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Most renal diseases have a common end inflammatory pathway, which is associated with a leukocytic infiltrate. Chemokines are small proteins that are responsible for the chemoattraction of leukocytes into areas of injury or insult. CX3CL1, also known as fractalkine, exists as a transmembrane protein as well as a soluble protein. It acts as a cell adhesion molecule in addition to its chemoattractant properties. This thesis firstly examines the distribution of CX3CL1 in renal tubular epithelial cells (RTEC) and in the second part of the thesis the regulation of CX3CL1 by stimulation of the thromboxane prostanoid (TP) receptor is examined. The localisation of CX3CL1 was initially demonstrated primarily on the apical surface of tubular epithelial cells in human renal biopsy specimens with histological diagnoses of acute tubular necrosis and acute allograft rejection. A cell model was then developed in MDCK cells to examine the distribution more closely. There are a limited number of mechanisms potentially responsible for the trafficking of CX3CL1 to the apical membrane and it was established that N-glycosylation of CX3CL1 is required for its presence on the apical membrane of RTEC. The mobility of CX3CL1 within the cell membrane was next assessed and it was shown to be relatively immobile. We hypothesized that this would promote cell adhesion and indeed further experiments confirmed that CX3CL1 in RTEC does promote adhesion of cells bearing the cognate receptor. Given that CX3CL1 and thromboxane A2 are both found in similar inflammatory conditions, are both present early in the inflammatory process and that stimulation of the TP receptor has been shown to regulate other chemokines, we next evaluated the effect of stimulation of TP on CX3CL1. We found that both total and surface cellular levels of CX3CL1 were reduced following stimulation of TP. A maximal nadir was present after 30-60 minutes and the levels returned to baseline by 4 hours. The mechanism for the loss of CX3CL1 was then assessed. CX3CL1 is known to recycle between the cell surface and an internal compartment. No effect of TP stimulation was seen on the endocytosis or exocytosis of CX3CL1. Stimulation of TP was however, shown to stimulate tumour necrosis factor-a converting enzyme (TACE) via ERK phosphorylation. TACE inducibly cleaves CX3CLI, releasing the soluble chemokine. TACE siRNA was used to knock down TACE gene expression and this prevented the loss of cellular CX3CL1, confirming that TP stimulation induces TACE cleavage of CX3CL1. T he results of further experiments are discussed in the discussion chapter.

Studies in humans have shown that 1 in 1800 infants are born with limb deficiencies and that 9% of these also suffers from renal abnormalities. Classification of these limb abnormalities revealed that the renal defects commonly associate with limb deficiencies such as micromelia (44%), amelia (18%) and radial/tibial (preaxial) deficiencies (27%). Additionally, experimental evidence has shown that the mesonephros, an important component of the primitive urogenital system, also could be responsible for inducing and maintaining limb development. These observations imply the existence of a developmental link between limb induction and early kidney development. However, this remains controversial since other experiments show that the mesonephros is not required for limb development. The work described in this thesis was prompted by the observation, that the mouse mutants dominant hemimelia (Dh) and luxate (Ix) disrupt both limb induction and kidney development. Furthermore, both mutants display preaxial abnormalities such as Polydactyly, oligodactyly and tibial hemimelia of the hindlimbs. In addition Dh also causes asplenia, microgastria, small pancreas, gut atresia. The studies presented here show that the hindlimbs in Dh animals are shifted 2-3 segments anteriorly coinciding with a lumbar-sacral transformation of the axial skeleton. In situ hybridisation shows that the anterior boundary of HoxclO in the flank mesenchyme has moved anteriorly in Dh embryos while the expression of HoxalO and HoxdlO appear to be unaffected. The spinal nerves innervating the hindlimbs also respond to the shift of the limbs. Analysis of the urogenital phenotype shows that the effect Dh has on the kidney abnormalities is indirect. It appears that the hydronephrosis observed in Dh mice is caused by blockage of the ureter, however, it is possible that this blockage is related to misexpression to HoxclO. Efforts were therefore made to determine if other traits related to the Dh mutation was caused by ectopic HoxclO expression, however, this does not appear to be the case. Nevertheless these studies led to the identification of a novel anatomical structure, the splanchnic mesothelial ridge (SMR). The SMR consists of a thickened mesothelium on the left side of the spleno-pancreatic region, which is required for asymmetric growth. In Dh mice the SMR is absent and as a result the splenopancreatic region asymmetric growth is impaired. The asplenic phenotype is also related to the absence of the SMR.

Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular disease to which conventional cardiovascular risk factors and co-morbidity contribute. Increased arterial stiffness and impaired endothelial function are common features of CKD and recognised markers of cardiovascular risk. In recent years, emerging cardiovascular risk factors - including inflammation, oxidative stress, and a shift in the balance of the vasodilator nitric oxide and vasoconstrictor endothelin systems - have become increasingly important as major contributors to increased cardiovascular complications and may also contribute to arterial stiffness and endothelial dysfunction in CKD. The overall aims of the work presented within this thesis were, therefore, to characterise the contribution of uraemia itself, and conventional and emerging cardiovascular risk factors, to arterial stiffness and endothelial dysfunction, as surrogates for cardiovascular risk, in a group of CKD patients, across a wide range of glomerular filtration rate (GFR) from normal to pre-dialysis, with relatively low comorbidity. Arterial stiffness and endothelial dysfunction were measured by carotidfemoral pulse wave velocity (CF-PWV) and flow-mediated dilatation (FMD), respectively. The first study aimed to assess the reproducibility for one observer with repeated measurements (intra-observer) and for two separate observers (inter-observer) of CFPWV and FMD. I have shown that both inter-observer and intra-observer measurements of CF-PWV and FMD are highly reproducible. Hence, these techniques are therefore suitable to be incorporated into clinical studies. The characteristics of the relationship of plasma and urinary endothelin-1 (ET-1) concentrations to renal function were studied. In this group of CKD patients, plasma ET-1 increased in a linear fashion, whereas fractional excretion of ET-1 increased exponentially as renal function declined. These findings support the role of renally derived ET-1 in renal pathophysiology. In the next study, I showed that arterial stiffness increases incrementally as GFR declines whereas endothelial dysfunction is a feature only of late stage CKD (GFR ≤ 20 ml/min/1.73m²). Age and blood pressure (BP) were the major determinants of both. However, GFR was not an independent predictor of either CF-PWV or FMD in this group of patients, suggesting that uraemia, on its own, is not the main driving force in the development of vascular complications in CKD. Therefore, I further explored the role of emerging cardiovascular risk factors to arterial stiffness and endothelial dysfunction. Whilst, BP remains the strongest determinant of arterial stiffness and endothelial dysfunction, inflammation and asymmetrical dimethylarginine, an endogenous nitric oxide synthase inhibitor, are independent predictors of CF-PWV, and oxidative stress and plasma ET-1 independently predict FMD. Thus, the cardiovascular complications that occur in CKD may be substantially driven by these emerging risk factors. Then, I examined the contribution of the metabolic syndrome to arterial stiffness and endothelial dysfunction in the same group of CKD patients. Irrespective of renal function, CKD patients with the metabolic syndrome have increased arterial stiffness and a trend to impaired endothelial function. Either the presence of the metabolic syndrome or the number of risk factors for it independently predicts CF-PWV and FMD. When risk factors for the metabolic syndrome are considered individually, BP remains an independent determinant of both CF-PWV and FMD. Additionally, waist circumference is also an independent predictor of CF-PWV. These findings suggest that the metabolic syndrome or its individual risk factors maybe targets for intervention to improve cardiovascular outcomes in all stages of CKD. The contributions of arterial calcification to arterial stiffness and endothelial dysfunction were also assessed. I showed that, irrespective of renal function, CKD patients with arterial calcification have increased arterial stiffness and a trend to impaired endothelial function. The findings from the observational studies presented in this thesis support the role of emerging cardiovascular risk factors on arterial stiffness and endothelial dysfunction in CKD. An interventional study using a peptide selective endothelin-A (ETa) receptor antagonist has confirmed this hypothesis. In a group of CKD patients, irrespective of renal function, selective ETA receptor antagonism lowered BP, reduced proteinuria, improved arterial stiffness on top of a standard BP lowering treatment with renin-angiotensin system blockade, and appeared to reduced arterial stiffness independent of its effect on BP. In summary, these studies show that in the absence of diabetes or established cardiovascular disease, CKD patients have increased arterial stiffness and endothelial dysfunction. Elowever, arterial stiffness and endothelial dysfunction are not predicted by renal function. Although the conventional risk factor, BP, is the strongest determinant of CF-PWV and FMD in CKD, the contribution of several emerging cardiovascular risk factors on arterial stiffness and endothelial dysfunction is observed. On the basis of these findings, chronic interventional studies, for instance with endothelin receptor antagonists are now needed.

