Reversible Ubiquitinproteasome Inhibitor Addition to UW Solution: A Strategy for Enhancing the Steatotic Liver Graft Preservation

Abstract

Introduction: During the last decade, the dramatic shortage of organs has obliged to consider the transplantation of liver graft with steatosis. Steatotic livers show a poor tolerance against ischemia reperfusion injury (IRI) due to exacerbated oxidation and microcirculation alterations responsible for the subsequent higher risk of graft dysfunction or primary nonfunction when steatotic grafts are used. Ubiquitin proteasome pathway (UPS) is the principal non-lysosomal proteolytic system responsible for the protein degradation but its role in liver IRI was not fully investigated, although recent studies have evidenced that UPS inhibition during hypothermia prolongs myocardial graft preservation. In addition, bortezomib (a reversible proteasome inhibitor) administered at non-toxic low doses in rats sed by chronic alcohol consumption, resulted to be protective by increasing liver antioxidant enzymes. Here, we evaluated and compared the BZ and MG132 addition to UW preservation solution at low and non toxic dose for fatty liver graft protection against cold IRI. Methods: Livers from lean and obese Zücker rats (11 weeks aged) will be stored in UW solution enriched with BZ (100nM/L) or MG (25 μmol/L) during 24 hours at 4°C and then subjected to 2 hour-normothermic reperfusion. (37°C). Liver injury (AST/ALT), hepatic function (bile output; vascular resistance); mitochondrial damage (GLDH); oxidative stress (MDA); nitric oxide (e-NOS activity; nitrates/nitrites); hemeoxygenase (HO-1), proteasome chymotrypsin-like activity and UPS (19S & 20 S) protein levels were measured, respectively. Results: BZ and MG132 addition to UW solution prevented similarly the liver injury (ASAT/ALAT) when compared to UW alone. However, BZ increased bile production more efficiently than MG132 but only BZ decreased vascular resistance in fatty livers. This well correlated with a concomitant increases in nitric oxide generation (through e-NOS activation) and AMPK phosphorylation. Also, this was accompanied by a significant prevention of mitochondrial damage (GLDH) and oxidative stress (MDA). In addition, BZ presence inhibited adiponectin, IL-1 and TNF alpha, only in steatotic livers. This well correlated with 20S-proteasome expression reduction (not 19 S moiety) and chymotrypsin-like activity inhibition. Conclusion: BZ at low and non toxic doses protects more efficiently fatty liver grafts against cold IRI than MG132 when used as additives in UW preservation. Interestingly, BZ benefits are mediated by nitric oxide. The results reported here shows the relevance of UPS in fatty liver preservationtroduction