Targeting brain death-induced injury
van Erp, Anne Cornelie
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Publication date: 2018
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van Erp, A. C. (2018). Targeting brain death-induced injury. Rijksuniversiteit Groningen.
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Summary
General discussion
Future perspectives
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SUMMARY
In Chapter 1, we provided a general introduction about organ donation and transplantation, describing how the organ graft is harmed during each step of the transplantation process (donor, preservation, recipient), followed by a more in depth look at the pathophysiology of donor brain death. Finally, two novel aspects of brain death pathophysiology – organ-specific metabolic changes and autophagy (dysregulation) – are introduced and current knowledge on these topics in relation to organ donation and transplantation are discussed. In Chapter 2, we reviewed all systematically tested clinical interventions in the brain-dead donor and their impact on graft function and/or survival following solid organ transplantation. Of the combined 33 studies that were included, only ascorbic acid treatment, ventilation with sevoflurane, and ischemic preconditioning improved short-term liver function; dopamine treatment and hypothermic cooling of the donor at 34-35°C improved short-term kidney function; and dopamine treatment improved long-term patient and graft survival following heart transplantation. Finally, hemodynamic stabilization with the colloid HES is not advised prior to kidney transplantations. Despite several promising studies, this review predominantly highlights the need for additional high-quality clinical studies before any particular treatment or intervention can be recommended as part of standard deceased donor care.
In Chapter 3, we showed that pre-treatment with immunosuppressive drug EA230, an oligopeptide of human chorionic gonadotropin, was unable to attenuate brain-death induced inflammation or hepatic and renal injury in rodents. This lacking immunosuppressive response might be explained by adrenal insufficiency or dysfunctional pituitary stimulation caused by brain death pathophysiology.
In Chapter 4, we revealed that pre-conditioning of brain-dead rats with free thyroid hormone 3,3’,5-Triiodo-L-thyronine (T3) attenuated brain death-induced injury and apoptosis in the liver. In contrast to prior studies that mostly focused on the hemodynamic and anti-inflammatory effects of T3, our data suggests that T3 treatment induced a transient boost of oxidative stress that preconditioned the liver for subsequent brain death-related injuries. In Chapter 5, we investigated whether brain death changes metabolism in the donor, as the metabolic status of the liver and kidney has been correlated to transplantation outcomes. We used magnetic resonance imaging to study in vivo, repetitive organ perfusion (ASL) and oxygenation (BOLD), high-resolution respirometry to assess mitochondrial function, and proteomics and gene expression to assess metabolic pathways in the liver and kidneys of brain-dead rats. Our findings show that the liver increased metabolic demands by enhancing
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aerobic metabolism, facilitating the use of alternative energy sources with functional mitochondria. In contrast, the kidneys shifted towards anaerobic energy production and suffered from oxidative stress while renal perfusion decreased. These results strongly support an organ-specific approach in which the liver is supported metabolically whereas renal protection against hypoperfusion and oxidative stress is advised.
In Chapter 6, we showed how hyperpolarization magnetic resonance imaging (MRI) can be used as a novel tool to assess real-time, in vivo catabolism of MRI-active 1-13Cpyruvate
into its metabolites. We showed an initial increase in lacate production in the liver and kidney, likely indicative of organ hypoperfusion during the onset of brain death. Our data also showed a different metabolic profile in the liver versus the kidney, with a preference for the production of alanine in the case of the liver, yet lactate in the case of the kidney. Using a reperfusion model, we showed significantly lower glucose oxidation in the kidneys of brain-dead compared to sham animals. Together, this pilot study shows that hyperpolarization MRI and the measurement of glucose oxidation during ex vivo renal reperfusion are two promising techniques that can be used to visualize the metabolic profile of the liver and kidney during brain death and afterwards during kidney reperfusion.
In Chapter 7, we reviewed the current knowledge on autophagy and the role it plays in each of the steps of the transplantation process: in the donor, during organ preservation, and in the recipient. Secondly, this review touches upon the complex relationship between oxidative stress and autophagy and includes clinically used therapies that target each of these processes. In our opinion, balancing the potentially beneficial properties of autophagy and moderate levels of oxidative stress, yet detrimental effects when excessive activation of each of these processes occurs, are essential to achieve optimal transplantation outcomes. In Chapter 8, we identified autophagy as a novel pathway that is affected in the brain-dead donor. Using a brain death model in rats, we showed that autophagy was inhibited in the liver and to a lesser extent in the kidney following brain death. mTOR inhibition using rapamycin was unable to induce autophagy or attenuate brain death-induced apoptosis and plasma injury markers, suggesting that autophagy regulation occurs in an mTOR-independent manner.
