Endotoxins or lipopolysaccharides (LPS), highly toxic component of the outer membrane of gram-negative bacteria, are normally present in the mammalian gut lumen.In this thesis, I investigated, in laboratory animals, whether these gut-derived endotoxins play a role in pathophysiology resulting from intestinal dysfunctions caused by intestinal ischaemia and heat-stress.In primates, reperfusion of the splanchnic region after a temporary ischaemia was followed by a rapid increase in LPS concentration, first in the hepatic portal plasma and, ten minutes later, in the systemic arterial plasma. Rises in plasma LPS concentrations during or following the temporary intestinal ischaemia was prevented by prophylactic administrations of corticosteroids, anti-LPS IgG antibodies and oral, non-absorpable, antibiotics agents which appear to stabilize cellular membranes, aid the reticuloendothelial system in removal of LPS from the circulation and destroy the intestinal aerobic gramnegative bacteria respectively. In addition, administration of therapeutic anti-LPS antibodies also rapidly reduced the plasma LPS concentrations to baseline during an endotoxaemia. In a control heat-stress model, elevations in plasma LPS concentration commenced at rectal temperatures greater than 41,SoC. Like the intestinal ischaemia model, this occurred first in the hepatic portal plasma, and 10-15 minutes later, in the systemic arterial plasma. Peak plasma LPS levels of about 0,3 ng/ml, measured in heat-stressed primates, have proved in previous studies, to be toxic. A rapid decline in mean arterial pressure was followed by increases in plasma LPS concentrations and heart rates. Reductions in splanchnic blood flow and consequent local ischaemia coupled with thermal injury to the intestinal wall and the liver, may have permitted rises in plasma LPS concentration. Furthermore, as in the ischaemia model, prophylactic administrations of corticosteroids, anti-LPS IgG antibodies, and oral, nonabsorbable antibiotics prevented a rise in plasma LPS concentration. Of importance, prophylaxis with intravenous corticosteroids and 'anti-LPS IgG antibodies increased the survival rates significantly in heat stroke in primates. In addition, monkeys having high titres of "natural" antiLPS IgG antibodies had lower plasma LPS concentrations and survived the induced-heat stroke. It is suggested that other pathophysiologic conditions which compromise the integrity of the gut wall would also lead to the development of an endotoxaemia, and that gutderived endotoxins contribute to the athogenesis of heat stroke and treatments with corticosteroids and anti-LPS IgG antibodies may prove beneficial in other endotoxinrelated disorders. / Thesis (Ph.D.)-University of Natal, Durban, 1988.

The phytocannabinoid cannabidiol (CBD) has a complex pharmacology which is thought to include, but is not limited to, an ability to act as an inverse agonist at the CB1 and CB2 receptors and an antagonist of GPR55. Moreover, is has been shown to reduce infarct size and ameliorate reductions in left ventricular function in vivo. These improvements in the pathogenesis of experimental MI are accompanied by a reduction in inflammatory cell migration to the area at risk. More recently it has been shown that CBD is anti-arrhythmic in acute experimental MI. Thus, it was suggested that the cardioprotective effects of CBD might be due to an anti-inflammatory action. In addition, GPR55 receptor activation is acknowledged to mediate mobilisation of intracellular Ca2+ (Ca2+i) which could potentially be pro-arrhythmic and so CBD, as an antagonist may confer cardioprotection via GPR55. However, the receptors and/or mechanisms responsible for mediating the cardioprotective effects of CBD are get to be determined. The present studies were therefore performed to; (1) better understand the pharmacology of CBD by assessing haemodynamic responses to CBD and other cannabinoids ligands in anaesthetised rats, (2) investigate the receptors involved in the anti-arrhythmic effect of CBD in a rat model of coronary artery occlusion (CAO), and (3) investigate if CBD can alter [Ca2+]i in isolated rat cardiomyocytes. The characterisation of the pharmacology of CBD in vivo showed that; firstly, CB1 receptor activation causes a hypotensive response which can be dose-dependently inhibited by AM251; secondly, both CBD and AM251 alone (a CB1 receptor antagonist and GPR55 agonist) can induce vasodepressor responses and finally, CBD can potentiate the AM251-mediated hypotension when co-administered, suggesting possible cross-talk between the CB1 and GPR55. Results from CAO studies showed that CBD and AM251 each have the capacity to reduce arrhythmias. Moreover, when CBD and AM251 were co-administered the anti-arrhythmic capacity of either alone was potentiated. However, the degree of potentiation was dependent on the order of administration, suggesting that more than one receptor is involved in the summative anti-arrhythmic effects. The investigation of cardiomyocyte [Ca2+]i suggested that AM251 can modulate [Ca2+]i at the level of the cardiomyocyte, while CBD cannot. These data give novel insight into the anti-arrhythmic effects of CBD and, moreover, for the first time demonstrate that AM251 is anti-arrhythmic. In addition, these data suggest a role for GPR55 in increasing [Ca2+]i via AM251.

