Download Role of the Sodium-Dependent Phosphate Cotransporters and

TOXICOLOGICAL SCIENCES 101(2), 254–262 (2008)
doi:10.1093/toxsci/kfm266
Advance Access publication November 12, 2007
Role of the Sodium-Dependent Phosphate Cotransporters and
Absorptive Endocytosis in the Uptake of Low Concentrations of Uranium
and Its Toxicity at Higher Concentrations in LLC-PK1 Cells
Dany S. Muller,*,1 Pascale Houpert,* Jean Cambar,† and Marie-Hélène Hengé-Napoli‡
*Institut de Radioprotection et de Sûreté Nucléaire, Laboratoire de Radiotoxicologie Expérimentale, BP-166, 26702 Pierrelatte Cedex, France; †GEPPR,
LSTE-EA 3672, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France; and ‡CEA/DEN/Valrhô-Dir-CVAR, BP 17171, 30207 Bagnols sur Cèze Cedex, France
Received July 31, 2007; accepted October 7, 2007
It has been suggested that uranium uptake and toxicity could be
mediated by endocytosis and/or the type IIa sodium-dependent
phosphate cotransporter (NaPi-IIa). The aim of this study was
therefore to characterize in vitro the role of these two cellular
mechanisms in the uptake and toxicity of low (200–3200nM) and
high (0.5 and 0.8mM) concentrations of uranium, respectively. At
low concentrations, uranium uptake in LLC-PK1 cells was
saturable (Vmax 5 3.09 ± 0.22 ng/mg protein) and characterized
by a K0.5 of 1022 ± 63nM and a Hill coefficient of 3.0 ± 0.4. The
potential involvement of endocytosis and NaPi-IIa in the uptake
of uranium was assessed by the use of various drugs and culture
conditions known to alter their relative activity, and 233uranium
uptake was monitored. Interestingly, the inhibitory effect of
colchicine, cytochalasin D, phorbol 12-myristate 13-acetate, and
chlorpromazine on endocytosis was highly correlated with their
effect on uranium uptake, a relationship that was not true when
the NaPi-IIa transport system was studied. Whereas the
competitive inhibition of the NaPi-IIa by phosphonoformic acid
(PFA) significantly decreased uranium uptake, this effect was not
reproduced when NaPi-IIa inhibition was mediated by the
replacement of extracellular Na1 with N-methyl-D-glucamine.
Uranium uptake was also not significantly altered when NaPi-IIa
expression was stimulated in MDCK cells. More surprisingly, we
observed by transmission electron microscopy that uranium
cytotoxicity was dependent upon the extent of its intracellular
precipitation, but not on its intracellular content, and was
suppressed by PFA. In conclusion, our results suggest that lowdose uranium uptake is mainly mediated by absorptive endocytosis, and we propose PFA as a potential uranium chelator.
Key Words: uranium uptake; endocytosis; phosphonoformic
acid; LLC-PK1; MDCK; uranium cytotoxicity.
The increasing use of uranium worldwide and the chronic
exposure of some populations to uranium in drinking water that
can reach several hundred to thousands of microgram per liter
1
To whom correspondence should be addressed at the b-Cell Development and
Function Group, School of Health and Biomedical Sciences, 2nd Floor, Hodgkin
Building, Guy’s Campus, King’s College London, London SE1 1UL, UK. Fax:
þ44 (0) 20-7848-6280. E-mail: [email protected].
(Kurttio et al., 2002; Moss et al., 1983) have led to questions
about its potential health effects.
Naturally occurring and depleted uranium both present
relatively low specific activities and are therefore chemotoxicants rather than radiotoxicants, although the potential
exists for a radiological toxicity. To date, the acute toxicity of
uranium has been extensively studied in in vivo and in vitro
experiments and is characterized by the development of acute
renal failure (ARF) due to tubular (Bulger, 1986; Leggett,
1989; Lopez et al., 2000; McDonald-Taylor et al., 1997; Sano
et al., 1998; Tyrakowski, 1979) followed by glomerular (Goel
et al., 1980; Kobayashi et al., 1984; L’Azou et al., 2002)
alterations. Uranium-induced tubular alterations are mainly
observed at the level of the second and third segment of the
proximal tubule and can develop with a uranium concentration
as low as 0.01 mg/kg of kidney. These alterations increase in
severity as either time or uranium concentration is increased
(Lopez et al., 2000) and are suggested to result in part from
transepithelial transport permeability defects (Goldman et al.,
2006; Leggett, 1989; Tyrakowski, 1979), inhibition of the
Naþ/Kþ ATPase activity, and mitochondrial injury (Brady
et al., 1989). At high concentrations, these alterations are
followed by necrosis (Bulger, 1986; McDonald-Taylor et al.,
1997) and/or apoptosis (Sano et al., 1998).
