THE KNOCKDOWN OF HEPATOCYTE AQUAPORIN-8 BY RNA INTERFERENCE INDUCES DEFECTIVE BILE CANALICULAR WATER TRANSPORT

M. CECILIA LAROCCA, LEANDRO R. SORIA, M. VICTORIA ESPELT, GUILLERMO L. LEHMANN, AND RAÚL A. MARINELLI

AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY

Año: 2008 p. 93 - 100

0193-1857

Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bilecanalicular membranes, are involved in water transport during bile formation.Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion intothe bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencingby RNA interference inhibits canalicular water secretion in the human hepatocytederivedcell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cellsexpress AQP8, and by confocal immunofluorescence microscopy that it is localizedintracellularly and on the canalicular membrane, as described in rat hepatocytes. Wealso verified the expression of AQP8 in normal human liver. Forty-eight hours aftertransfection of HepG2 cells with RNA duplexes targeting two different regions ofhuman AQP8 molecule, the levels of AQP8 protein specifically decreased by 60-70%.We found that AQP8-knockdown cells showed a significant decline in the canalicularvolume of about 70% (p < 0.01) suggesting an impairment in the basal(nonstimulated) canalicular water movement. We also found that the decreased AQP8expression inhibited the canalicular water transport in response either to an inwardosmotic gradient (-65%, p < 0.05) or to the bile secretory agonist dibutyryl cAMP (-80%, p < 0.05). Our data suggest that AQP8 plays a major role in water transportacross canalicular membrane of HepG2 cells, and support the notion that defectiveexpression of AQP8 causes bile secretory dysfunction in human hepatocytes.