PI3Kγ MEDIATES Mrp2 ACTIVITY IMPAIRMENT INDUCED BY IL-β IN SANDWICH CULTURED RAT HEPATOCYTES

SCHUCK, VIRGINIA S.; ANDERMATTEN, ROMINA B.; FAZZI, AGUSTINA; SALAS, GIMENA; MEDEOT, ANABELA C.; BAROSSO, ISMAEL R.; SANCHEZ POZZI, ENRIQUE J.

Mar del Plata

Congreso; REUNIÓN CONJUNTA SAIC SAB AAFE AACYTAL 2023; 2023

Sociedad Argentina de Investigación Clínica

In intrahepatic cholestasis, IL-1β is involved in altering canalicular secretion by causing changes in the localization and function of canalicular transporters such as Mrp2, this action promotes transporter disinsertion through activation of kinase-mediated signaling proteins. We have previously shown that the PI3K/AKT pathway is involved in this process. Therefore, our aim was to identify which isoform of PI3K is involved in Mrp2 alterations. Since there is evidence of a role of PI3Kγ in Mrp2 expression in sepsis, we analyzed whether this PI3K isoform participate in IL-1ß action in sandwich-cultured rat hepatocytes (SCRH). Methodology: SCRHs were transfected by adding 5 μL of lipofectamine with 70 nM of PI3Kγ siRNA per well or scrambled siRNA, followed by a 6 h incubation at 37 °C. After transfection, hepatocytes were washed and overlaid with gelled collagen for 1 h at 37 °C to obtain a collagen sandwich configuration. SCRHs were exposed to IL-1β (10ng/ml) for 20 min. After treatments, Mrp2 transport function was evaluated by the pseudo-canalicular accumulation of its substrate glutathione-S-methylfluorescein (GSMF). Chloromethylfluorescein diacetate (CMFDA) 2,5 μM for 15 min, was added to the medium and time lapse imaging was done every minute during 8 min with a fluorescence microscope. Between 70 and 100 pseudocanaliculi were selected in each image, and the average of time fluorescence of CMFDA-metabolite GSMF was measured. The slope of GSMF fluorescence vs. time was used as an estimate of initial rate of transport (IRT). Result: (% of Control ± SEM; n=3-4) IL-1ß decreased the initial rate of Mrp2 transport (49%±1a). This was partially prevented by knockdown of the PI3Kγ protein (68%±2ab). a different from control, b different from IL-1ß. Conclusion: The cytokine IL-1ß produces a reduction in Mrp2 function, associated with internalization of the transporter, mediated in part by the activation of the PI3Kγ protein.