

ions by I

strongly inhibited the Cd

2+
-
induced Cl

currents and caused a signicant shift of Erev toward

1020 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
positive values. (Erev for I

=+19.15.2 mV, n=7 monolayers from3

different mice). Further experiments (Fig. 1E) showed that the Cd
2+
-
induced Cl

current recorded at+100 mV in cftr

+/+
PCT cells was
insensitive to DIDS (1 mM) and blocked by glibenclamide (100 M),
I

, and CFTR
inh
-172 (5 M). These observations indicated that in
cftr
+/+
PCT cells, the Cl

conductance stimulated by Cd
2+
is related
to CFTR. As expected Cd
2+
did not modify the currents recorded in
cftr
/
PCT cells (Figs.1B, D, and E right). Experiments were also
performed to test the effect of a ROS scavenger (NAC, 10 mM) on the
Cd
2+
-activated CFTR Cl

currents. NAC did not modify the CFTR Cl

current already activated by Cd

2+
exposure (Fig. 1E). By contrast when
the cells were pre incubated with NAC (2 h) the addition of Cd
2+
failed to activate a CFTR Cl

current probably because NAC chelated

Cd
2+.
(Fig. 1F).
Mechanism of CFTR-like Cl

currents activation by Cd
2+
In several epithelia, it is well established that CFTR Cl

currents are
activated by cAMP-dependent protein kinase A (PKA) phosphorylation.
To determine the involvement of PKA in Cd
2+
-induced Cl

con-
ductance, the effect of H89 (10 M, an inhibitor of PKA) was examined.
H89 exposure (30 min prior to the addition of Cd
2+
) did not prevent
the development of the Cd
2+
-induced Cl

conductance (Figs. 2A and

C). Similar to Fig. 1, the Cd
2+
-induced Cl

currents measured at +100

mV in the presence of H89 were inhibited by substitution of Cl

ions of
the bath by I

(Figs. 2C and E). Moreover, incubation of the cells with

forskolin (10 M) 15 min prior to the addition of Cd
2+
did not modify
the amplitude of the Cd
2+
-activated Cl

currents (Fig. 2E). In the

absence of Cd
2+
, the incubation with forskolin alone did not stimulate
Fig. 2. Regulation of Cd
2+
-induced whole-cell Cl

currents in primary culture of PCT cells from cftr

+/+
mice. (A,B) Whole-cell Cl

currents recorded in the presence of H89 or PD

98059 and in the concomitant presence of Cd
2+
and H89 (A, 10 M) or PD 98059 (B, 10 M). A 30-min preincubation period was performed for H89 and PD 98059 experiments. (C)
Mean current-voltage (I/V) relationships corresponding to the experiments performed in (A) with cftr
+ /+
PCT cells in the presence of H89 (squares) and after Cd
2+
exposure (10
min, circles, n=3) or after substitution of the Cl

from the bath by I

(triangles, n=3). (D) Mean current-voltage (I/V) relationships corresponding to the experiments performed
in (B) with cftr
+ /+
PCTcells in the presence of PD 98059 (squares) and after Cd
2+
exposure (10 min, circles, n=3). (E) Histograms of whole-cell Cl

current amplitudes measured at

+100 mV in PCT cells from cftr+/+mice in the presence of H89 (10 M) or PD 98059 (1 0 M) or forskolin (FK, 10 M) before and after Cd
2+
exposure (810 min) and when
replacing the extracellular Cl

ions of the bath by I

. Values are meansSE; n, number of monolayers from 3 different mice.

1021 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
Cl

currents (data not shown). Altogether, these experiments excluded a

role of cAMP in the development of Cl

currents during Cd
2+
application.
Another signaling pathway was therefore tested. Since mitogen-activated
proteinkinases (MAPK) have beenidentied as mediators ina wide array
of physiological processes, we have examined their role as potential
mediators of Cd
2+
-induced CFTR activation. For this purpose, PD 98059
(10 M), a strong inhibitor of the mitogen-activated protein kinase kinase
(MAPKK/MEK), was tested on the Cd
2+
-induced Cl

currents. As
illustrated in Fig. 2B, the preincubation of the cells with PD 98059
completely prevented the development of Cl

currents induced by Cd
2+
exposure (Figs. 2B, D and E).
The inhibitory effect of PD 98059 suggests an important role of ERK
1/2 in the cascade of events leading to Cd
2+
-induced Cl

current. The
effect of Cd
2+
on ERK 1/2 phosphorylation was then tested by Western
blots analysis. Cd
2+
(5 M) exposure induced a signicant increase of
phosphorylation of p44/p42 (ERK 1/2) within 8 min with a maximum
reached after 60 min (Figs. 3A and B). The increased phosphorylation of
ERK 1/2 was completely prevented by pretreatment with PD98059 (10
M). Interestingly, ERK 1/2 phosphorylation was also stimulated by
Cd
2+
exposure in cftr
/
proximal cells. Thus the activation of ERK 1/2
by Cd
2+
was independent of CFTR and preceded the stimulation of
Cl