Acute kidney injury (AKI) induced by renal ischaemia reperfusion injury (IRI) is characterised by renal failure, acute tubular necrosis (ATN), inflammation and microvascular congestion. Apoptotic cell administration reduces inflammation in experimental models of acute inflammation in the lung, joints and peritoneum. Preliminary data suggested that administration of 20x106 apoptotic thymocytes to mice 24-hours prior to renal IRI ameliorated renal function without affecting ATN 24-hours following IRI. This thesis attempted to validate these finding and explore underlying hypothetical mechanisms. These studies examined if functional protection was conferred by apoptotic cell modulation of (a) circulating IgM antibodies or (b) coagulation status leading to improved intrarenal microvascular blood flow. Pathogenic IgM antibodies bind ischaemic cardiac or skeletal muscle and the intestine leading to complement activation and worse injury. We examined IgM binding to human renal (HK-2) cells by flow cytometry and to ischaemic murine kidney tissue. H2O2 or Antimycin A treated HK-2 cells incubated with human serum (IgM source) exhibited no IgM binding. Medullary IgM deposition assessed by immunofluorescence was minimal following IRI. We also assessed IgM deposition by immunohistochemistry following hepatic IRI and discovered dramatic deposition. These data suggest that IgM antibodies exhibit differential binding to injured tissues and are not directly involved in renal IRI, but may have a role in hepatic IRI. To support our second hypothesis we studied apoptotic cell modulation of coagulation. A thrombin generation assay revealed that early apoptotic cell-treated mice exhibited delayed thrombin generation. Furthermore, in vitro studies confirmed direct apoptotic cell-platelet binding. To replicate apoptotic cell derived functional protection Balb/c mice underwent 20, 24 or 25-minutes of ischaemia to induce mild, moderate or severe kidney dysfunction. Renal function and injury was determined 24-hours following IRI by plasma creatinine measurement and ATN scoring. Unexpectedly, intravenous pretreatment of mice with apoptotic thymocytes conferred no protection. Indeed, apoptotic thymocytes further impaired renal function depending upon injury severity. Impairment of renal function was not secondary to increased microvascular congestion, inferred by fibrin and platelet deposition, neither increased ATN nor inflammation, assessed by neutrophil infiltration. These data indicate that apoptotic cell administration does not protect from subsequent renal IRI and that apoptotic cells are thus not inherently anti-inflammatory in all models of acute inflammation. Unable to replicate apoptotic cell derived functional protection we explored the binding of IgM antibodies to apoptotic cells which acts to facilitate dead cell clearance. We characterised IgM binding to non-apoptotic and apoptotic murine thymocytes and human Jurkat cells using flow cytometry, confocal and electron microscopy. We demonstrated specific IgM binding to a subset of late apoptotic cells. Electron microscopy indicated that IgM+ apoptotic cells exhibited marked plasma membrane disruption, suggesting that access to intracellular epitopes was required for IgM binding. Binding of IgM to permeabilised non-apoptotic and apoptotic cells suggested that IgM bound epitopes are ‘apoptosis independent’ such that IgM may bind any cell with profound plasma membrane disruption. Interestingly, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognise and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption.

Inflammation in the absence of infection (sterile inflammation) is a crucial host defence response to tissue injury, but is also considered to contribute to the pathogenesis of many diverse disease states, including stroke. Sterile inflammation is initiated by damage associated molecular patterns (DAMPs) which are endogenous molecules released from necrotic cells or that are modified during disease. The pro-inflammatory cytokines IL-1α and IL-1β are key mediators of inflammation. IL-1β release is controlled by caspase-1 which, in turn, is regulated by the inflammasome. The NOD-, LRR-, pyrin domain-containing 3 (NLRP3) inflammasome is most typically associated with sterile inflammation and the recognition of DAMPs. Thus, understanding the mechanisms of NLRP3-activating DAMP-induced inflammation may lead to the identification of novel therapeutic targets with which to treat inflammatory diseases. This thesis sought to determine how NLRP3-activating DAMPs affect the pro-inflammatory response of glia, the immune cells of the brain. Experimental models in vitro typically use a pathogen associated molecular pattern (PAMP) such as LPS to prime cells before observing their response to NLRP3-activating DAMPs. As the brain is protected by the blood brain barrier (BBB), it is unlikely glia would be exposed to PAMP priming. However it remains unclear as to how glia respond to NLRP3-activating DAMPs in the absence of priming, or what the source of endogenous priming is. Therefore, the initial hypothesis was to investigate the pro-inflammatory response of mixed glia in vitro to NLRP3-activating DAMPs in the absence of PAMP priming. It is shown here for the first time that NLRP3-activating DAMPs can initiate an IL-1-NLRP3-independent inflammatory response in mixed glia in the absence of PAMP priming. Moreover, it is shown that the acute phase protein serum amyloid A is elevated in plasma after stroke and may act as an endogenous priming signal to allow IL-1β-dependent inflammation to contribute to the damage after breakdown of the BBB.Inflammation following acute sterile injury such as stroke is augmented by persisting cell death. It was therefore hypothesised that NLRP3-activating DAMPs released after the initial injury, may initiate a form of programmed cell death that continues to drive inflammation. Using inhibitors of specific types of cell death, it was identified that NLRP3-activating DAMP induced cell death is likely to be necrosis and not programmed cell death. Further investigation into the biological importance of DAMP-induced IL-1-independent inflammation and the specific contribution of acute phase proteins to brain pathology may aid the identification of new therapeutic targets.

