Background Steatotic liver grafts tolerate ischemia-reperfusion (I/R) injury poorly, contributing to increased primary graft nonfunction following transplantation. Activation of nuclear factor kappa-B (NFκB) following I/R injury plays a crucial role in activation of pro-inflammatory responses leading to injury. Methods We evaluated the role of NFκB in steatotic liver injury by using an orthotopic liver transplant (OLT) model in Zucker rats (lean to lean or obese to lean) to define the mechanisms of steatotic liver injury. Obese donors were treated with bortezomib to assess the role of NF-κB in steatotic liver I/R injury. Hepatic levels of NF-κB and pro-inflammatory cytokines were analyzed by ELISA. Serum transaminase levels and histopathological analysis were performed to assess associated graft injury. Results I/R injury in steatotic liver results in significant increases in activation of NF-κB (40%, p<0.003), specifically the p65 subunit following transplantation. Steatotic donor pretreatment with proteasome inhibitor bortezomib (0.1 mg/kg) resulted in significant reduction in levels of activated NF-κB (0.58±0.18 vs. 1.37±0.06 O.D./min/10μg protein, p<0.003). Bortezomib treatment also reduced expression of pro-inflammatory cytokines MIP-2 compared with control treated steatotic and lean liver transplants respectively (106±17.5 vs. 443.3±49.9 vs. 176±10.6 pg/mL, p=0.02), TNF-α (223.8±29.9 vs. 518.5±66.5 vs 264.5±30.1 pg/2μg protein, p=0.003) and IL-1β (6.0±0.91 vs. 19.8±5.2 vs 5±1.7 pg/10μg protein, p= 0.02) along with a significant reduction in ALT levels (715±71 vs 3712.5±437.5 vs 606±286 U/L, p=0.01). Conclusion These results suggest that I/R injury in steatotic liver transplantation are associated with exaggerated activation of NFκB subunit p65, leading to an inflammatory mechanism of reperfusion injury and necrosis. Proteasome inhibition in steatotic liver donor reduces NFκB p65 activation and inflammatory I/R injury, improving transplant outcomes of steatotic grafts in a rat model.

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BackgroundThe molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software.ResultsThe relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1was significantly upregulated in steatotic liver following I/R.ConclusionDown-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.

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