Although much less is known about the effects of long-term
exposure to low concentrations of uranium, its chronic
ingestion in drinking water has also been reported to affect
the renal function of the proximal tubule in humans (Mao et al.,
1995; Zamora et al., 1998), but these observations remain
controversial (Kurttio et al., 2006). In rodents, chronic
exposure to low concentrations of uranium in drinking water
revealed a dramatic alteration in expression of more than 200
genes expressed in the proximal tubule (Taulan et al., 2004).
Among these genes, those encoding for oxidative stress–related
proteins confirm previous observations showing that in vitro
DNA exposure to low concentrations of depleted uranium can
generate oxidative DNA damage and induce the formation of
hydroxyl radicals (Miller et al., 2002). Interestingly, the
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URANIUM UPTAKE AND TOXICITY
development of ARF in mice induced by 5 mg/kg uranyl nitrate
has also been associated with a dramatic alteration in expression
of the genes encoding the same oxidative stress–related proteins
(Taulan et al., 2006), which suggests that the chemical toxicity
of both low and high uranium concentrations might share, at
least in part, similar cellular and molecular mechanisms. In
accordance with this hypothesis, we recently demonstrated by
computer-assisted simulation that uranium speciation at low
concentration (1lM) is comparable with its speciation at high
concentration (1mM; Muller et al., 2006). For example,
similarly to what can be theoretically observed at low uranium
concentration (1lM), increasing the extracellular phosphate
concentration at high uranium concentration (1mM) is only
associated with an increase in the levels of the uranyl-phosphate
complexes. Taken all these observations into account, it is
therefore evident that identifying the cellular and molecular
mechanisms which are involved in the uptake of uranium at the
level of the proximal tubule epithelium has the potential for
reducing its accumulation and protecting from its toxic effects
in the kidney.
To date, there are several lines of evidence suggesting that
the proximal tubule uptake of uranium occurs by endocytosis
and/or by the type IIa sodium-dependent phosphate cotransporter (NaPi-IIa) system. The endocytosis hypothesis comes
from previous studies using electron microscopy of proximal
tubule cells from rats intoxicated with uranyl nitrate (Galle,
1974), as well as in vitro contaminated cell lines (Carrière et al.,
2006; Mirto et al., 1999a,b) and cultured kidney cells
(Ghadially et al., 1982), where the occurrence of urchin-like
electron-dense structures in lysosome-like bodies has been
reported. The hypothesis that uranium uptake is mediated by
the NaPi-IIa is based on the recent findings showing that
the toxicity of uranium is phosphate dependent, correlated to
the level of the uranyl-phosphate species UO2PO
4 and/or
UO2PO4(aq) present in the culture medium, and entirely dependent upon the expression and activity of the NaPi-IIa (Muller
et al., 2006). However, these lines of evidence are circumstantial, and the exact mechanisms by which uranium is taken up
by proximal tubule cells are still not clear and remain to be
characterized.
The aim of the current study was therefore to determine
in vitro the role of endocytosis and of the NaPi-IIa in the uptake
and toxicity of uranium using the LLC-PK1 and NaPi-IIa–
expressing MDCK cell lines. The role of absorptive endocytosis was assessed indirectly using fluorescein isothiocyanate
(FITC)–labeled albumin as a molecular marker. Uranium
uptake was performed using the high–specific activity isotope
233
uranium.
MATERIALS AND METHODS
Chemicals. Minimal essential medium (MEM), fetal bovine serum (FBS),
streptomycin, penicillin, phorbol 12-myristate 13-acetate (PMA),
L-glutamine,
255
cytochalasin D, colchicine, concanavalin A, bisindolylmaleimide (Bim),
phalloidin, chlorpromazine, sucrose, FITC-albumin, 3-(N-morpholino) propanesulfonic acid (MOPS), phosphonoformic acid (PFA), N-methyl-Dglucamine (MGA), and N-[2-hydroxyethyl] piperazine-N#-[2-ethanesulfonic
acid] (HEPES) were obtained from Sigma (L’Isle d’Abeau Chesnes, France).
The 233 isotope of uranium was obtained from Cerca (Pierrelatte, France;
specific activity 3.57 3 108 Bq/g). Uranyl nitrate (depleted uranium 1.6 3 104
Bq/g) was obtained from Merck Eurolab (Lyon, France). Ringer solution was
composed of (in mM) 122.5 NaCl, 5.4 KCl, 1.2 CaCl2, 0.8 MgCl2, 0.8
Na2HPO4, 0.2 NaH2PO4, 5.5 glucose, and 10 HEPES (titrated to pH 6.0 or 7.4).
