Bladder dysfunctions associated with overactive bladder are highly prevalent in the aging population and impose a huge financial cost, however the underlying mechanisms are poorly understood. The newly recognised sensory role of the urothelium provides a new direction of research into the pathogenesis of overactive bladder. As the activity of this sensory structure is highly important for correct bladder function, it is imperative to identify new regulators of this tissue. A role for transient receptor potential vallinoid 4 (TRPV4) channels in bladder function has been recently evidenced. This work aimed to explore the role of TRPV4 in urothelial ATP release, the key step in urothelial sensory transduction, elucidate the underlying mechanisms and assess changes to TRPV4 expression and function in aging and overactive bladders. Immunohistochemistry and western blotting assessed the expression and localization of TRPV4 throughout the bladder. Bladder strips from animals and humans were challenged with the selective TRPV4 agonist GSK1016790A and ATP release and tissue contractility measured. Various antagonists were employed to uncover the mechanisms. The role of Ca2+ in TRPV4-mediated responses was explored using live-cell Ca2+ imaging. The effect of TRPV4 activation on reactive oxygen species production was also measured. The interaction between TRPV4 and the principal urothelial receptor P2Y was assessed, supplemented with P2Y2 knockout mice. The responses between young and aging guinea pigs and normal and overactive human bladders were compared. This is the first study to establish a role for TRPV4 in mucosal ATP release and the underlying mechanisms. This study demonstrates increased TRPV4 expression with age and a functional link between TRPV4 and P2Y receptors as a result of aging. This work also provides preliminary evidence for an increased TRPV4-mediated ATP release in overactive human bladders. These novel findings identify a fundamental role for TRPV4 in bladder physiology and pathophysiology and present experimental evidence that pharmacological manipulation of this receptor may provide a novel method for treatment of overactive bladder.

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I have conceived, undertaken and published a body of work in Urology which has applied an evidence-based approach to different aspects with widely varying success in modifying the impact on treatment choices and outcome. On the positive side, the research work I have led has demonstrated that the era when death statistics could be used to the occurrence of benign prostatic hyperplasia was gone and that we had moved to an epoch where symptomatic disease and quality of life were the key issues. I have worked on the creation of the questionnaire-based approach necessary for evaluating the presence of various urological conditions for use in different populations clearly identified and quantified the extent and inter-relationships between the various benign urological conditions in communities. This work has made it quite clear that such benign conditions as benign prostatic hyperplasia, erectile dysfunction, incontinence, prostatitis and cystitis are remarkably common conditions world-wide in ageing populations. Since 1990, treatment options for men and symptomatic BPH have moved from an essentially surgical approach to an increasing introduction of pharmacologic options and less invasive approaches to disease management. The meta-analysis of the Phase III clinical trials of finasteride which I undertook, demonstrated that this drug was effective only in men with enlarged prostates and justified the biological approach taken in the development of this drug which was an inhibitor of 5-alpha reductase, the enzyme which converts Testosterone (T) to Dihydrotestosterone (DHT) the metabolite which made the prostate hyperplastic. I then demonstrated that serum PSA was a good indicator of prostate volume thus making identification of men who would most likely respond to this drug easily identifiable. Using this same dataset, I was able to demonstrate that finasteride reduced the risk of Acute Urinary Retention (AUR) and that it was superior to alpha-blockers, the other major class of drugs used to treat symptomatic BPH, in this regard. I designed the phase III trials of dutasteride, a new five-alpha reductase inhibitor, and the findings lay on the regression line demonstrated in the meta-analysis of finasteride and also had the identical effect on reducing the risk of AUR. I was able to develop a method of predicting individual risk of AUR in men who were diagnosed with benign prostatic hyperplasia.

Successful kidney transplantation is a life-saving procedure to patients with irreversible chronic renal failure. Despite the presence of various obstacles facing this surgery, preserving donor kidney and consequent ischemia reperfusion injury (IRI) are still major challenges affecting renal function as well as prognosis of transplant surgery. This study pursued two main aims: firstly, characterising changes in damage associated inflammatory gene expressions through developing, and analysis of an in vitro model of proximal tubular epithelial cells (PTEC) of normal human kidney mimicking renal IRI in vivo. The second aim was to simulate the concurrence of factors relevant to human intervention (renoprotective anaesthesia, peri- and postoperative analgesia, volume substitution) in mice deficient of properdin and congenic controls and to allow long-term observation of renal outcome after IRI. In this study, a reproducible and standardisable in vitro model was developed. It demonstrated the complexity of signalling where a multitude of factors affects the target cells. Secondly, the use of congenic properdin deficient mice showed that properdin has a significant role to play in renal injury (and recovery). There was significant impairment in renal function (and structure) compared to wildtype mice after IRI.

