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INTRODUCTION |
Intrahepatic bile duct epithelial cells (i.e.
cholangiocytes) play an essential role in bile formation, and by
integrated absorptive and secretory processes, they contribute up to
40% of daily bile production in humans (1, 2). Because bile is a
complex fluid composed of >98% water, cholangiocytes like other
water-transporting epithelial cells are required to rapidly transport
large amounts of water in response to osmotic gradients generated by
transported ions and solutes, a situation in which specific water
channel proteins (i.e. aquaporins
(AQPs))1 are probably involved
(3-6). Indeed, our initial observation (7) that isolated rat
cholangiocytes are capable of rapid mercury-sensitive, temperature-independent transmembrane water transport in response to
osmotic gradients was consistent with transport via water channels rather than by diffusion through the lipid bilayer. Our more recent molecular studies have demonstrated that rat cholangiocytes express six
AQPs (i.e. AQP 0, 1, 4, 5, 8, and 9) from the known 11 AQPs in mammals (5, 7-11). Moreover, at least two of them (i.e. AQP1 and AQP4) contribute to the water permeability of both the apical
and basolateral cholangiocyte membrane domains, AQP1 facilitating mainly the apical transport of water and AQP4 modulating its
basolateral movement (7-9, 11). Nevertheless, direct studies of the
contribution of AQPs to water transport in intrahepatic bile ducts and
other tissues have been severely hampered by the lack of specific AQPs inhibitors.
Recently, several groups have described post-transcriptional gene
silencing or RNA interference in a wide variety of organisms using double-stranded RNAs of ~200-1000 nucleotides in length that specifically suppress the expression of a target mRNA
(reviewed in Refs. 12-18). According to the prevailing model,
double-stranded RNA is processed into small interfering double-stranded
RNAs (siRNAs) of 19-25 nucleotides in length, which act as guides for
the RNA-induced silencing enzymatic complex required for the cleavage
of the target mRNAs (15-18). Although the physiological
significance of post-transcriptional gene silencing and RNA
interference is still under study, powerful new technology for
selective inhibition of specific gene expression employing siRNAs is
rapidly evolving (19-26).
In this study, we present data demonstrating that AQP1 gene expression
in cholangiocytes is specifically suppressed by AQP1-siRNAs, resulting
in a significant decrease of water transport by this cell type. These
data show the feasibility of utilizing siRNAs to specifically reduce
the expression of AQPs in epithelial cells and provide direct evidence
of the contribution of AQP1 to water transport in biliary epithelia.
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EXPERIMENTAL PROCEDURES |
Materials--
All of the chemicals were of highest purity
commercially available and were purchased from Sigma unless otherwise indicated.
Animals--
Male Fisher 344 rats (225-250 g) were obtained
from Harlan Sprague-Dawley (Indianapolis, IN), housed in
temperature-controlled room (22 °C) with 12-h light-dark cycles, and
maintained on a standard diet with free access to water. All of the
experimental procedures were approved by the Animal Use and Care
Committee of the Mayo Foundation.
Solutions--
The composition of isotonic (290 mosM) Ringer-HCO3 buffer was (in
mM): 120.0 NaCl, 5.9 KCl, 1.2 Na2HPO4, 1.0 MgSO4, 25.0 NaHCO3, 1.25 CaCl2, and 5.0 C6H12O6, pH 7.4. Hypotonic (90 mosM) solution was prepared by decreasing the concentration
of NaCl. The precise osmolality of Ringer-HCO3 buffer
solutions was determined with a freezing point osmometer (The Advanced
Micro-Osmometer, Model 3300, Advanced Instruments, Inc., Norwood, MA).
For water transport experiments with forskolin, the perfusate contained
140 mM NaCl and 5 mM
Na2HPO4, pH 7.2. The composition of lysis
buffer was 50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.1% sodium Nonidet P-40, 0.1% sodium deoxycholate, and 0.1% sodium
dodecyl sulfate. The composition of phosphate-buffered saline (PBS) was
(in mM): 137 NaCl, 2.7 KCl, 10 NaHPO4, and 1.8 KH2PO4.
