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Targeting Mitochondrial ROS Production in Kidney Transplantation 10.17863/CAM.52095 Beach, Timothy Elliott Saeb-Parsy, Kourosh Ischaemia reperfusion injury (IRI) is an inevitable consequence of transplant practices but is associated with reduced levels of graft function and survival. In addition, concerns regarding the severity of IRI has restricted the greater use of organs from the available donor pool. Critically, no pharmacological therapies currently exist to ameliorate the effects of IRI in organ transplantation (or other IRI-related pathologies), partly due to an incomplete understanding of the underlying pathophysiology. Recently, a specific mechanism of mitochondrial reactive oxygen species (ROS) production, thought to initiate many of the downstream pathways resulting in IRI, has been described. This mechanism has identified a number of new therapeutic targets within mitochondria, including the respiratory complex succinate dehydrogenase (SDH). The aim of this thesis was to determine whether malonate ester prodrugs, which competitively inhibit SDH, may reduce mitochondrial ROS production and ameliorate IRI in models of kidney transplantation. Herein, I show that the metabolic changes required for mitochondrial ROS production on reperfusion, including succinate accumulation and the depletion of adenine nucleotides, occur in grafts retrieved from both DBD and DCD donors, despite differences in their exposure to warm ischaemia. This may partly relate to difficulties in efficiently cooling organs and suggests grafts from both donor types may benefit from therapies aimed at reducing mitochondrial ROS production. I describe a translational model of kidney transplantation in the pig and human as well as a model of renal IRI in the mouse. I show the mitochondrial ROS probe, MitoB, may be limited in its ability to accurately quantify the burst of mitochondrial ROS production that occurs during IRI in the kidney; however mitochondrial ROS production may instead be inferred indirectly in mouse, pig and human models by comparing the metabolic changes that occur on reperfusion to those previously described to drive mitochondrial ROS production in vitro. In addition, I identify key markers of oxidative damage, cell death and kidney function in the mouse, pig and human and subsequently show malonate ester prodrugs administered at reperfusion (but not prior to ischaemia) may reduce IRI in the mouse. Finally, I present pilot data in the pig providing important dosing and timing information for the use of malonate ester prodrugs in this model. Further work is needed to determine whether malonate ester prodrugs may inhibit mitochondrial ROS production in kidney transplantation; however, this thesis has provided important inroads into the use of these compounds in a transplant setting as well as characterising a number of translational models that may pave the way to their use in future clinical trials. University of Cambridge 2020-04-28 Thesis Doctoral Doctor of Philosophy (PhD) en https://www.repository.cam.ac.uk/handle/1810/305014 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ea55c7a5-00bd-4ece-b4fc-ff559291e91c/download 783cae58640462a7f0cb8fc6eb27e9f8 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/71ee34c0-2ec1-4951-a5b4-59380bc6311b/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Ischaemia reperfusion injury Transplantation Succinate Mitochondria Kidney Malonate reactive oxygen species