Transcriptomic Analysis during Normothermic Machine Perfusion Reveals Similar Responses in Steatotic and Lean Discarded Human Livers

Abstract

Chronic liver disease is the 12th leading cause of death in the U.S., with end-stage liver disease alone accounting for 60,000 deaths in the U.S. each year.The only available treatment for liver failure is orthotopic liver transplantation.However, the severe shortage of viable donor organs results in less than 30% of patients on the wait list receiving transplants.The morbidity and mortality associated with liver disease could be dramatically reduced by expanding the donor organ pool by rendering marginal donor livers, especially steatotic grafts, transplantable.Steatotic livers are more susceptible to ischemia-reperfusion (I/R) injury, the major complication leading to graft dysfunction.Normothermic machine perfusion (NMP) combined with adjunct therapies may be able to rehabilitate discarded steatotic livers, thereby increasing the supply of organs for transplant.The goal of this study was to develop an understanding of the transcriptomic changes associated with NMP in steatotic compared to lean livers. Twelve discarded livers (6 lean, 6 steatotic), rejected by local transplant centers, were obtained and underwent 12 hours of NMP with oxygenated blood. Serial tissue and plasma samples were collected. Clinical viability criteria were used to determine if livers would have been transplantable (viable) or not (nonviable). Transcriptome sequencing was performed on biopsies taken at 0 (pre-), 3, and 6hr of perfusion. Comparative analysis between transcriptomes of individual conditions was performed using R. Ingenuity Pathway Analysis (IPA) was performed on significant genes to identify canonical pathways and upstream regulators. Three of the six livers from each group met viability criteria for transplant. IPA comparing viable livers at 0 and 3hr of perfusion demonstrated robust activation of innate immune responses, such as Toll-like receptor, IL-10, and IL-6 canonical pathways (P<10E-7) in both lean and steatotic livers, compared to nonviable livers. However, many of the same canonical pathways identified after 3 hours of perfusion in viable livers were not significant in the nonviable livers until 6hr of perfusion in both lean and steatotic livers, suggesting a delayed transcriptomic response in nonviable livers of both groups. There was significant overlap in upstream regulators identified in both viable and nonviable livers. However, these shared regulators demonstrated remarkably divergent downstream target activation.This is the first study to demonstrate activation of regenerative signaling in discarded lean and steatotic human livers during NMP. Comparison of gene expression profiles between lean and steatotic viable livers were similar as were the transcriptomic profiles of the nonviable livers from both groups.These data suggest that the response to NMP is similar in lean and steatotic liver.Our analysis provides numerous potential therapeutic targets for improving function and transplantability of nonviable lean and steatotic livers during ex vivo machine perfusion.