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GENERAL DISCUSSION
The global shortage of donor organs leaves over 117.00 people in the United States and almost 15.000 people in Europe on the organ waiting list1. These numbers highlight the need
to increase the number of transplantable organs. One strategy to enlarge the donor organ pool is to improve the quality of suboptimal donor organs. However, in order to achieve this, we first must expand our understanding of what harms these organs prior to transplantation. Most organs worldwide are obtained from brain-dead donors2. Despite extensive efforts to
treat the pathophysiological effects of donor brain death, these grafts remain suboptimal when compared to living donor grafts3. The aim of this thesis was to expand on the current
knowledge of brain death pathophysiology and to use this knowledge to target brain death-induced injury. In the first part of this thesis, we provided an update on the current status of deceased donor management in the clinical setting and investigated the effects of two different interventions targeting brain death-induced inflammation and hormonal perturbances. In attempt to broaden our understanding of brain death pathophysiology, we then investigated two novel aspects of donor brain death: metabolism and autophagy. Our results portrayed a distinct, organ-specific metabolic response as well as autophagy dysregulation during brain death. Altogether, this thesis expands on the currently available knowledge and identifies novel pathways that can be targeted to improve brain death-induced injury.
Despite extensive preclinical and clinical studies, current Intensive Care protocols for brain-dead donor care vary per center and are based on low level-evidence4. This is reflected by our
systematic review in Chapter 2, which shows that most of the clinically tested compounds or interventions were unable to improve graft quality or survival rates after transplantation. Not only does this highlight the need for additional studies to solidify the few potentially promising interventions - including hypothermic donor cooling, dopamine treatment, and ischemic preconditioning - it also underlines the need to improve our understanding of the brain death condition and to search for new therapeutic targets. Part of the issue is that the working mechanism of several clinically used therapeutic interventions remains unclear. This is exemplified by the question whether hormone replacement therapy should be included in standard deceased donor care. Studies on thyroid replacement therapy have shown mixed results in both preclinical and clinical studies5-9. Nevertheless, the use of thyroid hormone
therapy in the management of brain-dead donors has steadily increased over the years5,10.
In light of this, we investigated the effects of free thyroid hormone triiodothyronine (T3) preconditioning in a brain death rodent model in an attempt to elucidate a possible working mechanism of this compound. In Chapters 4 and 7, we show that T3 preconditioning was beneficial in the liver but not the kidney following brain death. In the liver, T3 reduced hepatic injury as well as apoptotic cell death. In the kidney, no attenuation of injury markers
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or apoptosis was observed. Interestingly, most studies that have reported beneficial effects of thyroid hormone replacement have focused on hemodynamic outcome parameters. Our data on the other hand, suggests that these positive effects might be related to a preconditioning effect involving a (transient) rise in oxidative stress and autophagy. As was discussed in Chapter 7, oxidative stress and autophagy are two processes that are tightly connected: transient, low amounts of oxidative stress and autophagy can be beneficial, whereas excessive activation of either process can be detrimental. Our data in Chapter 4 supports this idea, as T3 induced low levels of oxidative stress as well as autophagy in the liver, suggesting that the reduction in apoptosis and injury markers was caused by a T3 -related autophagy induction. This idea is supported by Sinha et al. who have shown that T3 induced autophagy via ROS-related pathways in the liver both in vivo and ex vivo11. In
these studies, T3 stimulated mitochondrial ATP production, which subsequently increased ROS production and activated autophagy11,12. Interestingly, we observed no such effects in
the brain-dead kidney, where T3 was neither protective, nor induced autophagy. This lacking response in the kidneys might be explained by brain death-induced oxidative stress and its effect on autophagy. In Chapter 6, we showed that as brain death progressed, levels of oxidative stress increased while renal perfusion decreased13. When oxidative stress
levels increase, this may push the balance from a protective towards a detrimental role for autophagy, as was reviewed in Chapter 7. Altogether, even though the exact mechanism of action of T3 has not yet been elucidated, our data suggests that both oxidative stress and autophagy might play a key role.