currents in cftr
+/+
cells (Fig. 3B). Additionally, H89 (10 M)
preincubation had no effect on the Cd
2+
-mediated activation of ERK in
both cell lines (data not shown, n=5) conrming that the CFTR
activation did not depend on PKA phosphorylation.
Consequences of CFTR activation by Cd
2+
Apoptosis vs necrosis
It is commonly admitted that cadmium induces cell death either by
apoptosis or by necrosis. The toxicity of Cd
2+
on both PCT cell lines was
then quantied using a uorescent costaining (Fig. 4A): Annexin-V-
Fluos labeling was used to estimate the apoptosis level of the Cd
2+
-
exposed cells while propidium iodide (PI) nuclear labeling was used to
quantify the necrosis level. In the cftr
+/+
cell line, addition of Cd
2+
to
the bath solution induced phosphatidylserine exposure at the cell
surface and a signicant increase of the level of annexin-positive cells
(33.8 3.2%, n=5, Fig. 4B). However, this Cd
2+
-induced increase of
annexin V binding was not correlated to a concomitant increase of
nucleus labeling by propidium iodide (Fig. 4C), suggesting that the
annexin-positive cells underwent the majority of apoptosis. By contrast
in the cftr
/
cell line, Cd
2+
did not enhance annexin-positive cell
numbers but increased signicantly the propidium iodide nucleus
labeling (49.2 10.8%, n=5; Fig. 4C). Altogether, these data revealed
that the Cd
2+
-induced apoptosis was related to the presence of CFTR in
the PCT cell line. To conrm this hypothesis the effect of Cd
2+
on
caspase-3 activity was monitored in both cell lines. Cd
2+
exposure (5
M) for 6 h induced a 2-fold increase in caspase-3 activity in the
cftr
+
/
+
cell line but was without effect in cftr

cells (Fig. 4D). This

Cd
2+
-induced caspase-3 activity was completely abolished in the
presence of CFTR
inh
-172 (5 M) conrming the crucial role of CFTR in
Cd
2+
-induced apoptosis. As observed for Cd
2+
-activated Cl

-currents
(Fig. 2E)forskolinexposuredidnotmodifytheCd
2+
-inducedcaspase-3
increase (Fig. 4D). Interestingly, incubating the cftr
+/+
cells with the
ROSscavengers NAC(10mM) or -tocopherol (100M) preventedthe
increaseof caspase-3activityinducedbyCd
2+
(Fig. 4D).
Apoptotic volume decrease
The process of apoptosis is generally associated with a reduction of
cell volume (apoptosis volume decrease, AVD). To check whether the
Cd
2+
-induced apoptotic process was related to an AVD phenomenon,
the time course of relative cell volume variation during Cd
2+
treatment
was measured in proximal cftr
+/+
and cftr
/
cell lines. In the cftr
+/+
line, the cells shrank signicantly 1 hafter the onset of Cd
2+
exposure (5
M, Fig. 5A). The maximal cell volume reduction (26 1%, n=5) was
reached 6 h after the application of Cd
2+
. After this time the volume
remained almost constant for at least 12 h (data not shown). Moreover,
in cftr
+/+
cell lines the Cd
2+
-induced AVD process was completely
blocked by the addition of CFTR
inh
-172 (5 M, Fig. 5A). Conversely
cftr
/
cells did not decrease their volume in the presence of Cd
2+
(Fig.
5B). Taken together these experiments strongly suggested the involve-
ment of CFTRin AVDand apoptosis inducedby cadmium. Incidentally as
it was observed for caspase-3 activity, the addition of NAC or -
tocopherol prevented the Cd
2+
-induced AVD process (Fig. 5A).
Ion conductances activated during the AVD process
The changes in cell volume during AVD are the consequence of an
exit of Cl

and K
+
ions from the cells. These conductances were
therefore analyzed during the time course of AVD induced by
cadmium. As far as the Cl

currents were concerned, the results

described above have suggested that soon after the application of
Cd
2+
the total Cl

conductance was driven by CFTR channels. The

experiments described in the Figs. 6A and B illustrated these data and
conrmed that the Cd
2+
-induced Cl

current recorded during the

rst 45 min of Cd
2+
exposure was completely inhibited by CFTR
inh
-
172. After this time the Cl

conductance increased and the addition of

CFTR
inh
-172 unmasked an outwardly rectifying Cl

current (Figs. 6C
and D) sensitive to DIDS (1 mM, not shown). This conductance
resembled the classical volume-sensitive outwardly rectifying Cl

current previously reported in the literature [15].