Benign prostatic hyperplasia (BPH) is a common disease of aging men and the development of lower urinary tract symptoms (LUTS) secondary to BPH is believed to be caused by both a static and dynamic component to bladder outflow obstruction (BOO). The static component is attributed to the mass effect of the enlarged adenoma, whereas the dynamic component is related to the smooth muscle tone within the prostate. The prostate is a heterogeneous organ and comprises three distinct anatomical "zones". The formation of stromal and glandular nodules is the histological hallmarks of BPH and occurs in the transition zone. The mechanical properties of benign prostate tissue obtained from transurethral resection (TURP) have been shown to reflect the underlying morphology with respect to smooth muscle content. To further investigate the structure-property relationship in benign prostate tissue, specimens from TURP and whole prostates from cystoprostatectomy were examined. The use of transverse sections of the whole gland allowed morphometric and mechanical studies to be performed in all regions of the prostate. A novel method of morphometric analysis representative of the local area of tissue that was subjected to mechanical testing was developed. Significant correlations were shown between the mechanical and morphological properties of glandular and stromal nodules. Glandular nodules may contribute to the pathophysiology of BPH by altering the dynamic characteristics of the prostate in addition to producing a mass effect. In parallel to the above aforementioned work in whole prostates, the effect of quinazoline and non-quinazoline based alpha-blockers upon prostate tissue obtained from TURP was examined. This work also investigated whether alpha blocker therapy influenced the mechanical measurements by their action within the prostate tissue. Significant correlations were shown between alpha blocker therapy and the underlying morphology and this is reflected in the mechanical measurements. These studies provide a greater understanding of the structure-property relationships within prostate tissue and it is likely that assessment of the mechanical properties of the prostate will be useful in the clinical evaluation and management of patients with benign prostate disease.

The Common Sense Model (CSM) of illness representations is a prominent psychological approach used to understand the motivation behind health behaviours. This thesis sought to understand the utility of the CSM by examining the relationship between facets of illness representations and health behaviours in individuals with Cardiovascular Disease (CVD) and Chronic Kidney Disease (CKD). Literature Review: CVD confers significant costs, which is mitigated by a physically active lifestyle. However, many CVD patients do not undertake sufficient exercise. This prompted studies to examine if illness representations underpin the motivation behind such health behaviours. Despite a growing body of research in this area, no review has systematically interrogated and synthesised the corpus of research evidence. The current review aims to systematically review the relationship between illness representations and indices of physical activity in CVD. Ten studies were elicited with overall findings being equivocal; the majority of the studies demonstrated a relationship between illness representations and indices of physical activities with domains of timeline, control and consequences of the CSM most often revealed associations. Future research exploring putative causal relationships between illness representations and indices of physical activity is warranted. Empirical Study: CKD is a debilitating condition, which is further exacerbated by depression. While being physically active have scope to mitigate adverse impacts, many do not meet the recommended physical activity guideline. The current study examined the relationship between illness representations and levels of physical activity in CKD. Seventy non-dialysing patients were recruited from an outpatient renal clinic. Results indicated that timeline cyclical of the CSM predicted levels of physical activity and was not moderated or mediated by depression while timeline cyclical, consequences, emotional representations, personal control and illness coherence were significantly associated with depression. Future research regarding the development of psychological interventions based on an illness representations framework is proposed.

Anti-neutrophil cytoplasm antibodies (ANCA) are associated with a severe form of small vessel systemic vasculitis, in which they target two specific auto-antigens, proteinase-3 (PR3) and myeloperoxidase (MPO) found within neutrophils and monocytes. Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are the main clinical syndromes, both characterized by kidney and lung disease, but granulomatous inflammation is almost exclusively found in GPA, and unlike other manifestations, remains difficult to treat. In GPA patients, PR3 is the predominant ANCA auto-antigen and neutrophil membrane PR3 expression is increased. There has been limited understanding of why granulomata are restricted to this patient subgroup. I have investigated the role of PR3 in driving giant cell and granuloma formation by generating a novel in vitro model. Using extensive tissue culture and microscopy techniques I have been able to demonstrate that PR3 induces both giant cell and granuloma formation in GPA patients’ cells. Giant cells are the precursors to granulomata and I have demonstrated that in GPA patients, monocytes firstly fuse with the persistence of PR3 and then later recruit lymphocytes to form an organized granuloma-like structure. I have developed a unique method of quantifying granuloma formation and I have been able to show that GPA patients show a statistically significant greater rate of PBMC aggregation both spontaneously and in the presence of PR3 compared to MPA patients and healthy controls. I have explored the potential mechanisms of granuloma formation in this patient subgroup. Specifically, IL-6 may be important in driving granuloma formation in GPA patients and supports the notion of PR3-mediated process. PR3 cleaves protease-activated receptor 2 (PAR2) and I have shown that the presence of a PAR2 agonist further augments cell fusion. These findings support the role of PR3-mediated monocyte activation and fusion with additional T cell aggregation. In summary, I have developed a novel system to test giant cell and granuloma formation in GPA patients, a potential platform to evaluate new therapeutic treatments.