Electrical excitation of the dorsal aspect of the rostral thoracic spinal cord imparts long-term therapeutic benefits to patients with angina pectoris. Such spinal cord stimulation also induces short-term suppressor effects on the intrinsic cardiac nervous system. The purpose of this study was to determine whether spinal cord stimulation (SCS) induces long-term effects on the intrinsic nervous system, particularly in the presence of myocardial ischaemia. The activity generated by right atrial neurons was recorded in 10 anesthetized dogs during basal states, during prolonged (15 min) occlusion of the left anterior descending coronary artery, and during the subsequent reperfusion phase. Neuronal activity and cardiovascular indices were also monitored when the dorsal T1-T4 segments of the spinal cord were stimulated electrically (50 Hz; 0.2 ms) at an intensity 90% of motor threshold (mean 0.32 mA) for 17 min. SCS was performed before, during and after 15-min periods of regional ventricular ischaemia. Occlusion of a major coronary artery, one that did not perfuse investigated neurons, resulted in their excitation. Ischaemia-induced neuronal excitatory effects were suppressed (-76% from baseline) by SCS. SCS suppression of intrinsic cardiac neuronal activity persisted during the subsequent reperfusion period; after terminating 17 min of SCS, at least 20 min elapsed before intrinsic cardiac neuronal activity returned to baseline values. It is concluded that populations of intrinsic cardiac neurons are activated by inputs arising from the ischaemic myocardium. Ischaemia-induced activation of these neurons is nullified by SCS. The neuronal suppressor effects that SCS induces persist not only during reperfusion, but also for an extended period of time thereafter. These long-term effects may account, in part, for the fact that SCS imparts clinical benefit to patients with angina of cardiac origin not only during its application, but also for a time thereafter.

Activation of A3 adenosine receptors has been shown to protect the myocardium from ischaemia reperfusion injury in a number of animal models. The PI3K - AKT and MEK1/2 - ERK1/2 cell survival pathways have been shown to play a critical role in regulating myocardial ischaemia reperfusion injury. In this study we investigated whether the A3 adenosine receptor agonist 2-CL-IB-MECA protects the myocardium from ischaemia reperfusion injury, when administered at reperfusion or post reperfusion and whether the protection involved the PI3K – AKT or MEK 1/2 – ERK1/2 cell survival pathways. In the Langendorff model of ischaemia reperfusion injury isolated perfused rat hearts underwent 35 minutes of ischaemia and 120 minutes of reperfusion. Administration of 2-CL-IB-MECA (1nM) at reperfusion significantly decreased infarct size to risk ratio compared to non-treated ischeamic reperfused control hearts. This protection was abolished in the presence of the PI3K inhibitor Wortmannin or MEK1/2 inhibitor UO126. Western blot analysis determined that administration of 2-CL-IB-MECA (1 nM) upregulated ERK1/2 phosphorylation. In the adult rat cardiac myocyte model of hypoxia/reoxygenation cells underwent 6 hours of hypoxia and 18 hours of reoxygenation. Administration of 2-CL-IB-MECA (1 nM) at the onset of reoxygenation significantly decreased cellular apoptosis and necrosis. Administration of 2-CL-IB-MECA (1nM) in the presence of the Wortmannin or UO126 significantly reversed this anti-apoptotic effect and anti-necrotic effect. Our data further showed that 2-CL-IB-MECA protects myocytes subjected to hypoxia/reoxygenation injury via decreasing cleaved-caspase 3 activity that was abolished in presence of the PI3K inhibitor but not in the presence of the MEK1/2 inhibitor UO126. Administration of 2-CL-IB-MECA (100nM) at the onset of reperfusion also significantly decreased infarct size to risk ratio in the ischaemic reperfused rat heart compared to controls that was reversed in the presence of Wortmannin or Rapamycin. This protection was associated with an increase in PI3K-AKT / p70S6K / BAD phosphorylation. 