K
+
currents were also recordedduring Cd
2+
exposure. No signicant
variation of K
+
conductance was observed during the rst 45 min of
incubation (data not shown). After this time a K
+
selective conductance
was recorded in all the cells tested (n=5 monolayers from2 mice). This
conductance was markedly decreased in the presence of an inhibitor of
TASK-2 K
+
current, clolium (10 M), suggesting the involvement of
TASK-2 channels in the Cd
2+
-induced AVD (Figs. 6 E and F).
Fig. 3. Cd
2+
induces ERK1/2 phosphorylation in proximal cell lines from cftr
+/+
and
cftr
/
mice. (A) Western blotting of PCT cftr
+ /+
cell line lysate using the phospho-
ERK1/2 antibody to detect the variation of phosphorylation of ERK1/2 when exposing
cells to Cd
2+
alone (5 M, 8 min) or to Cd
2+
+PD 98059 (10 M, 8 min of incubation).
An antibody revealing the total ERK1/2 level was also used to control the total expression
of ERK1/2. (B) Histograms representing the level of ERK1/2 phosphorylation obtained for
different times of Cd
2+
exposure (8, 30, and 60 min) obtained in cftr
+/+
and cftr
/
cell
lines. Western blots were quantied using PCB as protocol. Results are expressed as
meanSE increases in ERK phosphorylation in arbitrary units (n=5).
1022 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
ROS production during Cd
2+
exposure
It has been reported that Cd
2+
produced reactive oxygen species
[3,4]. Moreover this ROS production could be an early process to
induce AVD [11]. Our study conrms the involvement of ROS as well in
the Cd
2+
-induced apoptosis (Fig. 4D) as in the Cd
2+
-induced AVD
(Fig. 5A). To further analyze the relationship among ROS production,
cadmium-induced apoptosis, and CFTR, the intracellular ROS varia-
tions were monitored in both cell lines using a ROS-sensitive
uorescent probe during cadmium exposure. As illustrated in Fig. 7A
the intracellular level of ROS increased rapidly after a Cd
2+
application
Fig. 4. Effect of Cd
2+
on the level of apoptosis/necrosis and caspase-3 activation in proximal cftr
+/+
and cftr
/
cell lines. (A) Phase contrast and uorescence micrographs
taken from cftr
+/+
and cftr
/
cell lines after 6 h of Cd
2+
exposure. Given images obtained in phase contrast and in uorescent mode correspond to the same eld of the
monolayer. Exposure of phosphatidylserine residues on the outer leaet of the plasma membrane of the apoptotic cells are stained with the annexin-5-uos. Propidium iodide
labeling of the nucleus revealed only the necrotic cells. Cells were visualized with a microscope in phase contrast (left) and in uorescent mode at 550 nm for annexin-5-uos
labeling (middle) and at N600 nm for propidium iodide labeling (right). (B,C) The induction of apoptosis and necrosis processes by Cd
2+
was estimated by using annexin-5-
uos (B) and propidium iodide (C) costainings. cftr
+/+
and cftr
/
cell lines were exposed to Cd
2+
(5 M) for 6 h, rapidly washed and costained with annexin-5-uos and
propidium iodide. Annexin-5-uos labeling revealed the apoptotic cells and cells positively stained by propidium iodide are considered necrotic. Ratios of annexin-positive cells
(B) and propidium iodide-labeled cells (C) are expressed in percentage of total cells. Values are means SEM of 5 independent experiments. (D) Measurements of caspase-3
activity in proximal cftr
+/+
and cftr
/
cell lines obtained under control conditions and in the presence of Cd
2+
(5 M) or in the concomitant presence of Cd
2+
(5 M) and
CFTR
inh
-172 (5 M), NAC (10 mM), -tocopherol (100 M), or forskolin (10 M). CFTR
inh
-172, -tocopherol, and NAC signicantly suppressed Cd
2+
-induced caspase-3
activation. Values are means SEM of 7 different experiments. Pb 0.005; Student's t test.
1023 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
in cftr
+/+
cells. The addition of CFTR
inh
-172 (5 M), NAC (10 mM), or
-tocopherol (100 M) completely inhibited this Cd
2+
-induced ROS
increase. Moreover in the cftr
/
cell line, Cd
2+
exposure did not
increase ROS production (Fig. 7B). In conclusion CFTR could be
involved in the control of ROS generated by Cd
2+
exposure.
In the literature, it has been demonstrated that ROS production can
directly activate Cl

channels [11]. If Cd
2+
exposure induced ROS
production then the recorded outwardly rectifying Cl

current (Figs.
6C and D) might be activated by the Cd
2+
-induced ROS generation
and not directly by cadmium. To test this hypothesis, cftr
+/+
cells
were exposed to H
2
O
2
(500 M) and Cl

currents were recorded in

whole-cell conguration. As illustrated in Figs. 7C and D, H
2
O
2
exposure induced in a few minutes (510 min) the activation of an
outwardly rectifying Cl

current insensitive to CFTR

inh
-172 (5 M, not
shown) and strongly inhibited by DIDS (1 mM). This result suggested
that Cd
2+
exposure might induce indirectly the activation of an
outwardly rectifying Cl

current through the elevation of intracellular

ROS concentration.
Reactive oxygen species can be converted by cellular catalyzing
enzymes to peroxynitrite and nally to NO. Previous reports have
already shown that NO activates CFTR via cGMP-dependent and-
independent mechanisms [2729]. Experiments were performed to
study a putative CFTR activation by NO and the involvement of NO
in the Cd
2+
-induced caspase activation. cftr
+/+
PCT cells were
preincubated 1 h in the presence of L-NAME (an inhibitor of NO
production, 100 M) and then exposed to 5 M Cd
2+
(in the
presence of 100 M L-NAME). As illustrated in Figs. 8A and B,
L-NAME did not prevent the activation of CFTR
inh
-172-sensitive Cl