Cell culture. LLC-PK1 cells were obtained from the European Collection
of Cell Cultures (Wiltshire, England), and NaPi-2–overexpressing MDCK cells
(MDCK/NaPi-2 transfectants) were generously provided by Professor H. Murer
and Dr J. Biber (Zurich, Switzerland). LLC-PK1 and MDCK cells were
maintained in MEM supplemented with 10% FBS, 10mM HEPES, 2mM
4
L-glutamine, 50 lg/l streptomycin, and 10 IU/l of penicillin at 37C in the
presence of 5% CO2. LLC-PK1 cells were used from the 1st to the 20th passage
after the cells were purchased, and MDCK/NaPi-2 transfectants were used
within 15 passages after their arrival. MDCK/NaPi-2 transfectants are MDCK
cells that had been transfected with cDNA coding for the proximal tubule NaPi
cotransporter NaPi-2 by the use of a dexamethasone-inducible vector (pLKneo; Quabius et al., 1996). Dexamethasone-induced NaPi-2 expression in
MDCK/NaPi-2 transfectants was performed by incubation with 1lM
dexamethasone for 16 h. Dexamethasone was added from a 10,000-fold stock
solution made in ethanol, and the same amount of vehicle was added to control
cells.
Inhibitors, activators, and culture conditions. The role of endocytosis in
the uptake of uranium by LLC-PK1 cells was assessed using culture conditions
and drugs that are well known to alter absorptive endocytosis. Maintaining the
cells in culture at 4C is known to be the most effective and noninvasive
method of inhibiting various transport systems by modulating the membrane’s
physical state (Mamdouh et al., 1996). The use of sucrose (1M) has been
described to inhibit receptor-mediated endocytosis by blocking the formation of
clathrin-coated pits (Heuser and Anderson, 1989). PMA (10nM) and
chlorpromazine (50lM) have been shown to be two potent inhibitors of
albumin uptake by the proximal tubule–derived OK cells (Gekle et al., 1997;
Hryciw et al., 2005). Destabilizing the actin and microtubular networks with
colchicine (10lM) and cytochalasin D (10lM) to depress endocytosis has been
extensively used and described in the literature. Their effects were compared
with the effect of phalloidin (25lM), a potent actin network–stabilizing agent.
Finally, concanavalin A (0.4lM) was also used in our study because this
compound was previously reported to stimulate albumin endocytosis in murine
epidermal Langerhans cells (Becker et al., 1995).
FITC-albumin uptake. Albumin uptake was measured using a previously
published method (Gekle et al., 1997). LLC-PK1 cells were preincubated in
serum-free MEM at 37C for 2 h prior to uptake studies. After three acidic
washes (pH 6.0), the cells were further preincubated in Ringer solution (pH 7.4)
for 30–60 min in the presence of vehicle, 10lM colchicine, 10lM cytochalasin
D, 25lM phalloidin, 0.4lM concanavalin A, 50lM chlorpromazine, 10nM
PMA, 1lM Bim, or 1mM sucrose. LLC-PK1 cells were exposed to 0–250 lg/ml
FITC-albumin either at 37C or 4C for 0–120 min in the continued presence of
the inhibitors or vehicle, then washed in Ringer solution (pH 6.0), and lysed in
MOPS buffer (20mM MOPS, pH 7.4, with 0.1% Triton X-100) at 37C for
45 min. Cell-associated fluorescence was measured using a spectrofluorimeter
(FLX 800; Bio-Tek, Saint-Quentin en Yvelines, France), adjusted for
background, and standardized to total cellular protein, and the corresponding
amount of albumin was calculated by comparison with a standard curve. The
amount of internalized substrate was determined by subtracting the fraction of
membrane-bound FITC-albumin (determined by the addition of 1000-fold
excess of unlabeled albumin) from total cell–associated FITC-albumin.
Uranium uptake. Uranium uptake by LLC-PK1 and MDCK cells was
assessed using a solution of 233uranium. Briefly, the incubation medium was
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MULLER ET AL.
removed and cells were equilibrated in 3 ml serum-free MEM (pH 7.4) at 37C
for 2 h prior to the uptake studies. The cells were then preincubated for 30–60 min
in the presence or absence of the same inhibitors as described above, followed by
a further incubation at 37C or 4C for 0–300 min in the presence of 0–3.2lM
233
uranium. Finally, the cells were washed six times with ice-cold Ringer
solution (pH 7.4) containing 5 mg/ml ethane-1-hydroxy-1,1-biphosphonate,
a powerful chelating agent (Henge-Napoli et al., 1999) used to remove
membrane-bound uranium, and then lysed in MOPS buffer (20mM MOPS,
pH 7.4, 0.1% Triton X-100) at 37C for 45 min. Cell-associated a emission was
assayed by liquid scintillation counting, and uranium uptake was adjusted
for background and standardized to total cellular protein. The amount of
233
uranium incorporated was calculated per microgram cellular protein.