2-CL-IB-MECA (100nM) administered at reoxygenation also significantly protected adult rat cardiac myocytes from hypoxia/reoxygenation injury 28 in an anti-apoptotic and anti-necrotic manner. This anti-apoptotic/necrotic effect of 2-CL-IB-MECA was abolished in the presence Wortmannin. Furthermore, that this protection afforded by 2-CL-IB-MECA (100nM) when administered at reoxygenation was associated with a decrease in cleaved caspase 3 activity that was abolished in the presence of the Wortmannin Interestingly, postponing the administration of 2-CL-IB-MECA to 15 or 30 minutes after the onset of reperfusion significantly protected the isolated perfused rat heart from ischaemia reperfusion injury in a Wortmannin and UO126 sensitive manner. This protection was associated with an increase in AKT and ERK1/2 phosphorylation. Administration of the A3 agonist 2-CL-IB-MECA 15 or 30 minutes after the onset of reoxygenation significantly protected isolated adult rat cardiac myocytes subjected to 6 hours of hypoxia and 18 hours of reoxygenation from injury in an anti-apoptotic/necrotic manner. This anti-apoptotic was abolished upon PI3K inhibition with Wortmannin or MEK1/2 inhibition with UO126. The anti-necrotic effect of 2-CL-IB-MECA when administered 15 or 30 minutes post-reperfusion was not abolished in the presence of the inhibitors. Delaying the administration of 2-CL-IB-MECA to 15 or 30 minutes after reoxygenation was associated with a decrease in cleaved-caspase 3 activity that was abolished in the presence of Wortmannin but not in the presence of the MEK 1/2inhibitor UO126. Collectively, we have demonstrated for the first time that administration of 2-CL-IB-MECA at the onset of reperfusion protects the ischaemic reperfused rat myocardium from lethal ischaemia reperfusion injury in a PI3K and MEK1/2 sensitive manner. Delaying the administration of 2-CL-IB-MECA to 15 or 30 minutes after the onset of reperfusion of reoxygenation also significantly protects the isolated perfused rat heart from ischaemia reperfusion injury and the adult rat cardiac myocyte from hypoxia/reoxygenation injury in an anti apoptotic / necrotic manner. Furthermore, that this protection is associated with recruitment of the PI3K-AKT and MEK1/2 – ERK1/2 cell survival pathways.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: It is well-established that transient activation of the β-adrenergic signalling pathway with ligands such as isoproterenol, formoterol and dobutamine, elicits cardioprotection against subsequent long periods of ischaemia. Initially the focus was on the β1- and β2-adrenergic receptors (β1-AR, β2-AR), but recently the β3-AR also emerged as a potential target in the treatment of heart disease. In heart failure, β1- and β2-AR are typically known to be down-regulated while β3-ARs, on the other hand, are up-regulated (Moniotte et al., 2001). Thus, it has become important to examine the significance of the β3-AR and its downstream signalling under similar states of stress. It has been shown that β3-AR stimulation is resistant to short term agonist-promoted desensitization in vitro and in vivo (Liggett et al., 1993) and after being activated, this receptor is able to convey continual intracellular signals (Lafontan et al., 1994). Thus, it could be an ideal target for therapeutic intervention, also in ischaemic heart disease. We hypothesized that selective β3-AR stimulation during ischaemia / reperfusion may be cardioprotective, whereas selective inhibition of this receptor may prove useful in the end stages of sustained ischaemia and early reperfusion. Methods: The isolated working rat heart, subjected to 35 min of regional ischaemia (RI) and 60 min reperfusion was used as model. The β3-AR agonist (BRL37344) (1 μM) or antagonist (SR59230A) (0.1 μM) were applied as follows: (i) before 35 min RI (PT), (ii) during the last 10 min of RI (PerT) and /or (iii) at the onset of reperfusion (PostT) and (iv) administration of BRL37344 during the last 10 min of RI BRL37344 (PerT) was followed by SR59230A during first 10 min of reperfusion SR59230A (Post). The contribution of nitric oxide synthase (NOS) in β3-AR was assessed, using the non-specific NOS inhibitor, L-NAME (50 μM). Endpoints were functional recovery and infarct size. In another set of experiments BRL37344 and SR59230A were applied according to the