AQP1-siRNAs Design, Synthesis, and Labeling--
Sequence
information regarding mature rat AQP1 mRNA was extracted from the
NCBI Entrez nucleotide data base. Two target sites within
AQP1 gene were chosen from the rat AQP1 mRNA sequence
(GenBankTM accession NM_012778). Following
selection, each target site was searched with NCBI BlastN to confirm
specificity only to AQP1. Two different siRNAs designated AQP1-siRNA(a)
and AQP1-siRNA(b), which target nucleotides 71-91 and 673-693 of the
rat AQP1 mRNA sequence, respectively, and two nonspecific siRNA
duplexes containing the same nucleotides but in irregular sequence
(i.e. scrambled AQP1-siRNA(a) and AQP1-siRNA(b)) were
prepared by a transcription-based method using the Silencer siRNA
construction kit (Ambion, Austin, TX) according to manufacturer's
instructions. The 29-mer sense and antisense DNA oligonucleotide
templates (21 nucleotides specific to AQP1 and 8 nucleotides specific
to T7 promoter primer sequence 5'-CCTGTCTC-3') were synthesized by the
Mayo Molecular Core facility. One of the constructed siRNAs,
AQP1-siRNA(b), was labeled with Cy3 following manufacturer's
instructions (Ambion). The efficacy of AQP1-siRNA labeling with Cy3 was
estimated by acrylamide gel analysis of the Silencer siRNA-labeling
positive control experiment (Ambion) and found to be ~20%.
IBDUs Isolation and Transfection with siRNAs--
IBDUs, which
are portions of intrahepatic bile ducts ranging in luminal diameter
from 100 to 125 µm and in length from 0.6 to 1.2 mm, were isolated
from normal rat liver as we described previously (27). IBDUs were
cultured from 0 to 24 h in normal rat cholangiocyte medium
containing 10 nM AQP1-siRNAs or corresponding scrambled
AQP1-siRNAs. Exogenous delivery of siRNAs to cholangiocytes was carried
out with or without a lipid carrier (i.e.
TransMesengerTM transfection reagent (Qiagen, Valencia, CA)).
Fluorescence Analysis of the siRNAs Uptake by IBDUs--
IBDUs
were incubated in normal rat cholangiocyte medium with 0, 0.5, 1, 5, 10, and 20 nM Cy3-AQP1-siRNA(b) with or without a lipid
carrier for 24 h at 37 °C. AQP1-siRNA(b)-Cy3 then was visualized in IBDUs by fluorescent microscopy, and Cy3 fluorescence was
measured by a method proposed for analysis of fluorescent antisense
oligonucleotides (28). After the incubation, IBDUs were washed three
times with PBS and then lysed in 200 µl of a lysis buffer. Total
cellular Cy3-AQP1-siRNA(b) fluorescent emission (
ex = 552;
em = 568 nm) was measured with a PerkinElmer LS 55 luminescence spectrophotometer. An aliquot of the cell lysate was taken
to measure the amount of total protein using the fluorescence assay,
and the Cy3 fluorescence was normalized to 100 µg of total protein.
For visualization of AQP1 suppression in IBDUs by AQP1-siRNA, IBDUs
were incubated with 10 nM Cy3-AQP1-siRNA(b) for 0, 12, and 24 h at 37 °C on poly-L-lysine-treated chamber
slides. Following the incubation, the IBDUs were fixed with cold 100%
methanol for 5 min and air-dried. The slides were then washed three
times with 1× PBS and permeabilized in 0.2% Triton-PBS for 2 min at
room temperature. IBDUs were blocked for 20 min in blocking buffer (10% normal sheep serum, 0.05% Tween 20 in PBS) at room temperature and incubated with affinity-purified AQP1 antibody (Alpha Diagnostics, San Antonio, TX) at a 1:50 dilution in blocking buffer overnight at
4 °C. Following the primary antibody incubation, the IBDUs were
washed with 1× PBS three times and incubated at a 1:100 anti-goat IgG
fluorescein isothiocyanate conjugate (Sigma) secondary antibody for
1 h at room temperature. AQP1-siRNA(b)-Cy3 and AQP1 fluorescence in IBDUs was then determined by using scanning laser confocal microscopy keeping the pinhole and detector gain setting identical while analyzing the different IBDUs and quantified using LSM 510 Image
Examiner software (Carl Zeiss, Thornwood, NY).