Phosphate uptake. The Naþ-dependent and -independent phosphate
transports were determined in cells grown to confluency on plastic dishes
(35 mm) as described previously (Muller et al., 2006). Briefly, the cells were
washed twice with 2 ml Ringer solution and equilibrated in serum-free culture
medium (pH 7.4) at 37C for 2 h. The uptake solution consisted of 137mM
NaCl, 5.4mM KCl, 2.8mM CaCl2, 1.2mM MgSO4, 10mM HEPES-Tris
(pH 7.4), and 0.1mM KH232PO4 (37 kBq/ml). For Naþ-independent uptake,
NaCl was replaced with MGA. The uptake experiments were performed at
room temperature for 6 min and then stopped by washing the cells six times
with cold ‘‘stop’’ solution (137mM NaCl, 10mM Tris-HCl, pH 7.2). Finally,
the cells were lysed and the cell-associated 32P activity was determined by
liquid scintillation counting.
Uranyl-NaHCO3 stock solution and media preparation. Uranyl-NaHCO3
stock solutions were prepared as previously described (Muller et al., 2006).
Briefly, Uranyl-NaHCO3 stock solutions ([U] ¼ 10mM and [HCO
3 ] ¼ 100mM)
were obtained by dissolving uranyl nitrate crystals (UO2(NO3)2.6H2O) in an
aqueous NaHCO3 solution, and the pH was adjusted to 7.2. These stock
solutions were diluted in serum-free MEM to obtain the desired final
concentrations. In each case, the NaHCO3 concentration was adjusted to
3.34mM. To simplify the reading of the manuscript, the term uranium will be
used throughout the text.
Uranium-induced cytotoxicity: cell death determination. Uraniuminduced cell death was measured by quantifying the amount of lactate
dehydrogenase (LDH) released from damaged cells into the culture medium.
This was performed using an enzymatic cytotoxicity detection kit (Roche
Applied Science, Meylan Cédex, France) in which the consumption of nicotine
adenine dinucleotide in the presence of pyruvate was monitored by
spectrophotometry at 490 nm (Legrand et al., 1992). Total cellular LDH
content was determined following an incubation time of 30–60 min in MEM
containing 1% Triton X-100, and the percentage of cell death (percent of
cytotoxicity) was quantified as the ratio between the amount of LDH released in
the medium by treated cells and the total cellular LDH content.
Transmission electron microscopy. LLC-PK1 cells were cultured in
35-mm Petri dishes and pretreated for 30–60 min with 10mM PFA, 10lM
cytochalasin D, 0.4lM concanavalin A, or vehicle (dimethyl sulfoxide). The
cells were then cultured for an additional 16 h in the presence of 500lM
uranium, washed three times in Ringer solution (pH 7.4), fixed in 1.5%
glutaraldehyde, buffered with 0.1M cacodylate (pH 7.4) for 1 h, postfixed in
1% osmium buffered with 0.1M cacodylate (pH 7.4), and ethanol dehydrated.
Each sample was then embedded in Epon, and 60-nm sections were prepared
using the Reichert Ultracut S microtome. These sections were finally mounted
on copper grids, stained with uranyl acetate, and analyzed under a transmission
electron microscope (TEM) (Philips CM 120).
Statistics. Data are presented as mean ± SEM, and n represents the
number of experiments. Each uptake experiment was performed in triplicate or
quadruple, and each cell viability test was performed in duplicate with eight
observations. The significance of differences was tested by one-way analysis of
variance followed by the Tukey honestly significant difference test. Differences
were considered significant if p < 0.05. The correlation between 233uranium
uptake and FITC-albumin uptake, shown in Figure 3C, was analyzed with
a linear regression test.
RESULTS
FITC-Albumin Uptake in LLC-PK1 Cells
As shown in Figure 1A, FITC-albumin uptake increased
linearly in a time-dependent manner for the first 15–20 min,
then slowed down, and reached an apparent equilibrium after
90 min of incubation with 50 lg/ml FITC-albumin. Consequently, we decided to use an incubation time of 15 min to
further characterize FITC-albumin uptake in LLC-PK1 cells,
when the time course of the transport is still in the linear phase.
Figure 1B shows the concentration dependence of FITCalbumin uptake in this cell line. The uptake was saturable, and
the determination of the Hill coefficient was consistent with the
existence of a single mechanism of transport (Fig. 1C; Hill
coefficient: 0.9 ± 0.2). Accordingly, the characterization of the
Michaelis and Menten constant (Km) and of the maximal
velocity (Vmax), which was performed using the Eadie-Hofstee
plot (Fig. 1D), confirmed that albumin uptake in LLC-PK1 cells
is functionally mediated by one transport system. The Km and
Vmax values, averaged from three independent experiments,
were 23 ± 7 lg/ml FITC-albumin and 0.79 ± 0.09 lg/mg
protein, respectively.