same protocols, but the left ventricle was dissected from the heart and freeze clamped at 10 min reperfusion for Western blot analysis of extracellular signal-regulated kinase (ERK p44/p42), protein kinase B (PKB/Akt), glycogen synthase kinase-3β (GSK-3β), and endothelial nitric oxide synthase (eNOS). Data were analyzed with one or two-way analysis of variance (ANOVA). Results: Administration of the selective β3-AR agonist (BRL37344) (1μM) before 35 min RI (BRL37344 (PT), significantly reduced infarct size when compared to the non-pretreatment group (NPT) (21.43±2.52 vs 43.17±1.20, p < 0.001). BRL37344 had similar effects on infarct size when applied during the last 10 min of regional ischaemia BRL37344 (PerT) (14.94±2.34, vs NPT, p < 0.001) or at the onset of reperfusion BRL37344 (PostT) (19.06±1.81, vs NPT, p < 0.001). When BRL37344 was applied as a (PerT+PostT) strategy, infarct size was once again significantly reduced (20.55±2.01 vs 43.17±1.20, p <0.001). In contrast, administration of the β3-antagonist SR59230A according to the same protocol did not reduce infarct size and values similar to those of untreated hearts (NPT) were obtained. Surprisingly, when BRL37344 was applied during the last 10 min of regional ischaemia followed by the administration of the β3-AR antagonist (SR59230A) at the onset of reperfusion, [BRL37344 (PerT) & SR59230A (PostT)], infarct size was significantly reduced to 20.78±3.02 (p <0.001 vs NPT and SR59230A (PerT + PostT). Involvement of nitric oxide (NO) was shown since the reduction in infarct size elicited by BRL37344 was totally abolished by, L-NAME, when administered in combination with BRL37344 for 10 minutes prior to RI or at the onset of reperfusion for 10 minutes (% infarct size: 41.48±3.18 and 35.75±3.54, p <0.001 vs BRL37344 (PT) and BRL37344 (PostT), respectively. Western blot results show that PKB/Akt is activated by BRL37344 regardless of the time of administration. The intervention BRL37344 (PerT+PostT), exhibited the most significant phosphorylation of PKB/Akt (fold increase: 14.2±3.71, p<0.01 vs NPT and p<0.05 vs BRL37344 (PostT). In addition, BRL37344 (PT), (PerT), (PostT) and [BRL37344 (PerT) +SR59230A (PostT)] showed significant activation of this kinase (2.92±0.22, 5.54±0.43, 4.73±0.47, and 6.60±0.78, respectively). ERKp44/p42 however, was not significantly activated by any of the treatments. Phosphorylation of eNOS and GSK-3β was significant only in the BRL37344 (PerT+PostT) and [BRL37344 (PerT) + SR59230A (PostT)] groups. The activation of eNOS-S-1177 in the BRL37344 (PerT+PostT) group was (2.82±0.46, p<0.01 and 0.05 vs NPT and BRL37344 (PostT), respectively) and in the [BRL37344 (PerT) + SR59230A (PostT)] group was (2.26±0.48, p<0.05 vs NPT). A very significant increased phosphorylation of GSK-3β was seen in the same two groups (68.8±7.73, p<0.001 vs NPT and 25.5±5.42 vs NPT, p<0.05, respectively). Conclusion: β3-AR has potent cardioprotective effects when administered either before, during and after ischaemia during early reperfusion as indicated by the reduction in infarct size as well as activation of PKB, GSK-3β and eNOS. These beneficial effects can be linked to NO production through activation of eNOS. / AFRIKAANSE OPSOMMING: Dit is bekend dat verbygaande aktivering van die β-adrenerge seinpad, met ligande soos isoproterenol, formoterol en dobutamien, die hart teen daaropvolgende lang periodes van iskemie beskerm. Aanvanklik was die fokus op die β1- en β2-adrenerge reseptore (β1-AR, β2-AR); maar onlangs is ook die β3-AR as 'n potensiële teiken in die behandeling van hartsiektes ge-eien. In hartversaking, is dit bekend dat β1- en β2-AR afreguleer word, terwyl β3-ARs, aan die ander kant, opreguleer word (Moniotte et al., 2001). Dit het dus belangrik geword om die belang van die β3-AR en sy stroomaf seinpad onder soortgelyke strestoestande te ondersoek. Dit is bewys dat β3-AR stimulasie teen korttermyn agonis geïnduseerde desensitisering in vitro en in vivo bestand is (Liggett et al., 1993) en wanneer geaktiveer, is hierdie reseptor in staat om intrasellulêre seine voortdurend oor te dra (Granneman, 1995). Dit kan dus ‘n ideale teiken vir terapeutiese intervensie wees, ook in iskemiese hartsiekte. Ons