Abstract
Background: Rifampin is a known inhibitor of organic anion transporter proteins, thereby interfering with hepatobiliary transport of bilirubin resulting in hyperbilirubinemia. In-Vivo multiphoton microscopy (IVM) allows for real time quantification of hepatic organic anion (OA) transport (detailed methodology abstract control# 782514). Aim: To use IVM to measure hepatobiliary transport in a model of Rifampin-induced hepatotoxicity. Methods: Sprague-Dawley rats were treated with Rifampin (250mg/kg/day) for 4 days by oral gavage. 12-24 hours post final dose animals were imaged using the fluorescent probes Hoechst (nuclear probe), sodium fluorescein (NaF) and 6-carboxyfluorescein diacetate (6-CFDA). NaF is fluorescent and is taken up by hepatocytes via basolateral OA transporters. 6-CFDA is cleaved to its fluorescent form intracellularly, and secreted into biliary canaliculi (image) via apical OA transporters. Normalized fluorescence intensity units (NFU) in hepatocyte cytoplasmic (NaF) and biliary canalicular (6-CFDA) signal were measured. Rates of hepatocellular NaF uptake and excretion, and canalicular 6-CFDA clearance were calculated. Serum hepatobiliary clinical chemistry parameters were measured at baseline and at imaging. Data were compared in treated and control animals. Results: Rifampin treated animals had higher ALT, AST and bilirubin levels compared with baseline and control animal levels. As expected Rifampin treatment resulted in diminished hepatocellular NaF uptake (2.8 fold). Unexpectedly, Rifampin treatment also suppressed hepatocellular 6-CFDA excretion (p = 0.12). Canalicular 6-CFDA clearance in Rifampin-treated animals was similar to that of controls. Conclusion: This pilot study demonstrates the ability of IVM to elucidate mechanisms of hepatic OA transport in drug-induced hepatotoxicity In-Vivo.
Published Version
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