MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN 2 (MRP2) INTERNALIZATION IS DIFFERENTIALLY MOD- ULATED BY PI3K AND MEK-ERK PROTEINS IN ACUTE TUMOR NECROSIS FACTOR ALPHA (TNF)-INDUCED CHOLESTASIS

CIRIACI, NADIA; ANDERMATTEN, ROMINA B.; SCHUCK, VIRGINIA S.; TABORDA, DIEGO; RUIZ, MARÍA LAURA; SANCHEZ POZZI, ENRIQUE J.

Mar del Plata

Congreso; Reunión Anual de la SAIC; 2020

Background: TNF induces internalization of Mrp2. At least, two pathways are involved: one includes NADPH oxidase, reactive oxygen species and MEK-ERK kinases (Biochem Pharmacol 164:311, 2019) and the other, PI3K-Akt. The aim of this study is to discern whether these pathways are involved in the initial endocytic internalization of Mrp2 or in the blockage of its subsequent reinsertion toward the canalicular membrane, using the isolated perfused rat liver model (IPRL).Method: In IPRL, TNF was administered by an intraportal injection (120 pg/100 g b.w.). The roles of PI3K and ERK were evaluated adding their inhibitors wortmannin (W, 200 nM) or PD980589 (PD, 4μM) to the reservoir 10 min before TNF. Bile was collected over 30 min and bile flow was measured gravimetrically. To evaluate Mrp2 transport activity, 1-chloro-2,4-dinitrobenzene (0.5 mmol/L) was added to the perfusate. Then dinitrophenyl glutathione (DNPG) biliary secretion was determined spectrophotometrically. Finally, Mrp2 localization was analysed by immunofluorescence and confocal microscopy.Results: (% of control±SE; n=3). Bile flow was decreased by TNF (to about 80% of control values within 20 min) and it did not recover throughout the perfusion period. This impairment of bile flow was restored by inhibitors: PD tended to protect from the beginning whereas W protected from 15 min onwards, suggesting different protection mechanisms. TNF-induced decrease of cumulative biliary DNPG excretion (71±6a) was countered by W (100±3b) and PD (109±2b). Both inhibitors prevented Mrp2 delocalization induced by TNF in confocal images. a p < 0.05 vs. Control; b p < 0.05 vs. TNF.Conclusion: Mrp2 internalization induced by TNF was prevented by W and PD. As W does not protect from the initial bile flow decay induced by TNF, PI3K is probably not implied in the initial transporter desinsertion but in preventing spontaneous reinsertion of the canalicular carrier, previously endocytosed via MEK-ERK.