The effects of donor brain death on autophagy had not previously been investigated. However, prior studies did show that autophagy becomes activated by a number of processes that are upregulated during brain death, including oxidative stress, apoptosis, and ATP depletion18-20. Therefore, it was surprising to observe in Chapter 8 that autophagy
was impaired following brain death, predominantly in the liver and to a lesser extent in the kidney. These results likely indicate defective autophagy regulation, as is also seen in cancer, cardiac disease, and aging21. Furthermore, as mTOR inhibition was unable to induce
autophagy, this suggests that autophagy regulation during brain death occurs via mTOR independent mechanisms. As mTOR normally serves as a sensor of cellular, energetic, and oxidative stress, a defect in the regulatory machinery of the mTOR complex or upstream machinery could hinder autophagy activation during brain death22. This might be part of
the explanation why we observed ATP and metabolite depletion in the liver (Chapter 5), as autophagy normally contributes to energy homeostasis in the face of stress and energy depletion. Further research into autophagy regulation, including mTOR-independent autophagy stimulators such as trehalose, is required to shed light on the role (beneficial vs. detrimental) of autophagy during brain death.
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Oxidative stress and autophagy are key influencers of organ quality during the transplantation process, with the first hit of injuries already occurring in the brain-dead donor. An increase in oxidative stress levels is of clinical importance as it correlates to transplantation outcomes: increased levels of oxidative stress marker MDA, both in the brain-dead donor as well as during machine perfusion, correlate with delayed graft function and acute rejection following kidney transplantation23,24. Oxidative stress is also tightly linked to metabolism.
Firstly, the mitochondria are considered major contributors to oxidative stress by means of increased radical oxygen species (ROS) production, as a positive correlation exists between the level of ROS production and the mitochondrial metabolic rate25. In the BD setting,
mitochondrial impairment had previously been observed in the hearts of brain-dead pigs26
and the muscle fibers of brain-dead, human subjects27. Furthermore, we showed in Chapter
5 that brain death resulted in ATP depletion in the liver and kidney as well as oxidative stress in the kidney, suggesting this might be related to mitochondrial dysfunction in the individual organs. Therefore, it was contrary to our expectations that brain death did not result in mitochondrial dysfunction in the liver and the kidney. Instead, the metabolic changes we observed were likely the results of decreased perfusion of the kidneys and increased metabolic demand in the liver (see Fig 1).
In the kidneys, renal perfusion decreased despite normal blood pressure levels, suggesting that our observations reflected changes in the renal microcirculation. Additionally, we observed in Chapter 6 that the acute hypoperfusion of the kidney as a result of brain death induction resulted in increased lactate production, likely reflecting a shift towards anaerobic metabolism (see Fig 1). Furthermore, we showed that following the brain death period, glucose oxidation in the kidney was significantly reduced during ex vivo reperfusion. These results suggest that the ischemic and metabolic stress during the induction phase of brain death may in part be responsible for the subsequent decreased perfusion, increased oxidative stress13, ATP depletion, and metabolic changes (Chapter
5). A similar situation is observed in models of acute kidney injury and sepsis, where an initial hypoxic hit results in microcirculatory failure and oxidative stress hours later28-31
Interestingly, the liver also responded in a similar manner to the onset of brain death, increasing lactate production. However, the liver did not suffer from subsequent oxidative stress and hypoperfusion, but instead responded to a higher energy demand by raising its metabolic capacity. During brain death, the liver increased oxidative glycolysis and facilitated a metabolic shift from carbohydrate to alternative metabolites with functional mitochondria. Furthermore, we show in Chapter 6 that the major metabolic pathways and enzymes remained intact in the brain-dead liver. Nonetheless, the reduction in ATP levels as well as metabolic changes during brain death we observed are of potential clinical importance, as prior studies indicate that the pre-transplant energy status of the liver is a predictor of graft survival and function after transplantation32,33. Together, our results
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underline the potential clinical impact that these brain death-related processes may have on the future organ grafts. Finally, the differences between the liver versus kidney suggest that optimization of graft quality requires an approach tailored to each organ individually.
