Premature death from cardiovascular disease is common amongst adult patients with chronic renal failure (CRF). The implication for paediatric patients, the majority of whom now survive into adulthood, is that they maybe similarly affected but at an earlier age. Children with CRF already show signs of arterial disease in the absence of classical risk factors. The aim of this research was to develop ways of improving the outcome for children with CRF by investigating the effect of interventions on vascular function. Vascular endothelial function, which is dependent on nitric oxide (NO) (an antiatherogenic substance) can be measured using a non-invasive technique of high resolution ultrasound. Abnormal responses represent the earliest change in the pathogenesis of vascular disease and may be reversible. Bioavailability of NO may be reduced in CRF due to low levels of precursors, increased levels of inhibitors and increased oxidant stresses. The hypotheses examined were: 1) Dietary supplementation with L-arginine (the substrate for NO synthetase) improves large vessel endothelium-dependent vasodilatation and NO bioavailability. 2) Dietary supplementation with folic acid reduces homocysteine levels and improves large vessel endothelium-dependent dilatation. 3) Haemodialysis improves large vessel endothelium-dependent dilatation by lowering homocysteine levels, removing inhibitors of NO synthesis, reducing oxidative stress and/or removing free radicals. Methodology was double blinded, randomized and placebo controlled in children with stable CRF in the absence of classical risk factors for atherosclerosis. Endothelial function was measured in the brachial artery. Biochemical measures of NO activity and oxidant stress were also measured, along with the acute effect of haemodialysis on endothelial function. We demonstrated that L-arginine was not beneficial as a therapeutic agent. Folic acid improved vascular function and is now used routinely in all children with CRF at Gt Ormond St Hospital. Haemodialysis had an adverse effect on vascular function-an observation that needs further study as the process in itself may contribute to the development of atherosclerosis.

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It is recognised that the most common cause of acute renal failure is ischaemic injury. I have therefore used an animal model to investigate the molecular mechanisms underlying renal cell death and cell regeneration that occur post-ischaemic injury. A number of genetic programmes are activated post-ischaemic renal injury, which mediate a variety of pathways including those leading to cell death and those enabling cell regeneration and repair. I identified the up-regulation of a number of genes following ischaemic renal injury and characterised the temporal and spatial expression of these genes using northern blotting, in-situ hybridisation and immunohistochemistry. Using differential display PCR I detected the up-regulation of calcyclin, a member of the S100 family of EF-hand calcium binding proteins, and propose that calcyclin may play a role in the regenerative phase following ischaemic renal injury. Conversely I report the induced expression of a pro-apoptotic gene, Siva, which may mediate cell death occurring immediately following ischaemia. Further work identified the up-regulation of a cell surface receptor, CD44, and its ligands osteopontin and hyaluronic acid suggesting an important role for CD44 during the recovery phase of ischaemic acute renal failure. The final piece of work in this thesis describes the use of a novel therapeutic strategy to ameliorate the course of ischaemic acute renal failure through the inhibition of the DNA repair enzyme, poly-ADP ribose polymerase (PARP). Excessive activation of PARP exhausts ATP reserves that are essential for cell regeneration following ischaemic injury. The inhibition of PARP preserves ATP levels and allows for a more robust proliferative response.

Intrarenal complement activation and production have pivotal roles in progressive renal injury in chronic nephropathies. Complement has recently been implicated as a candidate mediator of inflammatory injury in the tubulointerstitial milieu during proteinuric states. A key target of the activated complement cascade 5 the proximal tubule, a site at which abnormally filtered plasma proteins and complement factors may concur to promote injury. This study investigated whether protein overloading of human cultured proximal tubular cells (HK-2) enhanced complement activation, possibly via impairment of complement regulation. HK-2 cells exposed to albumin, transferrin or IgG followed by human serum (HS) as a complement source, showed increased apical C3 and membrane attack complex deposition compared to HS exposure alone. Complement deposition occurred through the activation of the alternative pathway. Albumin and complement challenge had additive proinflammatory effects, shown by greater increases in fractalkine and TGF-p mRNA expression compared to each stimulus alone. Factor H binds to the cell surface through heparan sulfate residues. Protein overload reduced heparan sulfate surface density, resulting in decreased factor H binding and increased active C3b deposition. Thus, protein overload alters tubular cell phenotype by reducing the cell's capability to counteract complement activation.

