Papel del transportador mrp3 en la circulación enterohepática de ácidos biliares y en la regeneración hepática

Resumen

BAs are secreted into the bile and are subsequently avidly absorbed from the intestine. BAs in the portal blood are taken up by the hepatocytes and are re-secreted to continue cycling between the intestine and the liver, establishing their enterohepatic circulation. The dynamics of BAs transport and metabolism is tightly regulated. This maintenance of bile formation and enterohepatic recirculation of BAs is indispensable for liver regeneration. Partial hepatectomy (PH), resulting in the removal of approximately 70% of the liver, is widely utilized to study liver growth in experimental animals. After PH the remnant liver is exposed to a high flux of BAs reaching the liver from the portal circulation that could cause hepatic injury. In this case, the concentration of BAs is regulated by a decrease in BA production, increased biliary excretion, decreased basolateral uptake and increased basolateral efflux. Basolateral BA efflux from liver cells is mediated in part by MRP3. MRP3 belongs to a group of nine related ATP-binding cassette transporters. MRP3 presents a basolateral subcellular localization in polarized cells and it is expressed in all the organs where the enterohepatic circulations of bile acids takes place. Mrp3 transports a variety of organic anions with a preference for glucuronosyl- and sulfate conjugates, including BAs. In view of this we wanted to evaluate the importance of this transport protein in a model where the enterohepatic circulation of BAs is modified, and also during liver regeneration. We treated Mrp3+/+ and Mrp3−/− mice with a cholic acid (CA)-supplemented diet for 5, 7 and 11 days. Mrp3−/− mice were quite tolerant to the toxic effects of CA in that CA-induced hepatotoxicity was dramatically attenuated in Mrp3−/− mice. When we analyzed the expression of liver genes wich are implicated in the biosynthesis or transport of bile acids (Shp, Cyp7a1, Oatp1, Oatp2, Bsep) in both groups of mice treated with the CA diet for five days we observed a lesser or abolished modulation in Mrp3−/− mice. Bile acids induced proliferative effects (Cdc25b, Cyclin D1, PCNA or Ki67 expression) much more pronounced in wild type than in Mrp3−/− animals. These findings suggested that the liver of Mrp3−/− mice was not receiving the same amount of bile acids from the intestine. When we evaluated the level expression of several bile acid transporters in the enterocyte (Osta , Ostb and Asbt) or genes clearly induced by bile acids (Fgf15, Shp) we observed that the response to the CA diet was more attenuated in Mrp3−/− mice. We used a FXR reporter plasmid to monitor hepatic activation of the principal nuclear receptor involved in the regulation of the bile homeostasis, and we found that Mrp3−/− mice showed an impaired FXR activity. The BA pool was analyzed and we observed that the lack of Mrp3 produced a marked alteration in the BA composition. In view of these results liver regeneration was examined in mice lacking Mrp3 in a PH model. Mrp3−/− mice showed a decrease in liver growth at 24 and 36 h post hepatectomy combined with delayed inductions of inmediate early genes (c-myc, c-fos) and proliferative genes (Cyclin D1, Cyclin E, PCNA, FoxM1b). The Mrp3−/− mice also showed a decrease in BrdU-positive nuclei (DNA synthesis). Importantly, at 36 h post PH the concentration of all BAs in liver was significantly decreased in Mrp3−/− mice when compared to wild type animals. This work shows for the first time the Mrp3 transporter is essential for proper homeostasis of BAs and the adequate development of the regenerative processes after PH. The impaired expression of Mrp3 in chronically injured human liver may therefore compromise the regenerative activity of this organ