hipotetiseer dat selektiewe β3-AR stimulasie tydens iskemie / reperfusie kardiobeskermende mag wees, terwyl selektiewe inhibisie van hierdie reseptor effektief kan wees in die eindstadia van volgehoue iskemie en vroeë herperfusie. Metodes: Die geïsoleerde werkende rothart, onderwerp aan 35 min van streeksiskemie (SI) en 60 min herperfusie, is as model gebruik. Die β3-AR agonis (BRL37344) (1μM) of antagonis (SR59230A) (0.1 μM), is as volg toegedien: (i) voor 35 min SI (PT), (ii) gedurende die laaste 10 min van SI (PerT) en / of (iii) tydens die aanvang van herperfusie (PostT) en (iv) gedurende die laaste 10 min van SI is BRL toediening BRL37344 (PerT) gevolg deur SR59230A tydens die eerste 10 min van herperfusie SR59230A (Post). Die rol van stikstofoksiedsintase (NOS) in β3-AR is met behulp van die nie-spesifieke NOS inhibitor, L-NAME (50 μM) ondersoek. Eindpunte was funksionele herstel tydens herperfusie en infarktgrootte. In 'n ander reeks eksperimente is BRL37344 en SR59230A volgens dieselfde protokolle toegedien, maar die linker ventrikel is uit die hart gedissekteer na 10 min herperfusie en gevriesklamp vir Western klad analise van ekstrasellulêre-sein gereguleerde kinase (ERK p44/p42), proteïen kinase B (PKB/Akt), glikogeen sintase kinase-3β (GSK-3β), en endoteel stikstofoksied- sintase (eNOS). Data is met een of twee-rigting variansie analise (ANOVA) ontleed. Resultate: Administrasie van die selektiewe β3-AR agonis (BRL37344) (1μM) voor 35 min SI BRL37344 (PT), het die infarktgrootte beduidend verminder vergeleke met die nie-behandelde groep (NPT) (21.43±2.52 vs 43.17±1.20, p<0.001). BRL37344 het ‘n soortgelyke effek op infarktgrootte wanneer dit gedurende die laaste 10 min van streeksiskemie BRL37344 (PerT) (14.94±2.34, vs NPT, p<0.001) of by die aanvang van herperfusie (BRL37344 (PostT) (19.06±1.81, vs NPT, p<0.001) toegedien word. Wanneer BRL37344 as 'n (PerT+PostT) strategie toegedien is, was infarktgrootte weereens beduidend verlaag (20.55±2.01 vs 43.17±1.20, p<0.001). In teenstelling hiermee, het administrasie van die β3-antagonis SR59230A volgens dieselfde protokol, nie infarktgrootte verminder nie en waardes soortgelyk aan dié van onbehandelde harte (NPT) is verkry. Interessant, wanneer BRL37344 gedurende die laaste 10 min van streeksiskemie toegedien is, gevolg deur die administrasie van die β3-AR antagonis (SR59230A) by die aanvang van herperfusie, [BRL37344(PerT) & SR59230A(PostT)], was infarktgrootte aansienlik verminder tot 20.78±3.02 (p<0.001 vs NPT en SR59230A (PerT+PostT). Die betrokkenheid van stikstofoksied (NO) is waargeneem deurdat die vermindering in infarktgrootte ontlok deur BRL37344, heeltemal deur L-NAME opgehef is, wanneer dit in kombinasie met BRL37344 vir 10 minute voor SI of by die aanvang van herperfusie vir 10 minute toegedien is (% infarktgrootte: 41.48±3.18 en 35.75±3.54, p<0.001 vs BRL37344 (PT) en BRL37344 (PostT) onderskeidelik). Western kladresultate toon dat PKB/Akt deur BRL37344 geaktiveer word ongeag die tyd van die administrasie. Die intervensie BRL37344 (PerT+PostT), toon die mees beduidende fosforilering van PKB/Akt (voudige toename: 14.2±3.71, p<0.01 vs NPT en p<0.05 vs BRL37344 (PostT). Daarbenewens het BRL37344 (PT), (PerT), (PostT) en [BRL37344 (PerT) + SR59230A (PostT)] ook beduidende aktivering van hierdie kinase tot gevolg gehad (2.92±0.22, 5.54±0.43, 4.73±0.47 en 6.60±0.78, onderskeidelik). ERKp44/p42 is egter nie deur enige van die behandelings geaktiveer nie. Fosforilering van eNOS en GSK-3β was net beduidend in die BRL37344 (PerT+PostT) en [BRL37344 (PerT) + SR59230A (PostT)] groepe. Die aktivering van eNOS-S-1177 was beduidend in die BRL37344 (PerT+PostT) en [BRL37344 (PerT) + SR59230A (PostT)] groepe. 'n Baie beduidende toename in fosforilering van GSK-3β is in dieselfde twee groepe (68.8±7.73, p<0.001 en 25.5±5.42, p<0.05 vs NPT onderskeidelik) waargeneem. Gevolgtrekking: β3-AR het kragtige kardiobeskermende effekte wanneer dit, hetsy voor, tydens en na iskemie gedurende vroeë herperfusie toegedien word, soos deur die vermindering in infarktgrootte sowel as die aktivering van PKB, GSK-3β en eNOS aangedui is. Hierdie voordelige effekte kan aan NO produksie deur aktivering van eNOS gekoppel word.

