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Kidney transplantation is the only therapy for patients with end-stage kidney failure. There is a shortage of suitable organs from living donors and heart-beating donors (HBDs) leading to an increased use of marginal organs, including those from non- heart-beating donors (NHBD) to expand the donor pool. However, such kidneys are exposed to periods of warm and cold ischaemia plus reperfusion (IR) injury which can be associated with acute tubular necrosis leading to primary non function (PNF), delayed graft failure (DGF) with an increased risk of acute and chronic allograft rejection. Therefore, minimising ischaemic injury will be beneficial to improve immediate allograft function and reduce the incidence of PNF and DGF. Perfusate solutions can ameliorate the damage caused by IR injury. This study examined the possible protective effect of heparinoids, low molecular weight (LMW) sodium heparin (HS-3400) and sodium pentosan polysulfate (NaP PS), as supplements in perfusate solutions. An in vivo rodent surgical model of NHB kidney donation was used to investigate the effects of heparinoids as immune modulators during kidney preservation. It was demonstrated that heparinoids significantly reduced oedema but did not have any effects on pressure, intra-renal vascular resistance (I RVR) or osmolarity of the perfusate solution. Microarray and qPCR analysis revealed that many genes involved in IR injury were differentially expressed after warm oxygenated perfusion. Network analysis revealed that the pro-inflammatory cytokine TNF-a was the common denominator and a potential key regulator of IR injury. Data obtained strongly demonstrate that these networks were removed by the addition of heparinoids. In vitro biological assays investigated the effects of heparinoids on class 11 MHC antigen expression on primary endothelial cells (ECs) treated with IFN-y. It was found that NaPPS and the majority of its derivatives were more efficient in antagonising IFN-y activity. In addition, NaPPS and its derivatives significantly inhibited transendothelial migration of leukocytes across an IFN-y-activated endothelial monolayer. The anti-inflammatory properties of heparinoids can be exploited for better preservation of NHBD organs to improve short and long-term allograft survival and function. Significantly, this is the first study to reveal the potential of heparinoids as TNF-a antagonists and additives to perfusate solutions.

The calcineurin inhibitors, cyclosporine and tacrolimus, are pivotal immunosuppressants in renal transplantation, but produce a cascade of acute (functional) and chronic (fibrotic) injurious events that contribute to chronic allograft nephropathy (CAN). Using the rat salt depletion model of calcineurin inhibitor toxicity, this study aimed to examine the effects of clinically relevant combinations of cyclosporine, tacrolimus and sirolimus on renal functional, structural and molecular markers of injury. Further, the effect of pirfenidone when added to these drug combinations was examined. There were differences in the effects of cyclosporine and tacrolimus on functional and molecular variables, with tacrolimus displaying more favourable results. As sole therapy, sirolimus had no effect on renal function or messenger RNA expression. Deterioration in renal function and a deleterious effect on molecular markers of fibrosis were seen when cyclosporine and sirolimus were combined at high doses; at lower doses, favourable outcomes for these end-points were elicited. When sirolimus and tacrolimus were combined, renal function worsened and the beneficial molecular effects of tacrolimus were reversed. Pirfenidone's actions were non-dose dependent and beneficial effects for renal function and molecular markers were demonstrated. The effect of pirfenidone on renal function has not previously been described. There were no differences in urinary protein or interstitial fibrosis measurements across the groups. Without fibrosis, it is impossible to say whether pirfenidone acted in a truly antifibrotic manner. However, the molecular changes possibly represent interim markers of fibrosis, suggesting such an effect may have been developing.

Aims of the study: 1. To assess the use of machine perfusion in predicting the severity of injury in porcine kidneys subjected to warm ischaemic damage. 2. To assess the use of machine perfusion in predicting the severity of injury in porcine kidneys subjected to warm plus cold ischaemic damage. 3. To assess the potential therapeutic effect of nitric oxide donors and nitric oxide synthase inhibitors on porcine kidneys subjected to prolonged periods of warm plus cold ischaemia. Results: In study 1, a significant relationship between the length of WIT and IRR was found. During pulsatile MP, the IRR reduced in all WIT groups. Nitrate concentration and eNOS expression tended to become higher as the WIT increased. In study 2, the kidneys subjected to the shorter WIT demonstrated a significant linear relationship between the CIT and the IRR. This relationship was not found in the kidneys subjected to a longer WIT. As the CIT increased the nitrate concentration tended to decrease, however the eNOS expression decreased. In study 3, manipulation of the NOS pathway with NO donors and NOS inhibitors did not significantly affect the IRR or eNOS expression. Conclusions: Of the indices measured, IRR had the closest and most convincing relationship with WIT. It would seem possible that this could be used as a viability assessment measure. The fact that in all studies IRR decreased over the six hours of pulsatile MP provides positive evidence of the potential benefit of MP in minimising the deleterious effects of warm and cold ischaemia. The relationship between CIT and IRR was not so clear, but certainly when the kidneys investigated were relatively ischaemically undamaged there was a linear relationship between the two. This study did not find any evidence of an association between ischaemic damage and the NO pathway.

Background: The shortage of kidneys for renal transplantation has prompted renewed interest in non-heart-beating donors (NHBD). While this may increase the number of transplants, it also increases the primary non-function (PNF) rate. This is caused by excessive warm ischaemic injury in some NHBD, and has hindered their more widespread use. A reliable pre-transplant test of organ viability, and a preservation method minimising additional iscahemic damage, would allow the PNF rate to be reduced. The aims of this thesis were to explore the potential of warm ex-vivo perfusion as a preservation method and a means of diagnosing viability pre-transplantation. Methods: Warm ex-vivo perfusion of ischaemically injured porcine kidneys with an oxygenated emulsion of a perflourochemical in tissue culture fluid was used to measure ex-vivo function and preserve kidneys. A cadaveric model was used to assess the relationship of ex-vivo function and warm ischaemic time. An autotransplant model was used to determine the relationship of ex-vivo to post-transplant function, and to compare the efficacy of preservation by warm perfusion with conventional hypothermic techniques of static storage and pulsatile perfusion. Post-transplant outcome measures were survival, renal function and histology. Results: WIT correlated well with ex-vivo function. Ex-vivo function correlated with post-transplant function in terms of survival (and therefore the immediate life supporting function of the kidneys), but not to the extent that it could be used to predict viability better than knowing the WIT alone. The efficacy of warm perfusion was indistinguishable from hypothermic static storage. However hypothermic pulsatile perfusion was slightly superior to both other techniques. Conclusions: Warm perfusion as used in this thesis was broadly equivalent in efficacy to conventional hypothermic organ preservation techniques. Although ex-vivo function correlated with post-transplant function, the correlation was not tight enough to support a diagnostic role for ex-vivo function in viability determination.