RNA Isolation and Analysis by Real-time Reverse
Transcriptase-PCR--
IBDUs were lysed in 1 ml of Tri-Reagent with 5 µl of Glyco-Blue (Ambion) added as a co-precipitant and stored at
room temperature for 5 min. After the addition of 0.1 ml of
1-bromo-3-chloro-propane, the samples were vigorously shaken, incubated
for 15 min at room temperature, and centrifuged at 12,000 × g for 15 min at 4 °C. The aqueous phase was transferred
to a new tube, and 0.5 ml of isopropyl alcohol was added. The samples
were mixed, stored for 10 min, and centrifuged at 12,000 × g for 15 min at 4 °C. After removing the supernatant, the
RNA pellet was washed with 75% EtOH and repelleted by centrifugation
at 12,000 × g for 15 min at 4 °C. RNA was
resuspended in RNA Secure solution (Ambion), and the concentration and
purity were determined by spectroscopy. Quantitation of AQP1 message
was accomplished by real-time PCR. A standard curve to AQP1 was
generated by amplifying AQP1 from freshly isolated rat cholangiocyte
cDNA using rat AQP1-specific primers (sense, 5'-AGTTGAGCACCAGGCATCC-3', and antisense,
5'-CACTGATGTGACCCACACTTTG-3'). The 259-bp amplicon was electrophoresed
on a 1.5% agarose gel, visualized with ethidium bromide, and
gel-extracted. The amplicon was subsequently diluted and used as
template for the PCR reaction. One-step reverse transcriptase-PCR for
AQP1 and 18 S ribosome (Ambion) was performed using the LightCycler RNA
Master SYBR Green I kit according to manufacturer's instructions.
Protein Analysis by Western Blotting--
Total lysates were
obtained by lysing the IBDUs with M-PERTM mammalian protein
extraction reagent (Pierce). The lysates were heated to 60 °C for 10 min in sample buffer containing 0.8 M dithiothreitol and
10% SDS for protein denaturation and solubilization. The samples were
then subjected to electrophoresis through a 12% SDS-polyacrylamide gel
and transferred overnight to a nitrocellulose membrane. The blots were
blocked with 5% (w/v) nonfat dry milk and 0.2% (v/v) Tween 20. After
blocking, the blots were incubated with affinity-purified rabbit
anti-rat antibodies to AQP1 (Alpha Diagnostics) at a dilution of 1:1000
overnight at 4 °C. The blots were washed and incubated for 1 h
with a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (1:2000 dilution) at room temperature. Protein bands
were detected using an enhanced chemiluminescence detection system (ECL
Plus, Amersham Biosciences). After exposing the nitrocellulose membranes to Kodak X-Omat AR film, the autoradiographs were scanned and
quantified by densitometry using Molecular Analyst software (Bio-Rad).
Measurement of Water Movement across Intrahepatic Biliary
Epithelia--
IBDUs were perfused with 1 mM of the
impermeable volume marker, fluorescein sulfonate
(fluorescein-5(6)-sulfonic acid trisodium salt (Molecular Probes,
Eugene, OR)) at a rate of 20-80 nl/min as described previously in
detail (3). Net water movement (Jv) and osmotic
water permeability coefficient (Pf) in response to
established osmotic gradient (200 mosM) or stimulated by
forskolin were measured as described previously (3).
Statistical Analysis--
All of the values are expressed as the
mean ± S.E. Statistical analysis was performed by the Student's
t test, and results were considered statistically different
at p < 0.05.