Renal ischaemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) which is in turn the leading cause of morbidity and mortality in hospitalized patients. The principle aim of this thesis was to evaluate potential new therapies that might afford protection against IRI in both in vitro and in vivo settings. Recent evidence suggests that nitrite (NO2-) may play an important role in protecting the myocardium from IRI. Our initial work into the role of NO2- in an in vitro model of renal IRI in proximal tubular epithelial cells provided evidence that NO2- can prevent apoptosis and preserve cell viability. This lead to an in vivo study where high NO2- concentrations (50 mg/L) were given orally to rats for 7 days prior to inducing renal IRI but no beneficial effects of this treatment were observed. Another potential treatment identified was thiamine (vitamin B1) and this, like NO2-was investigated to see if it had the potential to protect rats from AKI injury. It has been previously recognized that in renal IRI the high energy phosphate ATP is found to be severely depleted whilst is is known that thiamine can play a pivotal role in generating ATP. Furthermore, thiamine has previously been demonstrated to protect against myocardial ischaemic injury and has the ability to reduce myocardial infarct size. In vitro, thiamine was found to reduce the degree of apoptosis in cultured HK-2 cells caused by ischaemia whilst in vivo it afforded protection against AKI caused by renal IRI by anti-apoptotic, anti-inflammatory and anti-oxidant mechanisms. Finally, a study into the possible therapeutic role of gene therapy with bone morphogenic protein 7 (BMP-7) in renal IRI was undertaken. Previous work has established that i.v. BMP-7 is able to protect against renal IRI but it has also been associated with ectopic bone formation at the site of injection. Therefore another method to increase circulating BMP-7 was sought. We initially found that BMP-7 gene therapy could attenuate apoptosis and preserves cell viability in an in vitro model of renal IRI. However, whilst in vivo gene therapy with electroporation of BMP-7 plasmid DNA increased BMP-7 expression in mice serum 2 days post electroporation, it was unable to protect the animals against IRI induced AKI. In rats the direct injection of naked DNA BMP-7 plasmid systematic 2 days prior to renal IRI was able to upregulate BMP-7 expression 4 days later in kidney tissue. Despite this it was unable to afford protection against renal IRI. Apoptosis and necrosis play a crucial role in the pathogenesis of renal IRI induced AKI. In this thesis we investigated the role of three putative therapeutic agents and their role in apoptosis and necrosis in vitro in PTECs and in vivo against renal IRI induced AKI. All three therapeutic drugs were able to attenuate apoptosis in PTECs but were unable to protect against necrosis, whilst against renal IRI induced AKI only thiamine was found to be protective. Thiamine appears to hold the most promise and more work needs to be undertaken so that its potential benefit in AKI can be realised.

Ischaemia-reperfusion (IR) injury makes a major contribution to graft damage during kidney transplantation and increases the risks of primary non-function, delayed graft function and rejection. Oxidative damage to mitochondria is a key early event in IR injury. The aim of this project was to examine the safety and efficacy of the mitochondria-targeted antioxidant MitoQ in reducing pig and human kidney IR injury using an ex vivo normothermic perfusion (EVNP) system. Over a range of 500 nM to 250 µM using a 150 pig kidneys and 80 declined deceased human kidneys, MitoQ was successfully taken up by pig and human kidneys in a concentration-dependent manner, resulting in stable tissue concentrations over 24 hours of cold storage followed by 6 hours of EVNP. The uptake of MitoQ was increased approximately 2-fold when MitoQ was administered to warm (rather than cold) kidneys and when kidneys were preserved using hypothermic machine perfusion (rather than cold static storage). 50 µM MitoQ, administered to pig kidneys at the end of warm ischaemia, significantly increased renal blood and urine output flow at the end of 6 h EVNP compared to the control group. Creatinine clearance was numerically higher in the 50 µM MitoQ group compared to the control group but the difference did not reach statistical significance. To test the safety and efficacy of MitoQ in human kidney IRI, pairs of declined deceased human kidneys were used, with one kidney in each pair used as control. The total urine output, creatinine clearance and percentage fall of serum creatinine were numerically higher in the 50 µM MitoQ group compared to the control group, although the differences did not reach statistical significance during 3 h of EVNP. There was a significant difference in the renal blood flow between the 50 µM MitoQ group and the control group at the end of the first hour of EVNP. The renal blood flow remained relatively stable during the first hour of EVNP in the 50 µM MitoQ group compared to a significant decrease in renal blood flow in the control group. There was no effect on fractional excretion of sodium or oxidative injury markers (protein carbonyl formation, lipid peroxidation) in pig or human kidneys, which is consistent with previous studies that demonstrated the requirement of >24 hour after reperfusion for manifestation of changes in these parameters. In this thesis, I was able to successfully demonstrate the safety and potential efficacy of MitoQ in ameliorating renal IRI using pig kidneys. While more declined deceased human kidneys need to be analysed to fully explore the potential efficacy of MitoQ in ameliorating renal IRI, this study provides important data that will help inform future studies and ultimately a clinical trial for assessing the efficacy of the mitochondria-targeted antioxidant MitoQ in human kidney transplantation. My findings suggest that MitoQ has the potential to increase the use of marginal kidneys and to improve graft and patient outcomes.

