Specific inhibition of AQP1 water channels in isolated rat intrahepatic bile duct units by small interfering RNAs

Patrick L. Splinter, Anatoliy I. Masyuk, Nicholas F La Russo

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)6268-6274
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number8
DOIs
StatePublished - Feb 21 2003

Fingerprint

Intrahepatic Bile Ducts
Aquaporins
Ducts
Small Interfering RNA
Rats
Water
Aquaporin 1
Epithelium
Hydraulic conductivity
Gene Silencing
Colforsin
Base Pairing
Liver
Transfection
Permeability
Proteins
Genes
Epithelial Cells
Messenger RNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Specific inhibition of AQP1 water channels in isolated rat intrahepatic bile duct units by small interfering RNAs. / Splinter, Patrick L.; Masyuk, Anatoliy I.; La Russo, Nicholas F.

In: Journal of Biological Chemistry, Vol. 278, No. 8, 21.02.2003, p. 6268-6274.

Research output: Contribution to journalArticle

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abstract = "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.",
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