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RESULTS |
Design of AQP1-siRNAs--
We selected two target regions of
rat AQP1 mRNA (i.e. 71-91 and 673-693
sequences) by scanning the length of the AQP1 gene for
AA-dinucleotide sequences and downstream 19 nucleotides without significant homology to other genes by using an appropriate genome data
base. The antisense strands of synthesized AQP1-siRNAs are the reverse
complement of the target sequences (Fig. 1).
The sense strands of the AQP1-siRNAs have the same sequences as the
target mRNA sequences with the exception that they lack the 5'-AA
sequence (Fig. 1). A uridine dimer was incorporated at the 3' end of
the sense strands siRNAs (Fig. 1). Thus, the end products are two double-stranded 21-mer siRNAs (i.e. AQP1-siRNA(a) and
AQP1-siRNA(b)) that theoretically should reduce the expression of AQP1
mRNA and protein and two siRNAs (i.e. scrambled
AQP1-siRNA(a) and AQP1-siRNA(b)) that theoretically should not be
effective in AQP1 gene silencing.

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Fig. 1.
Sequences and expected duplexes for siRNAs
(A) and sites of targeting (B) within
AQP1 mRNA. A, the sense (top) and
antisense (bottom) strands of siRNAs targeting AQP1 message
and the scrambled AQP1-siRNAs are shown. B, the partial
mRNA sequence of rat AQP1 (GenBankTM accession number
NM_012778). Two potential sites of targeting by siRNAs are
underlined. The start codon is in a
boldface.
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Uptake of AQP1-siRNA by IBDUs--
IBDUs incubated for 24 h
in normal rat cholangiocyte culture medium containing various amounts
(i.e. 0-20 nM) of Cy3-labeled AQP1-siRNA(b) in
the absence or presence of the lipid carrier took up AQP1-siRNA in a
dose-dependent manner (Fig. 2).
However, no lipid carrier-dependent uptake of siRNA by
IBDUs was observed. Given that only 20% AQP1-siRNA(b) transfected into
cholangiocytes was labeled with Cy3 (see "Materials and Methods"
for details), we conclude that the amount of AQP1-siRNA taken up by
cholangiocytes was 5 times greater, suggesting that IBDUs could be
effectively transfected with siRNAs in the absence of the lipid
carrier. Based on this observation and given that a transfection
reagent could potentially affect cholangiocytes function, studies of
AQP1 gene suppression in IBDUs were performed by using naked
AQP1-siRNAs.

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Fig. 2.
Uptake of AQP1-siRNAs by IBDUs.
A, visualizing of AQP1-siRNA in IBDUs. IBDUs were incubated
24 h in normal rat cholangiocyte medium containing naked
Cy3-labeled AQP1-siRNA(b) from 0 to 20 nM. IBDUs analyzed
by fluorescent microscopy showed a significant increase in
Cy3-AQP1-siRNA(b) fluorescence (red). B, IBDUs
take up AQP1-siRNAs in a dose-dependent manner. 24 h
after incubation with Cy3 labeled AQP1-siRNA(b) in the absence
(white bars) or presence (black bars) of the
lipid carrier, the fluorescence intensity of IBDUs was measured and
normalized to 100 µg of protein. The uptake of Cy3-AQP1-siRNA(b)
increased with increasing concentrations of siRNA(b) independent of the
presence of the lipid carrier. Values are the mean ± S.E. of
three separate experiments with 4-6 IBDUs in each group.