current by Cd
2+
. Additionally, L-NAME (100 M) remained without
effect on the basal level of caspase-3 and did not prevent the
activation of caspase-3 after 6 h of Cd
2+
exposure (Fig. 8C).
Variations of intracellular total GSH level during Cd
2+
exposure
Glutathione (GSH) is present in high amounts in proximal tubule
cells and reacts to neutralize ROS. Because Cd
2+
increase ROS
production, it was interesting to measure the variation of intracellular
GSH concentration during cadmium exposure in both cftr
+/+
and
cftr
/
cells. For this purpose total glutathione content (GSH/GSSG)
was determined. Fig. 9A demonstrates that Cd
2+
induced a rapid
decrease of intracellular GSH content in cftr
+/+
cells. This decrease
was strongly inhibited in the presence of CFTR
inh
-172 and was not
observed in cftr
/
cell lines. To further demonstrate the involvement
of glutathione, we performed experiments on primary cultures of PCT
loaded with the uorescent probe CMFDA which forms a S-conjugate
with GSH(GS-CMFDA). As illustrated in Fig. 9B, addition of Cd
2+
(5 M)
on CMFDA-loaded cftr
+/+
cells induced an intracellular decrease of the
uorescent CMFDA-conjugate (uorescence decrease rate: 5.3 0.2%
min
1
, n=5). This decrease was related to the presence of intracellular
GSH since it was strongly inhibited in the presence of a specic inhibi-
tor of glutathione production (DL-buthionine-S,R-sulfoximine; BSO,
uorescence decrease rate: 1.15 0.3%.min
1
, n=3). Interestingly the
Cd
2+
-induced decrease of the uorescent CMFDA conjugate was
inhibited in the presence of CFTR
inh
-172 (1.85 0.1% min
1
, n=3,
Figs. 9B and D) and was not observed in the cftr
/
cell line. These
results suggested that CFTR was essential for the reduction of
intracellular GSH content induced by Cd
2+
. The reduction of intracel-
lular GSH content observed in Fig. 9B corresponded to a concomitant
increase of extracellular GSH. Measurements of intracellular and
extracellular contents of the uorescent GS-CMFDA conjugate during
Cd
2+
exposure conrmed this hypothesis (Fig. 9C). However, the
increase of extracellular GS-CMDA content triggered by Cd
2+
was
strongly reduced in the presence of CFTR
inh
-172 (Fig. 9C). This
reduction was probably due to an inhibition of the Cd
2+
-induced
CFTR-dependent GSH efux across the cell membrane.
Additional experiments depicted in Figs. 9B and D demonstrate
that PD 98059 (10 M, 3 h preincubation) inhibited the Cd
2+
-
induced GSH efux while H89 (10 M, 3 h preincubation) remained
without effect.
Measurement of Cd
2+
efuxes
It has already been proposed in the literature [30] that GSH can
react with free Cd
2+
to form a GS-Cd conjugate. This newly formed
conjugate might be extruded from cells to reduce the intracellular
Cd
2+
concentration. To test this putative outowof intracellular Cd
2+
,
efuxes of Cd
2+
were measured in both cftr
+/+
and cftr
/
cells. For
this purpose, cftr
+/+
cell monolayers were loaded in the presence of
Cd
2+
(5 M, 10 min) and washed and fresh medium was added and
collected every 10 min for 1 h. The Cd
2+
content of each incubation
time was determined using furnace Zeeman spectrophotometry. As
illustrated in Fig. 10, the cumulative Cd
2+
amount measured in the
extracellular medium increased with time, suggesting a permanent
efux of Cd
2+
. Interestingly, CFTR
inh
-172 reduced signicantly this
efux (Figs. 10AC). Additionally, a 1-day preincubation with BSO (0.5
mM) to reduce the intracellular GSH content induced a strong
decrease of the Cd
2+
efux. In cftr
/
cells (Figs. 10B and C), the
basal efux of Cd
2+
was strongly reduced compared to cftr
+/+
cells
and CFTR
inh
-172 remained without effect. The histogram seen in Fig.
10C summarizes the rates of Cd
2+
efux variations measured under
the different experimental conditions in cftr
+/+
and cftr
/
cells.
Altogether, these experiments suggested that: (1) CFTR is directly
involved in the efux of Cd
2+
and (2) the Cd
2+
-efux rate is
correlated to the intracellular concentration of glutathione.
Discussion
In recent years a large number of studies have examined the renal
effects of cadmium intoxication. The nephrotoxicity of cadmium
results mainly fromits adverse effects on the proximal tubule because
this segment accounts for major portion of cellular Cd
2+
uptake [2,31].
With the use of in vitro models deleterious effects of Cd
2+
have been
described on several solute transporters as ion channels [32] and
transporters [3335]. Moreover Cd
2+
may cause nephrotoxicity by
Fig. 5. Effect of Cd
2+
on cell volume variations in proximal cftr
+/+
and cftr
/
cell
lines. (A) Measurements of cell volume variation in proximal cftr
+/+
cell line for
various Cd
2+
exposure times. Cell volume was measured by an electronic sizing
technique with a CASY 1 cell counter (Schrfe System). cftr
+/+
cells were suspended
in Casyton solution (NaCl isotonic solution), and relative volume was measured every
hour (for each time point, cells are isolated by trypsinization a few minutes before
measuring). Cell volume measurements were performed in the absence (circles) or
presence of Cd
2+
(5 M, squares) and in the concomitant presence of Cd
2+
and
CFTR
inh
-172 (5 M, diamonds), NAC (10 mM, triangles), -tocopherol (100 M, gray
symbols), forskolin (10 M, black stars). Values are expressed as the percentage of
cell volume variation measured before Cd
2+
exposure. Values are means SEM of 5
different experiments. (B) Measurements of cell volume variation in proximal cftr
/
cell line were performed as in (A) in control condition (circles) or in the presence of
Cd
2+
(5 M, squares). Values are means SEM of 5 different experiments.
1024 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
generating free radicals [4,36] and by inducing necrosis and apoptosis
[3]. As far as apoptosis is concerned it is known that the process
leading toward this cell death is coupled to normotonic cell shrinkage
called apoptotic cell volume decrease (AVD). The changes in cell
volume during AVD are the consequence of an exit of Cl

and K
+
from
the cells and the question arises as to whether Cd
2+
could induce
Cl

