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Modulation of Multidrug Resistance-Associated Protein 2 (Mrp2) and Mrp3 Expression and Function with Small Interfering RNA in Sandwich-Cultured Rat Hepatocytes

Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina

Canalicular multidrug resistance-associated protein 2 (Mrp2) and basolateral Mrp3 mediate the excretion of organic anions, including conjugated and unconjugated xenobiotics and bile acids, from the liver. The utility of RNA interference to specifically knock down the expression and function of transport proteins was demonstrated in sandwich-cultured rat hepatocytes, which exhibit functional and properly localized Mrp2 and Mrp3 over time in culture. Specific knockdown of Mrp2 (50% decrease in expression) resulted in an 45% decrease in the biliary excretion index of carboxydichlorofluorescein (CDF) (9.3% versus 16.5%), but did not affect Mrp3 or radixin expression. Specific Mrp3 knockdown (50% decrease in expression) resulted in significantly higher accumulation of CDF in cells + bile canaliculi (32.3 ± 2.5 versus 24.4 ± 4.3 pmol/mg of protein/10 min), but no change in cellular accumulation (13.7 ± 2.2 versus 15.6 ± 4.0 pmol/mg of protein/10 min), consistent with an 60% increase in the biliary excretion index of carboxydichlorofluorescein. The extent of protein knockdown was in good agreement with changes in carboxydichlorofluorescein disposition. In conclusion, modulation of drug transporters in sandwich-cultured rat hepatocytes by small interfering RNA treatment is a feasible in vitro approach to study the expression and function of drug transport proteins.

Address correspondence to: Dr. Kim L. R. Brouwer, University of North Carolina School of Pharmacy, CB #7360, Room #2312 Kerr Hall, Chapel Hill, NC 27599-7360. E-mail: kbrouwer{at}unc.edu

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