Urinary tract infections (UTI) are among the most prevalent infectious diseases worldwide, leading to significant morbidity and mortality and wreaking a substantial economic cost. Uropathogenic Escherichia coli (UPEC) has been shown to invade the urothelium in murine models of acute UTI, forming intracellular reservoirs that are thought to evade conventional antibiotic treatment and the immune response, allowing recurrence at a later date. However, the role of intracellular infection in chronic UTI causing more subtle lower urinary tract symptoms (LUTS), a particular problem in the growing elderly population, is not well understood. Moreover, the species of bacteria involved remains largely unknown, the model systems used to study them need improvement, and treatment options are not currently optimal. This thesis addresses these important research aims with a view towards improving the situation for LUTS patients who have an underlying UTI. In the first research section, we found strong evidence of intracellular Enterococcus faecalis harboured within urothelial cells shed via an innate immune response from the bladder of LUTS patients. Furthermore, these patient-isolated strains of E. faecalis showed robust invasive properties in a bladder cell line. However, E. coli only formed surface biofilms in these patients, suggesting that the murine UPEC model may not apply to patients with chronic LUTS. In the second section, we addressed the issue that the murine and human urinary bladder differ structurally and functionally, which may be hindering our understanding of UTI pathogenesis in humans. We therefore 4 designed and characterised a human three-dimensional (3D) bladder mimetic differentiated from primary urothelial progenitors, and showed that it closely resembles human tissue. Moreover, infection in this organoid model resulted in outcomes similar to those seen in LUTS patients. In the future we aim to use this 3D culture as a platform for modelling chronic infection and tissue regeneration in the presence of novel therapeutic agents. Finally, in the third section we tackled the issue that traditional oral antibiotic regimens for UTI fail in a high proportion of cases. This recurrence of disease post-treatment could be explained in part by the lack of cellular penetration of orally administered antibiotics, which are not able to accumulate to an effective concentration within intracellular bacterial niches. Meanwhile, oral antibiotics may also lead to antimicrobial resistance and systemic side effects. Using our human urothelial organoid, we tested the ability of novel liposome-coated ultrasound-activated lipid microbubbles to deliver drugs into the cortex of the apical cell layer. Ultrasound-activated intracellular delivery of gentamicin using microbubbles was over twice that achieved by liposomes alone. Moreover, little cell damage was detected and this therapeutic technology exhibited very encouraging antimicrobial activity, showing great promise as a more efficacious alternative to traditional oral antibiotic regimens. In conclusion, these collective results have implications for both the diagnosis and treatment of chronic UTI.

Background: Both urgency and stress urinary incontinence are heritable, with genetic factors contributing approximately half of total susceptibility. Aims: The overall aim of this project is to identify known and novel genetic polymorphisms associated with urgency and stress incontinence in women. Design: We systematically reviewed prior genetic association studies of incontinence, and other pelvic floor disorders. We then conducted a two stage GWAS, using women enrolled in NFBC1966, UK Twins, and ALSPAC for discovery, and women in six separate cohorts for replication. To prioritise likely susceptibility genes we measured gene expression in bladder biopsies, using whole genome microarrays, and PCR using custom microfluidic plates. Results: From prior studies of incontinence, and the related condition of prolapse among women, we conducted 13 meta-analyses for different polymorphisms, finding a single moderately credible association for a common variant in the ADRB3 gene associated with overactive bladder. From prior studies of lower urinary tract symptoms in men, we conducted 35 meta-analyses for different polymorphisms, finding a single moderately credible association for a common variant in the VDR gene associated with a composite of symptoms. For the GWAS discovery phase 8,997 women provided both incontinence phenotypes and genome wide genotypes. In meta-analysis, five loci included at least one genome-wide significant variant (p < 5x10⁻⁸). Twelve loci were taken forward for replication, with two demonstrating robust replication. In bladder biopsies we identified 1,115 significantly differentially expressed genes between stress and urgency incontinence. In the context of the previous literature, these results suggested EN1 and EDN1 as the most likely causal genes within the two replicated GWAS significant loci. Conclusions: This work highlights many of the challenges of identification of risk variants for complex conditions such as incontinence. The discovery of two novel risk loci for incontinence represents a significant advance in understanding the pathophysiology of these conditions.

Cadherins are a family of trans-membrane junctional proteins important in maintenance of cell-cell junction, phenotype regulation, tissue organisation and embryonic development. The proteins form calcium dependent homophilic cell junctional complexes and bind internally to the actin cytoskeleton and regulate intracellular signalling via the p- catenin pathway. Altered cadherin expression is essential for embryonic development, tissue repair or healing, fibrosis, cancer and metastasis. Much interest has developed in cadherin expression and its regulation along with signalling in renal proximal tubule epithelial cells (PTECs), an important cell type in the development of tubulointerstitial fibrosis and a potential source of myofibroblasts.