233
Uranium Uptake in LLC-PK1 Cells
Using a concentration of 5lM 233uranium, we observed that
uranium uptake at 37C increased linearly in a time-dependent
manner for the first 30–60 min and then slowed down but
without reaching equilibrium even after 5 h (Fig. 2A). As we
also observed that uranium uptake was significantly reduced
when these experiments were performed at 4C, we can
conclude that the mechanism through which uranium is taken
up by LLC-PK1 cells is not a diffusion process.
The concentration dependence of uranium uptake is shown
in Figure 2B, where a sigmoidal curve can be observed. First,
uranium uptake increased slowly for concentrations ranging
from 0 to 600nM. Then, it increased in a more dramatic manner
from 600nM to 2lM (uranium content at 600nM and 2lM:
0.41 ± 0.27 and 2.64 ± 0.26 ng/mg protein, respectively). An
apparent saturation was obtained for 233uranium concentrations
ranging from 2 to 3.2lM, and the determination of the Hill
coefficient (3.0 ± 0.4; Fig. 2C) was consistent either with the
presence of an allosteric mechanism or the existence of several
systems of transport for uranium. Overall, the maximal velocity
(Vmax) and apparent affinity (K0.5) were estimated as
3.09 ± 0.22 ng/mg protein and 1022 ± 63nM, respectively.
The complexity of the process by which uranium is taken up by
LLC-PK1 cells is further demonstrated by the Eadie-Hofstee
representation (Fig. 2D) showing an atypical plot. The
curvature observed for uptake values ranging from 0.4 to
URANIUM UPTAKE AND TOXICITY
257
FIG. 1. Characterization of the kinetic parameters of albumin uptake in LLC-PK1 cells. (A) Time course of albumin uptake (50 lg/ml FITC-albumin).
(B) Concentration dependence of albumin uptake measured after 15 min. (C) Hill plot representation obtained with the data from (B). The averaged Hill coefficient
obtained from three separate studies was 0.9 ± 0.2. (D) Eadie-Hofstee plot showing the existence of a single system of transport for albumin. The averaged Km
and Vmax values from three different experiments were 23 ± 7 lg/ml and 0.79 ± 0.09 lg/mg protein, respectively. Points shown in (A) and (B) are mean ±
SEM, n ¼ 3.
2.8 ng/mg
systems.
233
uranium is characteristic of cooperative transport
Inhibition of Absorptive Endocytosis Reduces
Uptake in LLC-PK1 Cells
233
Uranium
To investigate the potential relationship that could exist
between absorptive endocytosis and uranium uptake, we
compared the effects of several drugs and conditions (known
to interfere with absorptive endocytosis) on both FITC-albumin
and 233uranium uptake in LLC-PK1 cells (Figs. 3A and 3B).
On the one hand, carrying out the uptake studies at 4C
resulted in a dramatic reduction of both FITC-albumin (79.2%)
and 233uranium uptakes (87.1%). In a similar manner, when the
cells were cultured in the presence of a hypertonic medium
(1M sucrose), FITC-albumin and 233uranium uptakes were
decreased by 76.5 and 88.4%, respectively. Destabilization of
the microtubular and actin networks using 10lM colchicine or
10lM cytochalasin D also significantly reduced both transport
systems, with FITC-albumin and 233uranium uptakes being,
respectively, decreased by 49.8 and 53.3% (colchicine) and
61.6 and 79.7% (cytochalasin D). When LLC-PK1 cells were
pretreated with 50lM chlorpromazine or a protein kinase C
(PKC) activator (10nM PMA), both FITC-albumin and 233uranium uptakes were also reduced by 47.6 and 53.4% and 64.8 and
65.9%, respectively. The involvement of PKC in the regulation
of uranium uptake by LLC-PK1 cells was further demonstrated
by the results showing that 1lM Bim, a PKC inhibitor, completely suppressed the effect of 10nM PMA. On the other hand,
neither stabilization of the actin cytoskeleton with 25lM
phalloidin nor pretreatment of the cells with 0.4lM concanavalin A significantly altered the uptakes of FITC-albumin and
233
uranium.
Interestingly, plotting the effects of the drugs and culture
conditions on FITC-albumin endocytosis against their effects
on 233uranium uptake showed the existence of a significant
linear correlation (Fig. 3C, p < 0.01).