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AQP1 Gene Suppression in IBDUs by siRNAs--
The levels of AQP1
mRNA and protein in IBDUs transfected with 10 nM each
of four different naked siRNAs (i.e. two siRNAs to different
regions of the AQP1 and two corresponding scrambled AQP1-siRNAs) are
shown in Fig. 3. Both siRNAs to different
sequences within the AQP1 gene effectively inhibited AQP1 mRNA
(Fig. 3A) and protein expression (Fig. 3B). AQP1
mRNA and protein levels were inhibited by AQP1-siRNA(a) by 76.6 and
57.9%, respectively. AQP1-siRNA(b) was more effective, suppressing
AQP1 mRNA and protein levels by 92.0 and 79.4%, respectively. In
contrast, two scrambled AQP1-siRNAs had no effect on AQP1 gene
expression. Moreover, AQP1-siRNAs had no effect on mRNA expression
for AQP4 (data not shown), an AQP, which is also expressed in rat
cholangiocytes (11), providing evidence that both AQP1-siRNA(a) and
AQP1-siRNA(b) were specific for AQP1. Because there is a strong
correlation between AQP1 mRNA and protein suppression by siRNAs
(Fig. 3C), these data suggest that AQP1 silencing in
cholangiocytes results from a reduction in the amount of AQP1 mRNA
available for translation. These data also suggest that AQP1-siRNAs
were highly specific and efficient in AQP1 gene silencing in rat
IBDUs.

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Fig. 3.
Suppression of AQP1 gene in
IBDUs by AQP1-siRNAs. IBDUs were transfected with 10 nM AQP1-siRNA(a), AQP1-siRNA(b), and corresponding
scrambled AQP1-siRNAs for 24 h. A, a significant
suppression of AQP1 mRNA level occurred in IBDUs transfected with
AQP1-siRNA(a) and AQP1-siRNA(b) but not with scrambled (scr)
AQP1-siRNAs. Representative immunoblots (B) and quantitative
analysis (C) show a significant suppression of AQP1 protein
levels by both AQP1-siRNAs but not scrambled siRNAs. D, a correlation
between AQP1 mRNA and protein expression levels was seen
(y = 298.34 + 32.079 × x;
r = 0.8821). Values are the mean ± S.E. of three
independent experiments with 4-6 IBDUs in each group (*,
p < 0.05).
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Visualizing of AQP1 Gene Suppression in IBDUs by
AQP1-siRNA--
IBDUs incubated with 10 nM Cy3-labeled
AQP1-siRNA(b) for 12-24 h were analyzed for AQP1 expression using
immunofluorescence as described under "Materials and Methods." The
data in Fig. 4 show that fluorescence
intensity of AQP1 (green) decreased in transfected IBDUs as
the fluorescence intensity of AQP1-siRNA(b) (red) increased.
12 and 24 h after IBDUs transfection with AQP1-siRNA, AQP1
immunofluorescence in cholangiocytes was reduced by 63 and 72%,
respectively, suggesting that AQP1 could be silenced in cholangiocytes as quickly as 12 h post-transfection.

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Fig. 4.
Visualizing of AQP1 suppression in IBDUs by
AQP1-siRNA. A, AQP1-siRNA(b) was labeled with Cy3 and
transfected into cholangiocytes of IBDUs. At the times indicated, IBDUs
were analyzed using confocal fluorescent microscopy for fluorescence
intensity of Cy3-labeled AQP1-siRNA(b) (red) and AQP1
(green). Top left patterns show higher
magnification of region in inset. B, in IBDUs
treated with AQP1-siRNA, an increase in Cy3 immunofluorescence
(white bars) and decrease in AQP1 immunofluorescence
(black bars) occurred during 12-24 h, reflecting
accumulation of AQP1-siRNA in cholangiocytes and rapid and effective
suppression of AQP1 expression in these cells, respectively. Values are
the mean ± S.E. of three separate experiments with 3-4 IBDUs in
each group. (*, p < 0.05 for AQP1
fluorescence compared with point 0 min).