Figure 1. Overview of metabolic changes in the liver and kidney following brain death. All changes depicted in green represents increased activity or levels, whereas all changes marked in red denote decreased activity or levels. Brain death resulted in decreased ATP levels in both the liver and kidney following brain death. In plasma, glucose levels decreased while fatty acid metabolites increased. In the liver, glycogen storage decreased and gene expression of of glycolysis-related gene Pfk-1 increased. Furthermore, O2 consumption in the liver increased as brain death progressed. In the kidney, anaerobic glycolysis-related gene Ldha increased, while gluconeogenesis-related Pck-1 decreased. In the kidneys, oxidative stress increased while perfusion decreased.
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FUTURE PERSPECTIVES
Despite differences in the response of the liver and kidney to donor brain death, the interconnectedness of autophagy, metabolism, and oxidative stress might allow targeting of multiple pathways with a single compound. Influencing the activity of these pathways, whether it be stimulation in case of low activity or attenuation in the case of overstimulation, can be used to treat or precondition the organs for transplantation-related injuries. However, this approach mandates a better understanding of how these pathways are regulated during brain death. Particularly in regard to autophagy, future studies on mTOR-independent autophagy stimulators will need to be performed to understand how autophagy is regulated during brain death. Our group has recently started pilot experiments to investigate autophagy stimulation using trehalose, a disaccharide with great potential stimulating autophagy in an mTOR-independent manner.
Besides studying autophagy in the donor, there are huge opportunities for improvement during organ preservation. As the extent of cold ischemia seems to predict whether autophagy is beneficial (in the case of shorter cold ischemia times) or detrimental (in the case of extended cold ischemia times) (as was reviewed in Chapter 7), measuring autophagy activation could potentially be used to predict organ quality prior to transplantation. However, as there is currently no literature on the effects of machine preservation on autophagy, research should first focus on elucidating effects of this technique on autophagy regulation. Following, future studies might then try to target autophagy and autophagy-related pathways as a way to treat or precondition the organ grafts.
The recent clinical implementation of ex vivo machine perfusion of the liver and kidney prior to transplantation has numerous benefits besides a mere reduction in cold ischemic injury. This new method of preservation also allows for targeted, organ-specific treatments. This is particularly of interest given the different responses of the liver versus the kidney during brain death. Our data suggests that treatment of the liver should focus on providing metabolic support, adequate oxygen availability and enough time to replenish energy stores, for example during preservation with machine perfusion. As the liver seems to adapt to brain death-induced injury by shifting metabolic gears, providing metabolic support should be considered already within the brain-dead donor. Conversely, the kidney becomes ischemically stressed and shuts down metabolically during brain death. As brain death progresses, the kidney becomes progressively hypoxic as evidenced by decreased perfusion and increased oxidative stress, despite normal systemic blood pressures. This suggests that renal protection should commence as soon as possible, preferably after brain death onset. Additionally, our data indicate that even though providing hemodynamic support to maintain normotension in the donor is important, it alone does not suffice. Additional protection against local microcirculatory changes is essential. This approach might include
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preconditioning the brain-dead donor with (remote) ischemic preconditioning techniques or treatments with vasodilators or anti-oxidants. Particularly the vasodilatory effects of dopamine might explain why this compound successfully improved renal function. Furthermore, ex vivo kidney perfusion techniques might be used to improve renal perfusion and reverse the metabolic status of the kidney.
Finally, combining machine perfusion with novel imaging techniques such as hyperpolarized MRI provides us with the opportunity to monitor processes such as metabolism or oxygenation in real-time using labelled MRI-active compounds such as pyruvate or oxygen. Another strategy might be to add labelled metabolites or nanoparticles to the perfusion medium in order to assess organ quality by measuring metabolism or visualizing gene expression of pro-survival or pro-death pathways. With these techniques, visualisation of the metabolic or oxidative status of an organ might allow us to predict the quality of an organ prior to transplantation. We have recently started studies in isolated rodent and pig kidneys to optimize the isolated perfused organ device in combination with (hyperpolarized) MRI techniques. When these models are set up, the first goal is to see if these techniques can be used to visualize differences in organ quality. The ultimate goal would then be to assess, target, and improve every available organ that becomes available and to use only those which use will result in good outcomes after transplantation. Particularly processes such as metabolism, autophagy, and oxidative stress lend themselves well to evaluate and target graft quality prior to transplantation.
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