233
Uranium Uptake Is Not Mediated by the NaPi-IIa, but It Is
Significantly Reduced by PFA
We recently demonstrated that uranium-induced toxicity was
not only phosphate dependent but also dependent upon the
expression and activity of the NaPi-IIa (Muller et al., 2006).
Accordingly, we proposed that uranium uptake might also be
mediated, at least in part, by this system of transport.
To test this hypothesis, we monitored the uptake of 3lM
233
uranium in the absence or presence of 0.1–10mM PFA,
a specific NaPi inhibitor, or when the activity of the NaPi
cotransporters was suppressed by replacing the extracellular
sodium with MGA. Surprisingly, whereas uranium uptake was
reduced in a concentration-dependent manner when LLC-PK1
cells were treated with PFA (233uranium uptake in ng/mg
protein/h: 3.05 ± 0.24, 2.54 ± 0.55, 2.10 ± 0.45, and
258
MULLER ET AL.
FIG. 2. Characterization of the kinetic parameters of uranium uptake in LLC-PK1 cells. (A) Time course of uranium uptake. The uptake of 5lM 233uranium
was monitored either at 4C (open circle) or 37C (filled circle). (B) Concentration dependence of uranium uptake measured after 60 min. (C) Hill plot
representation by extrapolating the data obtained in (B). The averaged Hill coefficient determined from three separate studies was 3.0 ± 0.4. (D) Eadie-Hofstee
plot. The K0.5 and Vmax values that were averaged from three separate experiments were 1022 ± 63nM and 3.09 ± 0.22 ng/mg protein, respectively. Points shown
in (A) and (B) are mean ± SEM, n ¼ 3.
0.75 ± 0.08 in the presence of 0, 0.1, 1, and 10mM PFA,
respectively), the suppression of the NaPi-driving force by the
use of MGA instead of sodium did not have any significant
effect (Fig. 4A).
Given this unexpected observation, we decided to better
characterize the role of NaPi-IIa in uranium uptake. This was
performed by making use of the distal tubule MDCK cell line
which does not normally express the NaPi-IIa but had been
stably transfected with cDNA encoding the rat NaPi-IIa (NaPi-2)
under the control of a dexamethasone-inducible promoter
(Quabius et al., 1996). The data that are displayed in Table 1
show that stimulation of the rat NaPi-IIa by 1mM dexamethasone significantly increased the uptake of phosphate when
compared to nonstimulated cells. Using this cell line, we
previously demonstrated the existence of a strong correlation
between the expression level and activity of the NaPi-IIa and
uranium-induced cytotoxicity (Muller et al., 2006). However,
we now report that stimulation of NaPi-2 expression and transport
activity plays, at most, a small role in uranium uptake. Indeed,
233
uranium transport was not significantly increased when NaPi-2
expression was stimulated in MDCK cells (Fig. 4B).
PFA Protects LLC-PK1 Cells from Uranium-Induced
Cytotoxicity
Since our data showed that PFA significantly decreases the
uptake of low concentrations of uranium (Fig. 4), we decided
to further assess the capacity of this compound in protecting
LLC-PK1 cells from uranium-induced toxicity.
The effect of PFA on the cytotoxicity of uranium was
studied using 800lM uranium (~LC50, Muller et al., 2006)
whose toxicity was assessed after 24 h by quantifying the level
of LDH released from damaged cells as described in the
‘‘Materials and Methods’’ section. As shown in Figure 5,
specific inhibition of the NaPi cotransport systems with 10mM
PFA completely suppressed the cytotoxic effect induced by
uranium, confirming in that experimental condition a direct
correlation between uranium uptake and toxicity. However, the
existence of such a correlation was not always true and, for
example, the cytotoxicity of uranium was increased to almost
its maximal when LLC-PK1 cells were treated with 10lM
cytochalasin D, a compound we defined as an inhibitor of
uranium uptake (Fig. 3B). In a similar manner, while our
results showed that 0.4lM concanavalin A has no significant
effect on the transport of uranium (Fig. 3B), our cytotoxicity
data clearly demonstrate that this drug significantly potentiates
its cytotoxicity (Fig. 5).
PFA Inhibits the Formation of Uranyl Phosphate Precipitates
in LLC-PK1 Cells
It is well established that after internal contamination with
uranium compounds, uranium accumulates in proximal tubule
cells into precipitates of uranyl phosphate (Mirto et al., 1999b)
259
URANIUM UPTAKE AND TOXICITY
FIG. 3. Correlation between uranium uptake and endocytosis in LLC-PK1
cells. Effect of various drugs and culture conditions on the uptakes of (A) 50
lg/ml FITC-albumin (15 min) and (B) 3lM 233uranium (60 min).