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Inhibition of AQP1-mediated Water Transport by siRNAs--
Water
transport characteristics (i.e. Pf and
Jv) in AQP1-siRNAs-nontransfected IBDUs and in IBDUs
transfected with 10 nM AQP1-siRNAs were determined from the
volume of water transported into the lumen of IBDUs either in response
to a 200 mOsM transepithelial osmotic gradient (lumen
osmolality > bath osmolality) (Fig. 5)
or in response to 100 µM forskolin (Fig. 6), an agent known to stimulate biliary
bicarbonate and water secretion (2-4). The Pf in
AQP1-siRNA-nontransfected IBDUs and IBDUs transfected with scrambled
AQP1-siRNAs ranged from 50 × 10
3 to 72 × 10
3 cm/sec (Figs. 5 and 6). When a transepithelial
osmotic gradient was established in IBDUs transfected with
AQP1-siRNA(a) and AQP1-siRNA(b), Pf was decreased by
29.4 and 58.8%, respectively (Fig. 5A). In IBDUs
transfected with AQP1-siRNA(b), Pf was also
decreased by 53.1% in response to forskolin (Fig. 6A). In IBDUs transfected with AQP1-siRNA(b),
Jv across biliary epithelia in response to an
osmotic gradient or forskolin was decreased by 42.8 and 87.6%,
respectively (Figs. 5B and 6B). A strong
correlation between AQP1protein expression and water transport in IBDUs
transfected with AQP1-siRNAs was shown (Fig. 5C), suggesting that a decrease of cholangiocyte osmotic water permeability is a result
of specific AQP1 gene silencing by AQP1-siRNAs.

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Fig. 5.
Water transport in response to
transepithelial osmotic gradient in IBDUs transfected with
AQP1-siRNAs. A, osmotic water permeability coefficients
(Pf) reflect rapid water transport across biliary
epithelia in response to an inward osmotic gradient (200 mosM) in nontransfected IBDUs and in IBDUs transfected with
scrambled (scr) AQP1-siRNAs. Pf values
decreased by 29.4 and 58.8% in IBDUs transfected with AQP1-siRNA(a)
and AQP1-siRNA(b), respectively. B, net water movement
(Jv) values in IBDUs transfected with AQP1-siRNAs
reflect a decrease of water secretion by 42.8% in IBDUs trans- fected with AQP1-siRNA(b) compared with nontransfected IBDUs.
C, a correlation between AQP1 protein expression and
Pf was seen (y = 0.014192 + 0.00039212 × x; r = 0.9254). Values
are the mean ± S.E. of 4-6 microperfused IBDUs in each group
(*, p < 0.05).
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Fig. 6.
Water transport in response to forskolin in
IBDUs transfected with AQP1-siRNA. A, osmotic water
permeability coefficient (Pf) in
AQP1-siRNA-nontransfected IBDUs (white bar) and in IBDUs
transfected with scrambled (scr) AQP1-siRNA(b) (gray
bar) stimulated with forskolin (100 µmol) showed rapid water
transport across biliary epithelia. In IBDUs transfected with
AQP1-siRNA(b) (black bar) for 24 h, osmotic water
permeability in response to forskolin decreased by 53.1%.
B, net water secretion (Jv) in IBDUs
transfected with AQP1-siRNA(b) (black bar) in response to
forskolin decreased by 87% compared with nontransfected IBDUs
(white bar) and IBDUs transfected with scrambled
AQP1-siRNA(b) (gray bar). Values are the mean ± S.E.
of three microperfused IBDUs in each group (*,
p < 0.05).
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DISCUSSION |
The major objectives of this study were to develop and utilize
siRNAs to directly investigate the importance of AQP1 in water transport by biliary epithelia. Although several lines of evidence indicate that AQPs are involved in water transport by cholangiocytes (4-11), the absence of specific AQP inhibitors has impaired direct testing of hypotheses related to this issue. Indeed, until this study,
no specific pharmacological inhibitors of AQPs had been reported.
Our results show that AQP1-siRNAs effectively (i.e. by
57.9-79.4%), specifically (i.e. AQP1-siRNAs but not
scrambled AQP1-siRNAs), and rapidly (i.e. during 12 h
after transfection of IBDUs) down-regulate endogenously expressed AQP1
but not AQP4 in cholangiocytes. Moreover, direct quantitation of water
transport in IBDUs transfected with siRNAs to AQP1 demonstrates that
Pf and Jv are significantly
decreased compared with IBDUs transfected with scrambled siRNAs. The
observed decrease of net water secretion in IBDUs transfected with
AQP1-siRNA(b) under different experimental conditions (i.e.
an inward osmotic gradient or a secretory agonist) suggests that as
much as 65% of water transported across biliary epithelia may be
AQP1-mediated. Taken together, these findings demonstrate for the first
time that inhibition of AQP1 expression in mammalian cells by siRNAs
results in inhibition of water transport.
