No description available.

No description available.

No description available.

The incidence and mortality from cardiovascular disease (CVD) in patients with chronic kidney disease (CKD) far exceeds that seen in the general population. Whilst a number of risk factors and associations have been identified in patients with CKD that may contribute to the increased risk of CVD, our understanding of the underlying pathophysiology remains poor. It has previously been reported that uraemic animals sustain larger myocardial infarcts and that this ‘reduced ischaemia tolerance’ may in part explain the excess mortality from CVD seen in CKD patients. The aim of this work was to establish an in vivo model of uraemic myocardial infarction in order to further explore the pathophysiology of uraemic CVD with particular focus on ameliorating myocardial ischaemia-reperfusion injury using ischaemic and pharmacological preconditioning. An increase in myocardial infarct size was demonstrated in the sub-total nephrectomy model of chronic uraemia, confirming previous reports in the literature. However, infarct size was not found to be increased in adenine diet induced renal failure. In addition, it was demonstrated for the first time, that the techniques of ischaemic preconditioning (IPC) and remote ischaemic preconditioning (RIPC) are both efficacious and not attenuated by chronic uraemia induced by sub-total nephrectomy or adenine diet (IPC only). Investigations were undertaken using an agent (a HIF stabiliser, FG4497) to induce pharmacological preconditioning in both animals with renal insufficiency and those without. These studies demonstrate that stabilisation of hypoxia inducible factor (HIF) may be a promising strategy to induce pharmacological preconditioning. It is hoped that this work may lay the foundations for future investigations to determine why sub-totally nephrectomised rats have larger infarcts whilst those with adenine induced renal failure, with a substantially greater degree of renal dysfunction, do not. Moreover, it is hoped that; by demonstrating that uraemia 3 does not prevent or attenuate the myocardial protection afforded by ischaemic preconditioning, the recruitment of patients with CKD will be encouraged to clinical trials of both ischaemic preconditioning and other therapies to limit myocardial infarction.

The plasma protein complement factor H (fH, 155 kDa) regulates the activity of the alternative pathway of complement activation. Factor H is monomeric, and its 20 CCP modules are arranged in a predominantly elongated conformation, joined by linking sequences that vary in length, with the longest linkers occurring in the central portion of the molecule. CCP modules 1 through 4 of fH host its capacity to act as a cofactor for fI-mediated proteolytic degradation of C3b and its ability to accelerate the decay of the C3 convertase, C3bBb, thereby regulating the so-called tick-over activation of the alternative pathway. Mutations in this part of fH might compromise its function and lead to underregulation of the alternative pathway. It is hypothesized that this can cause predisposition to diseases such as atypical haemolytic uraemic syndrome (aHUS) and age-related macular degeneration (AMD). In the current work, the known disease-associated mutations R53H and R78G were compared to wild-type in terms of fluid-phase cofactor assays, C3b-binding affinity and the ability to accelerate the decay of the convertase. In addition, the protective variant, I62, was also inspected because its protective role might be explained by an increased regulatory activity. The second, linked, aim of this project was to employ Forster resonance energy transfer (FRET) to study the link between conformation and function in fH. FRET is valuable for obtaining long-distance restraints up to a maximum of 100 °A and is therefore particularly useful for inferring domain orientations within multidomain proteins. This approach to measure long-range inter- and intramolecular distances is a convenient way to complement NMR-based structural investigations, which rely on short-range restraints. It is also a valuable complement to X-ray crystallography since it is a solution technique that can be conducted under physiological conditions. By using site-directed mutagenesis in the current work, free cysteines were introduced into CCP modules 1-4 at strategic points, which were then used for attachment of fluorescent tags. C3 possesses an internal thioester which can be labelled with a fluorophore upon activation to C3b. Intermolecular FRET measurements were thus undertaken to gain information about the interaction between the two proteins that is crucial for understanding functional activity. The CCP modules in the centre of fH may be responsible for introducing a bend into fH that brings the N-teminus close to the C-terminus (the latter is important for host versus non-host discrimination) joined by the longest linkers occurring in the whole molecule. This coincidence of two relatively small CCP modules, 12 and 13, with the highest number of eight amino acids between them, is hypothesised to reflect some unique architectural features. To explore the structural details of this portion of fH by FRET, single-labelled cysteine mutants were further modifed to provide a recognition site for transglutaminase (TGase), which can be enzymatically labeled with a second fluorophore. This stoichiometrically-labelled protein was used for intramolecular FRET studies.