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Modulation of multidrug resistance-associated protein 2 (Mrp2) and Mrp3 expression and function with small
Xianbin Tian1, Maciej J Zamek-Gliszczynski, Peijin Zhang
1Division of Drug Delivery and Disposition, University of North Carolina, Chapel Hill 27599-7360, USA.
Abstract:
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 (approximately 50% decrease in expression) resulted in an approximately 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 (approximately 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 approximately 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.
Insights
RNA interference effectively reduced multidrug resistance-associated protein 2 (Mrp2) and Mrp3 expression in rat hepatocytes. This validated a method for studying liver drug transporter function and xenobiotic excretion.
Area of Science:
- Hepatology
- Molecular Biology
- Pharmacology
Background:
- Canalicular multidrug resistance-associated protein 2 (Mrp2) and basolateral Mrp3 are key transporters for organic anion excretion from the liver.
- Understanding their function is crucial for drug metabolism and toxicity studies.
Purpose of the Study:
- To demonstrate the utility of RNA interference (RNAi) for specifically targeting Mrp2 and Mrp3 in cultured rat hepatocytes.
- To investigate the functional consequences of Mrp2 and Mrp3 knockdown on xenobiotic transport.
Main Methods:
- Utilized sandwich-cultured rat hepatocytes, maintaining functional and localized Mrp2 and Mrp3.
- Employed RNA interference (small interfering RNA) to achieve specific knockdown of Mrp2 and Mrp3 expression.
- Assessed the biliary excretion index and cellular accumulation of carboxydichlorofluorescein (CDF) as a model organic anion.
Main Results:
- Specific Mrp2 knockdown (approx. 50%) decreased CDF biliary excretion by approx. 45%.
- Specific Mrp3 knockdown (approx. 50%) increased CDF biliary excretion index by approx. 60% with altered cellular accumulation.
- Protein knockdown levels correlated well with observed changes in CDF disposition.
Conclusions:
- RNA interference is a feasible in vitro method for modulating drug transporter expression in sandwich-cultured rat hepatocytes.
- This approach enables effective study of drug transporter expression and function in a controlled cellular environment.
- Findings highlight the distinct roles of Mrp2 and Mrp3 in hepatic xenobiotic transport.
