NAFLD in donor organs is an increasingly important problem in liver transplantation. Hepatic steatosis increases the susceptibility of the allograft to CIR injury leading to increased rates of allograft failure and/or slow functional recovery. Many centers currently utilize mildly steatotic livers for transplantation with good results; however, even with mild steatosis there is an increase in primary delayed function and increased resource utilization post-transplant compared to lean donor allografts (4
). It has been demonstrated that the pathophysiology of CIR injury in the steatotic liver differs from that in the lean organ, and few successful strategies have been developed to attenuate this injury (5
). Considering that hepatic steatosis is closely linked to the metabolic syndrome in the donor (1
), the exploration of factors linking the steatosis to systemic insulin resistance and inflammation could yield important information on the behavior of the steatotic allograft following a CIR injury.
Hepatic ER stress is recognized as an important mechanistic link between obesity, insulin resistance and hepatic steatosis (15
). Although, ER stress is, in general, an adaptive response, prolonged or sudden increases in ER stress can initiate pathways that result in cell death (22
). There are multiple pathways though which ER stress is known to mediate cell death (21
). Although their mechanisms are not entirely clear, drugs such as TUDCA are classified as chemical chaperones due to their ability to enhance ER protein folding and thus decrease the ER stress response (43
). Studies using chemical chaperones to target the PERK and IRE1α pathways of hepatic ER stress have demonstrated improvements in the insulin resistance of obese rodents (15
). In this study, we investigated the ER stress response in the steatotic liver allograft as a component of the post-reperfusion injury observed in severely steatotic livers. Our results demonstrate that acute ER stress plays an important role in the CIR injury of the steatotic allograft.
An ER stress response occurs following liver transplantation in both lean and steatotic allografts. While lean allografts showed an ER stress response, the magnitude of the response seen in the steatotic liver was far greater and more prolonged. The ER stress response in the steatotic allograft peaked at 12 hours but remained significantly elevated at 24 hours following reperfusion. The magnitude of ER stress gene induction and the prominence of CHOP expression suggested the hypothesis that ER stress associated cell death pathways may play a mediating role in the CIR injury incurred by the steatotic allograft. Because chemical chaperones are well accepted ER stress inhibitors, we initially treated the obese donor animals to determine if decreasing chronic hepatic ER stress in the steatotic liver would affect the post-CIR injury. When TUDCA was given to donor animals for 7 days, we observed an improvement in the animal’s insulin resistance accompanied by a decrease in the hepatic expression of IRE1α and CHOP. These findings are similar to previous reports of TUDCA administration in ob/ob
). Despite the successful decrease in the chronic hepatic IRE1α and CHOP levels, the steatotic allografts procured from the TUDCA treated donors had similar post-transplant ER stress responses and allograft injuries as donors treated with vehicle. These results suggest that the acute ER stress response in the steatotic liver is not directly related to chronic ER stress levels.
In contrast, the addition of TUDCA to the cold storage solution in addition to delivery at the time of reperfusion was a successful strategy to decrease the acute post-transplant ER stress response in the steatotic allograft. While this effect could be measured at 6 hours following transplant, the greatest improvement was seen at 12 hours. The effect on allograft injury followed the pattern of ER stress change, with a small improvement in serum ALT noted at 6 hours and a greater change at 12 hours. While TUDCA was able to decrease HMGB1 production in the steatotic allograft, qualitatively there was not a clear change in histologic necrosis. The histologic changes following TUDCA treatment were improvement of decreased portal and lobular inflammation. This suggested that TUDCA decreased a secondary component of the CIR injury. The improvements in histologic allograft inflammation are consistent with the observed decreased NF-κB activation, and decreased IL-6, and IL-1β levels in the TUDCA treated steatotic allografts. This data implicates ER stress as a mediator of the inflammatory component of CIR injury in the steatotic allograft.
Because the component of injury which seemed to be improved by TUDCA was inflammatory, we suspected that the IRE1α pathway with its well described ability to stimulate NF-κB and pro-inflammatory cytokines was a mediating pathway. However, we observed no difference in downstream IRE1α pathway activation (XBP-1 splicing) between the lean and steatotic allografts. Further, TUDCA had no effect on XBP-1 splicing. Together, these observations suggest that the IRE1α pathway may not be an important mediator of allograft injury in our model.
CHOP, a specific cell death mediator, was the most prominently upregulated ER stress marker in the steatotic allograft following CIR injury, and the improvements observed in allograft injury were accompanied by an almost 50% reduction in CHOP expression at 12 hours of reperfusion. The correlation of CHOP’s over-expression with CIR injury and the decrease of this expression with TUDCA suggested that the CHOP was an important effector of ER stress mediated cell death in the steatotic allograft. Our observation that caspase 11 activity was inhibited by TUDCA supports this hypothesis as caspase 11 links CHOP to IL-1β production (34
). This is also one of many pathways linking CHOP to cell death (36
). This observation, combined with the lack of support to implicate the IRE1α pathway, suggest that the ER Stress-CHOP pathway plays a mediating role in steatotic liver allograft injury via induction of inflammation associated caspases.
The relationship of ER stress mediated cell death and CIR injury in the steatotic liver will require further definition. However, this study serves to implicate ER stress on a broad scale as an important factor in this injury. The rodent transplant model is a good clinical approximation of the clinical scenario of steatotic liver transplantation, but it has limitations in the definition of specific cellular mechanisms involved in this multi-faceted problem. The non-arterialized model is useful for making observations about acute CIR injury such as in this study, but is less useful for more long term survival studies. In addition, this is a severely steatotic model; the extent of the reperfusion injury can often obscure the potential benefit of an intervention. This potentially explains the lack of correlation between histologic necrosis and decreased HMGB1 release. In this study we utilized a leptin deficient model of obesity, metabolic syndrome and hepatic steatosis. Steatotic livers from leptin deficient rodents and high fat diet treated rodents have been shown to have similar ER stress responses and it is unlikely that the leptin mutation in our model had any significant effect on hepatic ER stress response following CIR injury (16
In summary, the steatotic liver allograft has an exaggerated ER stress response following CIR injury. These data implicate ER stress as a mediating factor in cell death and inflammation of the steatotic liver following transplantation. The definition of the exact mechanisms involved in ER stress mediated steatotic allograft injury will require further investigation, but the results of this study suggest that the ER stress pathways, specifically the CHOP-caspase 11-IL-1β pathway, are potential targets to improve steatotic liver allograft function following liver transplantation.