(C) Correlation between uranium uptake and endocytosis obtained by plotting
all the results obtained in (B) versus those obtained in (A). The use of a linear
regression test showed a p value < 0.01. Bars shown in (A) and (B) are
mean ± SEM, n ¼ 4–7, *p < 0.05 versus control.
that are also known as urchin-like structures. Accordingly, to
better understand why PFA but not cytochalasin D nor
concanavalin A reduced both uranium uptake and toxicity,
we monitored by TEM the effect these three compounds have
on the intracellular precipitation of a subtoxic concentration of
uranium (500lM).
As expected, TEM analysis of sections obtained from
uranium-treated LLC-PK1 cells confirmed our cytotoxicity
data; whereas the addition of 10mM PFA in the culture
FIG. 4. Effect of MGA, PFA, and of the sodium-dependent phosphate
cotransporter NaPi-2 on uranium uptake. (A) Effect of inhibition of the sodiumdependent phosphate cotransporters on 233uranium (3lM) uptake in LLC-PK1
cells. Inhibition was obtained by replacement of Naþ with MGA or by the
addition of 0.1–10mM PFA. (B) Role of NaPi-IIa in the uptake of uranium
using MDCK cells that were stably transfected with the rat NaPi-IIa (NaPi-2)–
expressing vector pLK-neo containing a dexamethasone-inducible element in
its promoter region. Transfected MDCK cells were pretreated for 16 h with
1lM dexamethasone to induce NaPi-2 expression (open circle) or with the
corresponding vehicle (control cells, filled circle), and uranium uptake was
determined using 3lM 233uranium. Each bar and point shown are mean ±
SEM, n ¼ 3, *p < 0.05 versus control.
medium completely abolished the intracellular precipitation of
uranium, LLC-PK1 treatment with 10lM cytochalasin D or
0.4lM concanavalin A resulted in a significant increase in the
number these urchin-like structures (Fig. 6).
DISCUSSION
So far, the renal toxic properties of uranium in experimental
animals have been extensively studied and provide a detailed
description of its pathophysiology where injury, including
necrosis and/or apoptosis of the terminal part of the renal
proximal tubule, is characteristic in all mammalian species
260
MULLER ET AL.
TABLE 1
Sodium-Dependent Transport of Phosphate in the NaPi-2 Stably
Transfected MDCK Cell Line
32
P uptake in MDCK cells (pmol/mg protein)
Naþ
MGA
NaPi-2 ()
NaPi-2 (þ)
539.0 ± 82.4
280.6 ± 28.0
2236.9 ± 182.6***
326.4 ± 10.9
Note. NaPi-2 expression was stimulated by 1lM dexamethasone (NaPi-2
(þ)). The sodium-dependent and -independent transport of phosphate was
assessed by monitoring the uptake of 32P in the presence of 145mM NaCl or
MGA, respectively. Each point represents the mean ± SEM, n ¼ 3,
***p < 0.01 versus NaPi-2 ().
(Bulger, 1986; Haley, 1982; McDonald-Taylor et al., 1997;
Sano et al., 1998).
The chemical nephrotoxicity of uranium is pH dependent
(Goldman et al., 2006) and characterized by the development
of transepithelial transport and permeability defects (Leggett,
1989; Tyrakowski, 1979), inhibition of the Naþ/Kþ ATPase
activity and mitochondrial injury (Brady et al., 1989), gene
expression alteration (Taulan et al., 2004, 2006), and possibly
oxidative DNA damage (Miller et al., 2002). Uranium-induced
toxicity is also dependent upon its speciation (Carrière et al.,
2004; Mirto et al., 1999a,b; Muller et al., 2006), and we
recently demonstrated the existence of a strong correlation
between its toxicity, the level of phosphate, and the expression
and activity of the NaPi-IIas (Muller et al., 2006). Interestingly,
while the cellular and molecular mechanisms by which
uranium mediates its toxic effects are currently studied, very
little is known about the cellular mechanisms that are
FIG. 5. Cytotoxicity of uranium in LLC-PK1 cells. Determination of the
percentage of cytotoxicity induced by 800lM uranium in the absence or
presence of 10mM PFA, 10lM cytochalasin D, or 0.4lM concanavalin A. Bars
shown are mean ± SEM, n ¼ 5, *p < 0.05 versus control.
responsible for its uptake. This is surprising since uranium
uptake by proximal tubule cells represents the first step in its
cytotoxicity, and knowing the cellular mechanisms involved in
uranium transport at this level could initiate new therapeutical
approaches aimed at decreasing its uptake with specific
inhibitors thereby reducing its toxic effects.
