Investigating the role of pharmacological preconditioning of organs from brain dead donors to improve the outcomes of kidney transplantation

Medical context of proposed research and its importance:
Organ transplantation saves the lives of thousands of patients every year. Most types of organ transplantation including liver, kidney, pancreas, heart, lung and even bowel transplants are now recognised as definitive treatments for patients with end stage organ failure. Despite the increasing awareness of transplantation both amongst medical professionals and members of the general public, a significant gulf still exists between organ availability and need. This is predicted to worsen over the course of the next decade making this disparity one of the biggest challenges facing the transplant community today. In an attempt to reconcile this difference researchers have been exploring how the "donor pool" can be expanded and how organs previously considered unsuitable for transplantation can be repaired or resuscitated.

Kidneys donated from brain dead donors have poorer short and long-term outcomes when compared to living donors, even when immunological factors and storage times are taken into consideration. Part of the reason for the poorer function of these organs is because of a reduction in the blood, oxygen and nutrient supply during the brain death process itself. This leads to toxic metabolite production which, when the blood supply is restored, results in dramatic tissue injury. In addition, the immune system appears to be overactive. I speculate that interventions made in the brain dead donor can help protect organs against damage. In doing so, I propose that interventions will make previously unusable organs transplantable, and also reduce the incidence of acute and chronic organ failure.

It is recognised that a short period of deprivation of blood supply (ischaemia) to an organ can prevent against future damage from prolonged ischaemic periods. The mechanism for this protection has not been fully delineated, however, Hypoxia Inducible Factor (HIF) and the genes that it regulates have been suggested as being responsible for some of the conferred protection. The HIF pathway is part of the cellular response mechanism to oxygen deprivation. HIF can be pharmacologically induced by administering dimethyloxaylglycine (DMOG). To date no published scientific research has investigated the role of the HIF pathway in preventing kidney injury following brain death and improving the outcomes of transplantation.

Goals of project and methods:
I propose to carry out experiments to explore the role of the HIF pathway in protecting against kidney injury following brain death. I will use a rat model I have developed during my Academic Clinical Fellowship. I will activate and sustain the HIF pathway using DMOG. I will be assessing the kidneys procured from brain dead rats treated with this agent using techniques including a rat model of renal transplantation.

By conducting this research I aim to establish the ability of DMOG to abrogate the kidney injury caused by brain death, and improve our understanding of the mechanisms of brain death induced kidney injury. In doing so, I aim to protect kidneys from brain dead organ donors, improving the quality of kidneys procured, but also salvaging previously unusable kidneys and thereby making the untransplantable transplantable.

Aims and objectives:
Exp1: To determine the effects of brain death (BD) on HIF and HIF downstream effector production in the donor kidney.
Exp2: To evaluate whether dimethyloxaylglycine (DMOG) administration can up-regulate the production of HIF downstream effectors in the BD setting. Establishing the optimal dose of DMOG (Exp 2.1) and time point for administration (Exp 2.2) in the BD rat. The pharmacodynamics of the agent will be further evaluated (Exp 2.3).
Exp3: To determine whether DMOG administration to the BD donor improves the outcomes of isogeneic rat renal transplantation?

Methodology:
Surgical preparation: BD is induced in anaesthetised adult male Fischer rats (250-300g) using slow inflation of an epidural catheter. After confirmation of BD, anaesthesia is terminated but ventilation continued. 4 hours after BD induction samples including kidney biopsies are obtained. In Exp 2 left kidneys will be explanted for evaluation on an isolated kidney perfusion apparatus.

In Exp 3 isogeneic renal transplantation will be performed on bilaterally nephrectomised rats using kidneys from BD rats after cold storage (20 hours). Blood samples will be taken on post-operative days 0, 3, 7 and 14. Transplant nephrectomy will be performed on day 14.

Drugs: DMOG (40/200mg/kg i.v.) will be administered prior to BD induction (Exp 2.1). Further experiments will elucidate the optimal time point for administration (30min prior to BD induction, post BD induction: 30, 60 and 120 min after BD induction, Exp 2.2) and the pharmacodynamics of DMOG in the BD setting (Exp 2.3).

Primary outcome measures: Exp 1 and Exp 2 HIF target genes (rtPCR: HO-1, AngPTL4, Glut-4, VEGF, EPO, GLUT1, NFkB, MAPK, E-selectin, VCAM). Exp 3 Day 3 post operative creatinine

Scientific and medical opportunities:
This project aims to increase our understanding of the role of the HIF pathway in protecting against kidney injury following BD and the effect of HIF up-regulation using DMOG.

Fore-mostly we hope that the proposed research will lead to improving the quality of organs and make previously unusable organs transplantable. The discard rate of organs is particularly high from extended criteria donors and we envisage the proposed donor interventions will have the greatest impact on these donors. Although we will be mainly exploring the benefit of these agents in the realms of kidney transplantation we hope the proposed research will impact on all transplantation sub-specialties.

Our goal is to ultimately improve the quality of organs procured for transplantation, which will lead to fewer episodes of primary non-function and acute rejection and chronic transplant dysfunction. Not only will this be life-saving in itself, but will also reduce the re-transplantation rate which will have obvious benefits for patients including fewer operations and less exposure to immunological sensitising events. As organ function may also be improved, this would mean less need for additional therapies such as post-operative dialysis. Overall this would result in fewer in-patient hospital days and have significant cost benefits for the NHS.

By providing further understanding of the molecular and physiological processes behind brain death induced organ injury, we will contribute to the research literature on this important topic.

If the proposed therapies are shown to be of benefit, pharmaceutical companies will also benefit. For communities this will result in increased manufacturing opportunities and act as a stimulus for the local, national and possibly even global economy.

For the country, as a whole, the research if successful will undoubtedly spark debate as to what is acceptable in the realms of donor management. We aim to provide informed evidence for the advantages of intervening in the donor and will be liaising with the UK Donation Ethics Committee throughout the course of the Fellowship.

Finally, the proposed research Fellowship will have a significant impact on my future career aspirations to become a transplant surgeon-scientist. The skills I particularly hope to acquire during the Fellowship include bench-side laboratory skills, the ability to understand and apply statistics and an opportunity to improve on both my verbal and written communication. I aim to learn more about the important aspects of developing clinical studies, as background to anticipated post-doctoral work as a Clinical Lecturer. This includes ethics applications, trial design and methods of data collection and analysis.