Abstract
Chronic kidney disease is a highly prevalent condition and renal transplantation is the definitive treatment for those with end stage renal failure. This life saving treatment is unfortunately hindered by a shortage of donor organs. In an attempt to boost the donor pool, the use of kidneys from Non Heart Beating Donors has become increasingly common, with Hypothermic Pulsatile Perfusion (HPP) of the donor kidney being employed in an attempt to improve outcomes from these extended criteria donor kidneys.
HPP provides a theoretical opportunity for the pharmaco-modulation of donor kidneys ex vivo providing a “therapeutic window” whereby bioactives may be added to perfusates with the goal of maximising post transplantation outcomes. A candidate molecule is carbon monoxide (CO) that has been demonstrated to be vasodilatory and anti-apoptotic. However, little is known about the vascular function of kidneys during hypothermia and accordingly no data exist as to the effect that the administration of CO during HPP to donor kidneys has upon post transplant outcomes.
Using an ex vivo isolated perfused rat kidney model this thesis provides robust data regarding renal vascular function during hypothermia; demonstrating that vasomotor responses to constrictors and dilators are preserved in addition to providing confirmation that autoregulation is present during HPP.
In addition, the potential toxicity of CO to renal tubule cells (administered as carbon monoxide releasing molecule-2 (CORM-2) or CO gas) is investigated. Results demonstrate that despite protecting against apoptosis, CORM-2 is toxic to renal tubule cells independent of its solvent and most likely secondary to its ruthenium based parent molecule. As a consequence of these novel findings, CO gas was used in subsequent experiments that investigated the impact that CO administration during HPP has on outcomes following transplant Ischemia-Reperfusion Injury (IRI).
Using a clinically applicable rat model of transplant IRI, it is demonstrated that persuflation of HPP solution with 0.001% CO gas provided protection against transplant IRI secondary to the anti-apoptotic and vaso-active properties of CO. Renal tubule damage and parenchymal oedema were diminished following CO treatment. Autoregulation was found to be absent in kidneys that had been subjected to the IRI protocol and CO administration did not improve this. The absence of any significant impact on mitochondrial energetics following CO administration in conjunction with an absence of increased ROS generation suggests CO is not toxic at the level of the cellular respiratory chain. Furthermore the impact that CO administration has appears to be independent of endogenous heme oxygenase-1 activity.
In summary the results of the experiments detailed in this thesis support the hypothesis that the ex vivo application of CO gas during HPP provides protection against transplant IRI. These data provides the first step in a “bench to bedside” approach supporting the use of CO gas during HPP to minimise the deleterious impact of IRI. The current study offers a “proof of concept” providing a sound scientific basis for further research designed to investigate the impact CO administration (during HPP) has on clinical transplant outcomes in an in vivo large animal model.