Although we cannot rule out the possibility that the observed
non-Michaelis-Menten type of kinetic in our uranium uptake
study could be due to a direct effect of uranium on its own
speciation, the corresponding Hill coefficient of 3.0 ± 0.4 that
was obtained for uranium concentrations ranging from 0 to
3200lM is consistent with the existence of more than one
binding site for uranium. One belief that comes from TEM
analysis of kidney cells from contaminated animals or cell lines
(Galle, 1974; Ghadially et al., 1982; Mirto et al., 1999a,b), where
uranium has been shown to precipitate within the cytoplasm, is
that uranium is taken up by endocytosis. Consistent with this
hypothesis, uranium is known to bind to the plasma membrane,
a chemical property used to contrast tissue sections for TEM
analysis, and can therefore be internalized in a nonspecific
manner by endocytosis. Here, we indirectly verified this
hypothesis by demonstrating for the first time the existence of
a strong correlation between absorptive-mediated endocytosis
and uranium uptake ( p < 0.01). Thus, like endocytosis-mediated
albumin internalization, uranium uptake was PKC dependent,
dependent on cytoskeletal integrity, and significantly reduced by
the use of drugs that are known to alter endocytosis.
However, while one might expect that if uranium uptake is
inhibited then its toxic effects would be reduced, here we
observed that uranium cytotoxicity was increased when endocytosis, and therefore uranium internalization, was inhibited
by 10lM cytochalasin D (Figs. 3 and 5). A similar dissociation
was observed with 0.4lM concanavalin A which increased
uranium toxicity without having any significant effect on its
uptake. One possible explanation for such unexpected results is
that uranium is taken up by at least one other transport system
whose expression and/or activity would be downregulated
by endocytosis and that this mechanism of transport would
be responsible for the uptake of a smaller but cytosolic fraction
of uranium. This cytosolic fraction of uranium would then
alter the cell metabolism (Brady et al., 1989; Bulger, 1986;
Goldman et al., 2006; Leggett, 1989; McDonald-Taylor et al.,
1997; Tyrakowski, 1979) and generate an oxidative stress
(Miller et al., 2002; Taulan et al., 2004, 2006). This would then
explain why inhibition of endocytosis produced a reduction in
uranium internalization but also an increase in its cytotoxic
effect. Consistent with this hypothesis, endocytosis plays
several central functions in cellular homeostasis and one of
them is the regulation of plasma membrane expression and
activity of channels, transporters, and exchangers (Chow et al.,
1999; Collazo et al., 2000; Hernando et al., 2001; Shimkets
et al., 1997).
Several lines of evidence suggest that the NaPi-IIa could be
the second transport system that would either mediate the
261
URANIUM UPTAKE AND TOXICITY
FIG. 6. Section analysis by TEM of LLC-PK1 cells treated with 500lM uranium. TEM analysis of sections obtained from LLC-PK1 cells treated with
a subtoxic concentration of uranium (500lM) in the absence or presence of 10mM PFA, 10lM cytochalasin D, or 0.4lM concanavalin A.
uptake of a smaller but cytotoxic fraction of uranium or
potentiate its cytotoxicity. Indeed, (1) NaPi-IIas activity is
downregulated by endocytosis (Murer and Biber, 1996); (2) all
the drugs used in this study which inhibited endocytosis and
reduced uranium uptake were previously shown to stimulate
the sodium-dependent transport of phosphate and increase the
toxic effect of uranium (Muller et al., 2006); (3) concanavalin
A, which did not alter uranium uptake and endocytosis, was
found to increase NaPi-mediated phosphate uptake and
uranium cytotoxicity (Muller et al., 2006); (4) while the
replacement of Naþ with MGA to suppress the transport
activity of the sodium-dependent system of transporters did not
altered uranium uptake, it was previously shown to significantly decrease its maximal toxicity by more than 70% (Muller
et al., 2006); and (5) finally, while uranium-induced toxicity
was very low in MDCK cells, overexpression of NaPi-2
resulted in a strong sensitization of this cell line to uranium
toxic effects (Muller et al., 2006) but did not significantly
increase its uptake.
Another important finding of this study is the observation
that PFA, a well-characterized competitive inhibitor of the
sodium-dependent phosphate cotransporters, is a potent inhibitor of uranium uptake, uranium intracellular precipitation,
and uranium toxicity.
In conclusion, our results suggest that absorptive endocytosis is the major mechanism of uranium internalization. We also
propose that the NaPi-IIas mediate directly or indirectly the
cytotoxic effect of uranium, a cellular event that can be
suppressed by the use of PFA. It would now be interesting to
test the therapeutical impact that this compound could have in
in vivo studies.
FUNDING
COGEMA; Institut de Radioprotection et sûreté Nucléaire.
ACKNOWLEDGMENTS
We would like to thank Professors H. Metivier, M. Fournier,
and P. Brochard for their helpful discussion. We also thank
Dr S. Persaud for critically evaluating and correcting the
manuscript.
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