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J. Biol. Chem., Vol. 278, Issue 8, 6268-6274, February 21, 2003
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From the Center for Basic Research in Digestive Diseases, Division
of Gastroenterology and Hepatology, Mayo Medical School, Clinic,
and Foundation, Rochester, Minnesota 55905
Cholangiocytes express water channels
(i.e. aquaporins (AQPs)), proteins that are increasingly
recognized as important in water transport by biliary epithelia.
However, direct functional studies demonstrating AQP-mediated water
transport in cholangiocytes are limited, in part because of the lack of
specific AQP inhibitors. To address this issue, we designed,
synthesized, and utilized small interfering RNAs (siRNAs) selective for
AQP1 and investigated their effectiveness in altering AQP1-mediated
water transport in intrahepatic bile duct units (IBDUs) isolated from
rat liver. Twenty-four hours after transfection of IBDUs with siRNAs
targeting two different regions of the AQP1 transcript, both AQP1
mRNA and protein expression were inhibited by 76.6-92.0 and
57.9-79.4%, respectively. siRNAs containing the same percent of base
pairs as the AQP1-siRNAs but in random sequence (i.e.
scrambled siRNAs) had no effect. Suppression of AQP1 expression in
cholangiocytes resulted in a decrease in water transport by IBDUs in
response to both an inward osmotic gradient (200 mosM) or a
secretory agonist (forskolin), the osmotic water permeability
coefficient (Pf) decreasing up to 58.8% and net
water secretion (Jv) decreasing up to 87%. A
strong correlation between AQP1 protein expression and water transport
in IBDUs transfected with AQP1-siRNAs was consistent with the decrease
in water transport by IBDUs resulting from AQP1 gene silencing by
AQP1-siRNAs. This study is the first to demonstrate the feasibility of
utilizing siRNAs to specifically reduce the expression of AQPs in
epithelial cells and provides direct evidence of the contribution of
AQP1 to water transport by biliary epithelia.
Specific Inhibition of AQP1 Water Channels in
Isolated Rat Intrahepatic Bile Duct Units by Small Interfering
RNAs*
*
This work was supported by Grant DK24031 (to
N. F. L.) from the National Institutes of Health, American
Gastroenterological Association/Elsevier Research Initiative Award (to
A. I.M.), and by the Mayo Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Center for Basic
Research in Digestive Diseases, Mayo Clinic, 200 First St., S. W.,
Rochester, MN 55905. Tel.: 507-284-1006; Fax: 507-284-0762; E-mail:
larusso.nicholas@mayo.edu.
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