and K
+
currents to trigger nal apoptosis of proximal tubular cells.
Therefore the rst aim of this study has been to verify the ability of
Cd
2+
to induce such ion currents. Surprisingly our results demon-
strate that in the proximal cells fromwild-type mice acute application
of 5 M CdCl
2
signicantly enhanced the Cl

conductance. This
conductance exhibited properties consistent with CFTR Cl

channels.
Thus the Cl

channels activated by Cd
2+
were blocked by glib-
enclamide, by I

substitution, and by CFTR

inh
-172, a potent and very
specic inhibitor of CFTR [37,38]. To further implicate CFTR in the
Cd
2+
-induced Cl

currents, experiments were performed in proximal

cells from cftr
/
mice. As expected these cells did not exhibit any
Cd
2+
-dependent Cl

currents. Only few studies have reported the

effects of Cd
2+
on Cl

transport in epithelial cells. However Faurskov

and Bjerregaard [39] have found that Cd
2+
administered to the
basolateral side of A6 cells induced a transient electrogenic Cl

secretion. Interestingly, Cd
2+
has also been shown to induce Cl

secretion in the shark rectal gland epithelia by a cAMP-dependent

mechanism [40]. No further indications were given on the molecular
basis of this Cl

secretion; however, it is now well established that in

this systemcAMPactivates a Cl

channel, whichis the sharkcounterpart

of the mammalian CFTR [41].
The emerging issue was therefore how Cd
2+
could activate CFTR
Cl

currents in proximal cells. One possibility would be that Cd

2+
directly activated CFTR but this appears unlikely because the
activation required minutes instead of seconds to increase Cl

conductance. Nevertheless a direct action of Cd

2+
on CFTR has been
previously shown [42]. In this study Cd
2+
interacts covalently with
CFTR to decrease both opening and closing rates of the channel.
Indeed, this conclusion is at variance with that in the present study:
the experiments were performed on CFTR molecules incorporated in
planar lipid bilayers with high Cd
2+
concentration (100 M). In our
experiments Cd
2+
concentrations higher than 50 M were lethal
Fig. 6. Effect of short andlong time Cd
2+
exposure onthe whole-cell Cl

andK
+
currents inthe proximal cftr
+/+
cell line. (A,C) Whole-cell Cl

current traces recordedinthe presence of

Cd
2+
(10 M) for short (A, time b to 45 min) and long exposure times (C, time N to 45 min). The records were obtained for each exposure time before (left traces) and after perfusion of
CFTR
inh
-172 (5 M). Records were performed withsymmetrical NMDGCl pipette and bath solutions. The membrane potential was held at-50 mVand currents were elicitedbya trainof
11 voltage steps (400 ms duration) between-100 and+100 mV. (B,D) The I/V curves corresponding to whole-cell traces illustrated in Figs. A and B corresponding to short and long
exposure times aredepictedrespectively inFigs. CandD. Squares illustratedthe means I/Vrelationships (SEM, n=34) recordedinabsence of CFTR
inh
-172while circles represented
the mean I/V relationships recorded in the presence of CFTR
inh
-172 (5 M). (E,F) Whole-cell K
+
current traces recorded after a minimum of 45 min of Cd
2+
exposure (10 M) in the
absence or presenceof clolium(10M). The pipettewas lledwitha 140mMK-gluconatepipette solutionandthe bathcontained145mMNa-gluconate. The membrane potential was
held at-50 mV and currents were elicited by a train of 12 voltage steps (400 ms duration) between-100 and+120 mV. The corresponding I/V curve is illustrated on the right (F). The
diamonds represented the mean I/V relationship (n=4) recorded in the presence of Cd
2+
while squares represented the mean I/V relationship (n=2) obtained after clolium
exposure.
1025 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
within minutes (data not shown) so that we cannot conclude
whether high Cd
2+
concentrations would modify directly the gating
of the CFTR channel.
Alternatively Cd
2+
could indirectly regulate CFTR by activating
signaling pathways as suggested by the critical role played by PKA
in CFTR opening [43]. In cftr
+/+
cells, H89 did not inhibit the
activation of CFTR Cl

currents by Cd
2+
. Moreover the pretreatment
of the cells with forskolin did not modify the amplitude of the Cl

currents activated by Cd
2+
. These results conrm our previous
observation which indicated that forskolin or cAMP permeant
derivatives did not directly stimulate CFTR Cl

conductance in
primary culture of rabbit [26] and mouse [44] proximal tubules
despite the presence of CFTR mRNA. Therefore it was important to
investigate the possibility that another signaling pathway was
involved in the activation of CFTR Cl

current by Cd
2+
. ERK/
MAPKK are candidates known to activate ion channels [45].
Treatment of cftr
+/+
cells with Cd
2+
induced a rapid and sustained
activation of ERK1/2. Furthermore preincubation of the cells with
PD 98059 reduced drastically the ability of Cd
2+
to activate ERK1/2
and to increase the CFTR Cl

currents. These data provide evidence

that ERK pathways could participate in CFTR activation by Cd
2+
. A
relation between ERK/MAPKK activation and butyrate-induced
expression of CFTR was previously demonstrated by Sugita et al.
[46]. According to their results the integrity R domain of CFTR could
be important in this regulation involving active ERK/MAPK in the
biogenesis and trafcking of the protein. Moreover, ERK might
phosphorylate proteins that interact and regulate CFTR. For example,
emerging data implicate MAP kinase signaling in the control of F-
actin [47] and it is also postulated that actin must be directly
associated with CFTR to elicit its activation [48]. One other
possibility would be that ERK directly phosphorylates CFTR. It is
evident that the exact mechanism of CFTR activation by ERK1/2
remains to be further explored. Whatever this mechanism, our data
suggest that Cd
2+
could induce the ERK1/2 phosphorylation with a
kinetic which was compatible with the activation of CFTR. Previous
studies have also documented that NO could stimulate Cl