Background and aims: End stage renal disease (ESRD), an increasing global major public health problem, has greatly increased cardiovascular disease risk (CVD) than the general population for unknown reasons. Preliminary data suggest that adipose tissue, shown to contribute to CVD risk in non-ESRD populations, may also play a part in ESRD. Adipose tissue is a complex organ which not only functions as a fat storing depot, but also plays an essential role in lipid metabolism. Recently, researchers have recognized that adipose tissue is also an endocrine organ since it secretes several cytokines and adipokines including leptin, adiponectin and interlukin-6 (IL-6). Alteration in circulating adipokines has been shown in ESRD patients. The objective of the study was to clarify the role that adipose tissue and lipoproteins may have in ESRD. The work presented in this thesis has focused on the role of adipose tissue in ESRD patients undergoing haemodialysis and peritoneal dialysis. Both the nutrient and endocrine functions of adipose tissue were addressed in this thesis. Materials and methods: Most studies were carried out on novel in vitro adipocyte assay system developed for these experiments. Novel approaches to investigate ESRD included isolating lipoprotein fractions (VLDL, LDL and HDL) by ultracentrifugation from ESRD and matched human controls followed by incubation with the normal adipocytes in the novel assay system developed for these experiments. One experiment involved collection of human adipose tissue samples. Given the scope of the thesis mainly gene expression data was collected on candidate genes analysed by quantitative real-time PCR in these novel pilot studies. Results: An atherogenic lipoprotein profile was seen in ESRD patients. Haemodialysis patients had increased serum triglycerides and decreased HDL- cholesterol compared to the control subjects. Both haemodialysis and peritoneal dialysis groups had increased VLDL. The lipoprotein pattern in the present study also indicated the peritoneal dialysis group had more atherogenic lipoproteins, IDL and small dense LDL. Expression of lipoprotein lipase, hormone-sensitive lipase and regulatory proteins of lipid metabolism complement protein 3 and low density lipoprotein receptor were downregulated when treated with uraemic lipoproteins in vitro. Serum concentrations of total adiponectin, high molecular weight adiponectin and leptin were significantly higher in the ESRD. Gene expression of adiponectin, IL-6 and leptin was lower in ESRD patients. The results showed a tendency towards an increased accumulation of macro phages in adipose tissue in ESRD patients. Discussion: These findings are strongly suggestive of a defective storage function as well as impaired ability to release fatty acids consistent with dysfunctional adipose tissue in ESRD. These data strongly suggest that normal adipocyte function is altered when exposed to a uraemic environment in vitro. Macrophage infiltration into adipose tissue has been suggested to play role in insulin resistance and systemic inflammation. The results from the modulation of this in vitro adipocyte assay system not only serves as a convenient and unique model to study mechanisms of disease such as ESRD and its treatments but has so far also provided an initial insight that strongly suggests several mechanisms that could contribute to the CVD risk associated with ESRD. Conclusion: An in vitro system to study the effect of uraemic lipoproteins and serum on adipose tissue nutrient and endocrine function through adipocyte gene expression was conducted and compared where available with the corresponding circulating protein measurements. Further studies in ESRD, both by broadening the number of adipocyte genes (and proteins) and undertaking a mechanistic approach to clarifying the reasons for the changes observed would be necessary and informative.

NHS clinical governance recommends that parents and children are involved in their healthcare and take an active role in making decisions. Existing research does not contribute to how this policy should be implemented by professionals in practice. In the context of paediatric ESRF, there is little evidence on how professionals should involve parents and Iren in their treatment and care, or if there are any barriers or facilitators to implementing shared patient involvement. Nor is there research indicating the views of the professionals regarding the involvement of parents and children in their care. The empirical questions of this research aim to generate a greater understanding of professionals' experiences of involving paediatric patients and their parents in ESRF care.