Aims There are few proven therapies that can protect against the inevitable ischaemia reperfusion injury (IRI) that occurs during renal transplantation. IRI increases the likelihood of delayed graft function (DGF), which negatively impacts on the long-term survival of a transplanted kidney. One enzyme of interest, heme oxygenase-1 (HO-1), degrades heme and protects against the oxidative stress that occurs secondary to IRI. Clinical renal recipients with higher HO-1 levels have improved graft function post transplant. Heme arginate (HA), a form of hemin, which has been used to treat porphyria for over 30 years, has repeatedly been shown to induce HO-1 in in vivo and in vitro macrophages. It is one of the few HO-1 inducers approved for clinical use and healthy volunteer studies confirmed that HA could also safely induce HO-1 in humans. Prior to the formal start of the MD, the University of Edinburgh successfully applied to NHS Blood and Transplant for funding to investigate whether giving HA to recipients of deceased donor renal grafts prior to transplant could upregulate HO-1 and whether this had any effect on the function and health of the grafts. This MD aims to explain the background behind the proposed study, the process of study approval, planning and trial logistics and protocol. This thesis then describes the methods of sample analysis, the results and future directions for the HOT (Heme Oxygenase-1 in renal Transplantation) study. Methods The HOT study planning and approval process took eight months and the first participant was randomised in January 2012. The study was sponsored by ACCORD, a joint company from University of Edinburgh and NHS Lothian, and recruited patients from the Edinburgh Royal Infirmary Transplant Unit. The protocol was followed to ensure that 40 recipients were randomised blind to either active (two doses 3mg kg-1 HA: pre-operatively, day 2) or placebo (NaCl: same schedule). To ensure that the primary outcome was fulfilled, recipient blood was taken daily for peripheral blood mononuclear cells (PBMC) extraction. After further blinding steps, the PBMCs were analysed for HO-1 protein and mRNA. The secondary outcome measures involved collecting urine for analysis of urinary biomarkers (KIM-1 and NGAL), taking renal graft biopsies pre-op and day 5 for renal HO-1 analysis and collecting renal function data. DGF was calculated daily. To ensure that all adverse event data was captured, the recipients were closely reviewed for 7 days and their renal function was monitored for 90 days. Results The final participant was recruited in May 2013 within the predicted timescale and to budget. This participant completed follow-up in August 2013. Of the 40 participants, three received the infusion but did not receive a transplant and therefore could not give primary outcome data. The remaining 37 did and this was analysed. Adverse events were equivalent between groups and there were no adverse reactions to HA. HA upregulated PBMC HO-1 protein at 24 hours compared to placebo: HA 11.1ng/ml (1.0- 37.0) vs. placebo 0.14ng/ml (-0.7- 0.3)(p= < 0.0001). PBMC HO-1 mRNA was also increased: HA 2.73 fold (1.8- 3.2) vs. placebo 1.41 fold (1.2- 2.2) (p=0.02). HA increased HO-1 protein immunopositivity in day 5 renal tissue compared with placebo: HA 0.21 (-24- 0.7) vs. placebo -0.03 (-76- 0.15) (p=0.02) and the percentage of HO-1 positive renal macrophages also increased: HA 50.8 cells per HPF (40.0- 59.8) vs. placebo 22.3 (0- 34.8) (p=0.012). Renal HO-1 mRNA was also increased in HA group: 2.02 (0.20- 4.03) fold increase compared to 1.68 (0.75- 10.39) fold in the placebo group but it was not significant (p= 0.451). Urinary biomarkers were reduced after HA but not significantly so. Histological injury and DGF rates were similar between the groups. Conclusion HA is safe and effective in renal transplant recipients as reported in this phase II, randomised, placebo controlled, blinded, single-centre study. The primary outcome was achieved and demonstrated for the first time that HA induces HO-1 in peripheral and renal macrophages in kidney transplant recipients. There was also evidence that HA increased HO-1 expression in renal tissue. There was no evidence that HA improved renal function or reduced injury as seen in animal models but it is recognised that the sample size was small and the study was not powered to these endpoints. Larger studies are planned to determine the impact of HO-1 upregulation on clinical outcomes and evaluate the benefit to patients at risk of IRI. The plans for HOT2 are expanded in this thesis.