secretion
across airwayepithelial cells by an increase of cGMP level leading to
activation of CFTR [27,28]. Such a mechanism could be also
involved in the Cd
2+
-activated CFTR Cl

current in PCT cells since

it is well known that Cd
2+
leads to increased production of reactive
oxygen nitrogen species [49]. However incubation of cftr
+/+
cells with
an inhibitor of NOproduction (L-NAME) did not prevent the activation
of CFTRCl

currents by Cd
2+
. This result indicates that rapid activation
of CFTR did not occur via Cd
2+
-dependent NO release.
Beside the activation of Cl

conductance, the question arises:

what is the consequence for the cell of CFTR activation by
cadmium? It is known that Cd
2+
kills the cells by apoptosis and/
or necrosis. Moreover it has been well established that CFTR could
be involved in the control of apoptosis [14,50]. This implication is
conrmed here because Cd
2+
induced an increase in caspase-3 and
apoptosis in proximal cells exhibiting functional CFTR only. As in
most mammalian cells, a cell volume decrease accompanies
apoptosis [8]. Apoptotic volume decrease is an early prerequisite
event for apoptosis and precedes caspase activation during
apoptotic processes [8]. In our study, it is noteworthy that Cd
2+
-
dependent AVD did not take place in the absence of CFTR. In cftr
+/+
Fig. 7. Cd
2+
-induced ROS production and Cd
2+
efux in PCT cftr
+/+
and cftr
/
cell lines. (A,B) Cd
2+
-induced ROS production in cftr
+/+
(A) and cftr
/
(B) cell lines. Cells were
incubated for 30 min in carboxy-H
2
DCFDA (10 M) and washed in serum-free culture medium. After trypsinization, isolated loaded cells were incubated in the absence (squares) or
presence of Cd
2+
(5 M, circles), Cd
2+
+CFTR
inh
-172 (5 M, diamonds), Cd
2+
+NAC (10 mM, triangles), or Cd
2+
+-tocopherol (100 M, gray symbols) or Cd
2+
+forskolin (10 M,
black stars). The emitted uorescence intensity was measured every 2 min at 538 nm. Values are expressed as the relative variations of the uorescence intensity SE of 5 different
experiments. (C) Whole-cell Cl

current traces recorded in the absence (left, control), presence of H

2
O
2
(500 M, middle), and after addition of DIDS (right, 1 mM). Records were
performed with symmetrical NMDGCl pipette and bath solutions. The membrane potential was held at 50 mV and currents were elicited by a train of 11 voltage steps (400 ms
duration) between 100 and +100 mV. (D) Corresponding I/V curves to whole-cell traces illustrated in panel C. Symbols represented the mean I/V relationships (SEM, n=3
4) recorded under control conditions (squares) in the presence of H
2
O
2
(circles, 500 M, 510 min) and after DIDS exposure (triangles, 1 mM).
1026 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
cells the Cd
2+
-dependent AVD resulted mainly from the loss of
cytosolic Cl

and K
+
(and osmotically obliged water) due to the
activation of Cl

and K
+
currents. Both conductances exhibited
similar biophysical properties as compared to those recorded
during RVD in response to a hypotonic shock (VSOR)
Cl

conductance and TASK2 K

+
conductance [21,22]. As expected
these Cl

and K
+
currents were not recorded in wild-type cells
treated with CFTR
inh
-172 or in cftr
/
cells. Concerning TASK2
K
+
channels it has been recently demonstrated that they are also
involved in staurosporine-induced AVD in proximal tubule cells [13].
However, in cftr
/
cells, TASK2 channels were always activated
during RVD [22] whereas they were inhibited during Cd
2+
-induced
AVD [15]. Finally, it is very likely that CFTR controls apoptosis by
modulating both the swelling-activated Cl

and the TASK2 K

+
channels activated during Cd
2+
-induced AVD. In RVD phenomenon
it has been postulated that CFTR controls the swelling-activated
Cl

channels by modulating autocrine adenosine production in

renal cells. The resulting exit of Cl

would provide the driving

force to activate the Cl

/HCO
3

exchanger, allowing an efux of

HCO
3

which activates the TASK2 channels [15,22,23]. By contrast

in the present study, the Cd
2+
-induced AVD was insensitive to
adenosine and to the activity of the Cl

