J. Kim1, A. Martin1, J. Yee1, L. Fojut1, M. A. Zimmerman1, J. C. Hong1 1Medical College Of Wisconsin,Transplant Surgery,Milwaukee, WI, USA
Introduction: Cholestasis is an inevitable consequence of severe hepatic ischemia-reperfusion injury (IRI), and no treatment is available to prevent it. The detrimental function of membrane bile transporters is presumed to be the underlying precedent condition of cholestasis that arises during ischemia. Nuclear factor erythroid 2-related factor 2 (NRF2) mediates antioxidant gene transcription following oxidative stress. Previous studies suggest that bile transporter genes are regulated by NRF2 in a drug-induced liver injury model. However, the role of NRF2 in bile metabolism during hepatic IRI and its therapeutic potential are unknown. Bile metabolism is mediated by membrane transporters, such as bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), MRP3, and anion exchange protein 2 (AE2). We investigated the validity of NRF2 activation by using bardoxolone methyl (BARD) as a treatment to increase the transcriptional activities of bile transporters thereby to improve bile metabolism in hepatic IRI.
Methods: An empty vehicle (control) or bardoxolone methyl (2 mg/kg) was infused into the inferior vena cava of Sprague-Dawley rats 5 min before the ischemia. Hepatic warm ischemia was induced by clamping the median and left lobes. Sham surgery, 60 min or 90 min periods of hepatic warm ischemia were applied, and samples were obtained after 24 h of reperfusion (n=5 per each group). Serum levels of bile acids were tested, and hepatic tissue was sampled to determine the quantitative polymerase chain reactions (qPCR) of bile transporters. The results of qPCR were presented as mRNA levels relative to those of the glyceraldehyde 3-phosphate dehydrogenase gene (Gapdh).
Results: After 60 min of ischemia and 24 hours of reperfusion, the hepatic tissue mRNA levels of Mrp2 and Mrp3 in the BARD group were higher when compared to those of the control (P=0.032 and 0.008, respectively; Figure A). After 90 min of ischemia and 24 hours of reperfusion, the mRNA levels of Bsep and Ae2 in the BARD group were lower than those of the control (P=0.008 and 0.016, respectively; Figure B). The serum bile acid levels were significantly lower in the BARD group after 60 min of ischemia and 24 hr of reperfusion (P=0.016, Figure A).
Conclusion: NRF2 contributes to the transcriptional activity relevant to bile metabolism during hepatic IRI. The activation of NRF2 at the time of ischemia can increase transcription activities of bile transporters to improve hepatic bile metabolism after reperfusion. The effect of NRF2 activation disappears when the warm ischemia time is prolonged. This study suggests a novel therapeutic target in hepatic IRI.