We found that cholangiocytes effectively take up siRNA duplexes if
IBDUs are incubated with AQP1-siRNAs either in the absence or presence
of transfection reagent. This observation is in contrast to published
data (29) that siRNAs are taken up by cultured HeLa, COS-1, and 293 cells only in the presence of transfection agent. However, our data are
consistent with recently published results that hepatocytes in
vivo can be effectively transfected with naked siRNAs (30, 31).
Although a precise explanation for these differences is not apparent,
they emphasize the importance of assessing both forms of siRNAs when
employing this gene-silencing approach.
In our study, we utilized two different siRNAs constructed against
different regions of AQP1 mRNA. Whereas both constructs inhibited
AQP1 expression and function in cholangiocytes, siRNA-designated AQP1-siRNA(b) was more effective. We have no explanation as to why
these two siRNAs differed in their inhibitory activity, but this
phenomenon has been observed for other genes. For example, several
siRNAs synthesized against different sites on the same target mRNA
demonstrated striking differences in silencing efficiency (29).
To our knowledge, this study is the first to successfully utilize siRNA
gene-silencing technology to achieve AQP gene suppression in epithelial
cells. AQP gene silencing has previously been achieved primarily by
developing transgenic null mice lacking AQPs and by using dominant
negative mutants or antisense oligonucleotides (ODNs). The phenotype
analysis of transgenic mice deficient in AQP1, AQP3, AQP4, and AQP5 has
provided new insights into their critical role in water transport in
the kidney, brain, eye, ear, salivary glands, skin, and
gastrointestinal organs (32-40). However, given that cholangiocytes
express numerous AQPs, deletion of a single AQP might not significantly
affect biliary water transport in vivo because other
cholangiocyte AQPs could undergo compensatory up-regulation. Dominant
negative mutants, which exert their effects in the presence of the
wild-type gene product, have also been employed in water-transporting
studies, demonstrating dominant negative effects of AQP2 and AQP4
mutants on AQP-mediated water transport in Xenopus oocytes
(41-43), LLC-PK1 cells (44), and mouse cholangiocytes
(45). This experimental approach also has its limitations being
applicable principally to studies utilizing cultured cell systems.
Antisense ODNs have been used to a limited degree to suppress AQP
function. For example, the Pf of Xenopus
laevis oocytes injected with poly(A)+ RNA from
cultured bovine corneal epithelial cells was inhibited by coinjection
with AQP5 ODNs (46). Also, the expression of AQP1 in human trabecular
meshwork cells was suppressed by corresponding ODNs (47). Although
useful, the specificity of this approach is somewhat limited because
nonspecific hybridization of ODNs with intracellular protein rather
than mRNAs has been reported previously (48). Moreover, the
efficiency of gene silencing by ODNs is relatively low (e.g.
only one of eight antisense oligonucleotides is expected to provide a
specific suppression of a targeted gene) (49). Our own experience has
been that AQP1 ODNs are not effective in inhibiting AQP1 expression and
function in isolated rat IBDUs (data not shown).
The advantages of siRNA technology for effective and specific
suppression of selected genes are becoming increasingly apparent. Recent studies (19-26) indicate that siRNAs directed against
endogenous genes can inhibit the expression of virtually any
protein-coding gene in any mammalian cells. Importantly, RNA and
protein analysis indicate that only the targeted gene is affected by
the corresponding siRNA (15-26).
In summary, we designed and utilized siRNAs to AQP1 that markedly
diminished the expression of this protein in IBDUs. As a result,
cholangiocyte osmotic water permeability and net water secretion were
significantly reduced, suggesting that at least 65% of water
transported across biliary epithelia is AQP1-mediated. These data are
the first to demonstrate the feasibility of utilizing siRNAs to
specifically reduce the expression of AQPs in epithelial cells. They
also provide further evidence of the importance of AQP1 in water
transport by biliary epithelia.