/HCO
3

exchanger (data not

shown), suggesting that CFTR controlled AVD by a different
mechanism. In a recent work performed in proximal cells, L'Hoste
et al. [13] have shown that the staurosporine-induced apoptosis
involved an interplay among AVD, ROS production, and TASK2
channels. It has been already reported that cardiomyocyte
apoptosis involved VSOR Cl

channel activity and intracellular

ROS production [10]. As commonly admitted, Cd
2+
(as other heavy
metals) increased production of ROS in the proximal tubule cells
[3,4]. In our study the production of ROS induced by Cd
2+
depends
strongly on CFTR activity as AVD and apoptosis. Therefore it is
tempting to conclude that the modulation of ROS concentration by
CFTR during Cd
2+
exposure is an important step in the activation
of swelling-activated Cl

and TASK2 K
+
currents responsible for
AVD and apoptosis. The interaction of ROS with ion channels is
further supported by the observation that H
2
O
2
application induced
both swelling-activated Cl

currents and TASK2 K

+
currents in cftr
+/+
and cftr
/
cells (data not shown). Such effects of ROS on ion
channel activities have been already reported in the literature
[11,51]. Altogether these results conrm that ROS serve as upstream
signals in activating ion channels involved in AVD caused by
mitochondrion-mediated apoptosis inducers. Experiments have
been then performed in order to elucidate how CFTR could control
the Cd
2+
-induced ROS production in the proximal cells. It is well
known that glutathione (GSH), normally present in high amounts in
tubular cells, can react with and neutralize ROS [18]. Moreover
several ndings have noted that the CFTR channel is not only
permeant to Cl

, but also to larger organic anions including GSH

[5254]. As demonstrated in the lung, the ability of GSH to
permeate through CFTR confers to this protein an essential role in
the antioxidant defense of the epithelial cells [55]. In the proximal
tubule cells, our results indicate that the decrease of total GSH
content induced by Cd
2+
correlated with an increased GSH efux
and was strictly related to the presence of CFTR. On the basis of
these results it appears reasonable to postulate that this CFTR-
dependent GSH efux depletes intracellular GSH leading to an
increase in the ROS level. As discussed above this ROS increase
probably triggers AVD and apoptosis. It is recognized that depletion
of GSH is associated with the initiation of apoptosis [56]. Moreover a
relation between this process and the CFTR was already suggested.
Thus using epithelial cells, Jungas et al. [57] have discovered that
the rate of apoptosis and the rate of intracellular GSH depletion
were higher in cell expressing normal CFTR than in mutant cell
lacking CFTR during H
2
O
2
incubation. Our present study is in
accordance with these results and establishes the crucial role of
CFTR in apoptosis via the control of GSH level during Cd
2+
exposure of proximal tubule cells.
Fig. 8. Effect of L-NAME on the Cd
2+
-induced Cl-current and Cd
2+
-induced caspase-3 increase fromPCT cftr
+/+
cells. (A) Whole-cell currents recorded in L-NAME preincubated cells
(1 h, 100 M) 10 min after Cd
2+
perfusion (left, 5 M) and after CFTR
inh
-172 addition (right, 5 M). Experiments were performed in cftr
+/+
primary culture cells. Currents were
recorded in symmetrical 140 mM NMDGCl bath and pipette solutions. The membrane potential was held at 50 mV and currents were elicited by a train of 11 voltage steps (400 ms
duration) between 100 and +100 mV in increments of +20 mV. (B) Mean current-voltage (I/V) relationships measured at 350 ms after the onset pulse corresponding to
experiments performed in (A) with cftr
+/+
cells preincubated with L-NAME and exposed for 10 min to Cd
2+
(squares, n=4) and after addition of CFTR
inh
-172 (circles, 5 M). (C)
Measurement of caspase-3 activity in the cftr
+/+
cell line under control conditions, in the presence of Cd
2+
(5 M), L-NAME (100 M), or in the concomitant presence of Cd
2+
(5M)
and L-NAME (100 M). Values are means SEM of 4 different experiments.
1027 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
A recent study suggests that Cd
2+
binds GSHvia deprotonation of a
sulfhydryl group to form Cd
2+
-glutathione conjugates (GSH-Cd) [30].
Given the fact that CFTR belongs to the ABC transporter family, which
includes MRP transporters, CFTR could mediate not only GSH but also
the xenobiotic GSH conjugates [17,54]. It was therefore interesting to
check whether CFTR could also be involved in the detoxication of the
proximal tubule by extruding GSH-Cd conjugates out of the cell. The
measurement of Cd
2+
efux performed in cftr
+/+
and cftr
/
cells
clearly indicates that Cd
2+
efux depended on CFTR (Fig. 10). Further,
we have demonstrated that the reduction of intracellular GSH with
BSO strongly reduced the efux of Cd
2+
in cftr
+/+
cells. The simplest
interpretation of these results would be that during Cd
2+
application
CFTR is able to drive the efux of GSH-Cd conjugates. Putative
transporters of GSH have already been identied in the brush-border
Fig. 9. Effect of Cd
2+
on the variation of intracellular GSH/GSSG content in proximal cftr
+/+
and cftr
/
cell lines or in primary cultures of proximal tubules from cftr
+/+
and cftr
/
mice. (A) GSH/GSSG concentrations measured as a function of time in proximal cftr
+/+
and cftr
/
cell lines in the absence or presence of Cd
2+
or Cd
2+
+CFTR
inh
-172.
The intracellular glutathione content was assayed before (arrow) and 10, 20, and 30 min after addition of Cd
2+
to the bath in cftr
+/+
(closed symbols) and cftr
/
cell lines (open
symbols). Values are means SEMof 5 individual experiments. (B) Time-response uorescence curves measured in primary culture of cftr
+/+
and cftr
/
cells loaded with CMFDA
(1 M) and exposed to Cd
2+
(5 M) in the absence or presence of CFTR
inh
-172 (5 M), BSO (500 M, 24 h preincubation), H89 (10 M), or PD98059 (10M). Values are expressed as a
percentage of the uorescence recorded at t =0. The absolute values of uorescence (gray levels) at time zero were 192.1 6.5 (n=3), 186.2 7.3 (n=3), 199.1 6.5 (n=5),
188.2 8.6 (n=5) for cftr
+/+
, cftr
/
, cftr
+/+
+ CFTR172, and cftr
+/+
+ BSO, respectively. (C) Relation between the intracellular decrease and the correlated increase in the
extracellular compartment of the uorescent glutathione probe (CMFDA) asafunctionof timeinthecftr
+/+
cell line. Opensymbols:intracellularuorescencedecreaseinducedby
Cd
2+
(5M)intheabsence(opensquares)orinthepresenceof CFTR
inh
-172(5M, opendiamonds). Closedsymbols: concomitantextracellularuorescenceincreasemeasuredinthe
absence (closed square) or in the presence of CFTR
inh
-172 (5 M, closed diamonds). Values are expressed in uorescent arbitrary units SE of 3 independent experiments. (D)
Fluorescence slope variations obtained in primary culture of cftr
+/+
or cftr
/
cells loaded with CMFDA in the presence of Cd
2+
(5 M) alone or in the concomitant
presence of Cd
2+
and CFTR
inh
-172 (5 M), BSO (500 M), H89 (10 M), or PD98059 (10 M).Values are expressed as the percentage of uorescence loss per minute SE of
3 to 5 independent experiments recorded as in B.
1028 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
membrane of the proximal tubule cells. Of these transporters, MRP2/4
probably mediates the efux of GSHor GSHconjugates. Interestingly it
has been reported that long-term exposures to low concentrations of
CdCl
2
resulted in a concentration-dependent increase in MRP2
function, suggesting a protective role for this transporter [19]. In the
present study, the observation that the blockade of MRP transporters
by MK 571 (data not shown) did not modify the efux of GSH induced
by Cd
2+
excluded a putative involvement of MRP2 during acute
Cd
2+
intoxication.
Another possibility could be that CFTR controls the activity of
cation channels through which Cd
2+
may leave the cells. We have
previously demonstrated that CFTR indirectly controls the activity of
a cation channel. Notably during regulatory volume decrease induced
by hypotonic challenge, CFTR controls K
+
channels (TASK2 and BK
channels) via a mechanism involving Ca
2+
inux driven by
mechanosensitive Ca
2+
channels [22]. Although TASK2 and BK
channels are not recognized to be permeable to Cd
2+
, the entry of
Cd
2+
through Ca
2+
channels belonging to the TRPM family has been
already reported [58]. These kinds of channels could participate in
the accumulation of Cd
2+
into the cell. However, under our
experimental conditions, the electrochemical cell membrane poten-
tial was against the passive exit of Cd
2+
through a cationic channel.
The present paper is the rst report showing the role of CFTR in the
cascade of events triggered by acute Cd
2+
intoxication in proximal
cells. As summarized in Fig. 11, application of Cd
2+
induces the
activation of CFTR Cl

currents. Once activated, CFTR might transport

GSH and GSH conjugates. As suggested in the literature it is likely that
Cl

and GSH share common permeation through CFTR [52,54]. In the

proximal cells, the increase of GSH permeation through CFTR could
have at least two consequences: (1) a direct exit of GSH-Cd conjugates
that could contribute to a rapid detoxication of cells by pumping out
a part of the cytosolic Cd
2+
; (2) a depletion of GSH that decreases the
capacity of the cell to scavenge ROS produced by free Cd
2+
. The
resulting ROS increase stimulates both swelling-activated Cl

and
TASK2 K
+
channels, leading to AVD and apoptosis. Under our
experimental conditions Cd
2+
application was maintained for the
entire duration of the experiment. It could be therefore probable that
Cd
2+
triggered both mechanisms suggested above. However, it could
be hypothesized that each mechanism depends on the level of
intoxication. At a low level, the toxic effect of cadmium could be
clamped by the formation of GSH-Cd conjugates. These conjugates
Fig. 10. Efux of Cd
2+
in cftr
+/+
and cftr
/
cell lines. (A,B) Conuent PCT monolayers fromcftr
+/+
(A) and cftr
/
(B) mice were loaded for 10 minwith Cd
2+
(10 M), washed, and
placedinPBS mediuminthe absence (control, squares) or presence of CFTR
inh
-172(5M, circles) or BSO(0.5 mM, diamonds). The extracellular mediumwas successivelyreplacedafter
10, 20, 30, 40, and 60 min of incubation. Cd
2+
content of each sample was determined by using atomic absorption spectrometry. Values are means SEM of 6 to 12 monolayers. (C)
Rates of Cd
2+
efux calculated between 10 and 20 min from experimental values obtained in A and B. Values are means SEM of 6 to 12 monolayers and are expressed in pmol of
Cd
2+
mg prot
1
min
1
.
Fig. 11. Model describing the mechanisms controlling ROS/GSH-GSSG intracellular
contents activated during cadmium exposure.
1029 S. L'hoste et al. / Free Radical Biology & Medicine 46 (2009) 10171031
could be secreted from the cells through a CFTR-dependent mechan-
ism. At high levels, the cadmium could additionally induce CFTR-
mediated ROS production, AVD, and apoptosis. Further work will be
required to establish whether the cascade of events induced by Cd
2+
in cultured proximal tubule cells is relevant in the intoxicated animal.
Nevertheless these data give us important indications for analyzing
mechanisms of the onset of free cadmium Cd
2
toxicity and highlight
the crucial role of CFTR in the cell defense mechanisms against heavy
metals intoxication.
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