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Hypertriglyceridemia and its associated increase in triglyceride-rich lipoproteins (TGRLs) is a great threat to today’s modern society. Sedentary life style and western type diet subject our bodies to repetitive influx of TGRLs following each meal which is being increasingly recognised as a risk factor for coronary artery disease (CAD). Moreover, a hyper-TGRL state complicates the course of various immunological and non-immunological conditions contributing to both morbidity and mortality. How TGRLs mediate their harmful effects is unclear. In view of their ideal location, monocytes (MOs) are at the front line against fluctuating levels of TGRLs, yet the influence of such an environment on their behaviour remain obscure. With the growing recognition of MO heterogeneity, the impact of this diversity on MO functions deserves more attention. In the mouse, two MO subsets are recognized; Gr1-high or “inflammatory” MOs, known to populate inflamed tissue giving rise to macrophages (MØs) and the Gr1-low or “patrolling” MOs, shown to crawl along the endothelial surface under steady-state conditions and rarely extravasate. To explore whether changes in TGRLs could alter MO behaviour, I used a compound, P-407, that induces a dose-dependent increase in TGRLs in C57BL/6 (B6) mice. The increase in plasma TGRLs led to a sharp drop in blood Gr1-low MOs subsets while levels of Gr1-high MOs remained unchanged. The drop in Gr1-low MOs was associated with accumulation of CD68+ MØs in the liver, heart, and kidney. In the absence of an inflammatory insult, CD68+ MØs did not trigger kidney injury. However, with an inflammatory insult (accelerated nephrotoxic nephritis), the kidneys of P-407 injected mice exhibited more renal damage. Collectively my findings demonstrate that an environment loaded with TGRLs influences the steady state behaviour of MOs and MØs, and primes the kidney for injury during inflammation.

Progression of glomerular diseases to chronic kidney disease or renal failure is a major diagnostic and therapeutic problem. In addition to finding new treatment, more reliable biomarkers, using near patient technology, are needed to improve early detection of patients at risk of progressive renal injury. To achieve this aim, two approaches were applied to study novel biomarkers of glomerular inflammation and fibrosis. Firstly, expression of specific inflammatory cytokines and growth factors, including two members of the CCN protein family which are connective tissue growth factor (CTGF)/CCN2 and nephroblastoma overexpressed gene (NOV)/CCN3, were studied in a rat model of crescentic glomerulonephritis (GN). CCN2/CTGF is now regarded as a major profibrotic growth factor of chronic renal inflammation and fibrosis. On the other hand, recently, emerging evidence suggests that CCN3/NOV acts as an endogenous negative regulator of extracellular matrix accumulation and is capable of counteracting the fibrogenic effect of CCN2/CTGF. This targeted approach may be helpful in providing a novel insight into the pathophysiological activities of these two CCN proteins involved in progressive kidney disease and development of novel therapy. With regard to Fourier transform infrared (FTIR) spectroscopy, it has been increasingly used in biomedical research but so far it has not been investigated for diagnosing and/or screening progressive kidney disease. Thus, the second approach was aimed to investigate whether FTIR technique could be employed as an unbiased method of detecting sensitive renal biomarkers. Using FTIR spectroscopy, several characteristic urinary and plasma spectral markers related to renal inflammation and chronic fibrosis were identified from the rat model of crescentic GN. In particular the urinary 1545 cm-1 spectral marker was also reliable in assessing therapeutic responses in corticosteroid-treated rats with GN and severe lupus nephritis in mice. In addition, this urinary spectral marker was translatable in assessment of crescentic GN in patients. In conclusion, analysis of specific cytokines/growth factors and FTIR spectroscopic technology are likely to improve or assist diagnosis and evaluate progression of glomerulonephritis.

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No description available.

The principle aim of this thesis is to examine the hypothesis that patients with acute renal failure (ARF) who are treated with high dose loop diuretics have a better prognosis in terms of survival, need for dialysis and speed of recovery than those who do not receive these drugs. A prospective, double blind, randomised, placebo controlled study was carried out on 94 patients with ARF in Glasgow Royal Infirmary. The use of loop diuretics caused a significant increase in urine output and fractional excretion of sodium in the first 24 hours, but had no effect on the final outcome (recovery, dialysis or death) at day 21. Patients who became non-oliguric (with or without loop diuretics) had a better survival but were less ill (APACHE II score 17.2 v 20.6, p=0.007, non-oliguric v oliguric) and had less severe renal failure (creatinine clearance 14ml/min v 4.5ml/min, p < 0.0001) than those who remained oliguric. Loop diuretics can lower cytosolic calcium in normal individuals and in those with hypertension. Because cell death has been shown to be accompanied by a rise in intracellular calcium, I postulated that cytosolic calcium levels would be high in patients with ARF. Further, the use of loop diuretics might lower cytosolic calcium in patients with ARF and thereby exert a beneficial effect on renal tubule cells. Intraplatelet calcium levels were high in patients with ARF compared to normal controls (109nm v 92.4nm, p=0.004). Administration of a loop diuretic had no effect on intraplatelet levels of calcium. 1 hypothesised that this rise in intracellular calcium might be related to the severity of illness and thus correlate with the APACHE II score, an objective scoring system used to stratify patients according to prognosis. No correlation was found. Finally, indirect calorimetry is an accurate, although painstaking, method of measuring energy expenditure in the critically ill patient. Metabolic rate may be related to clinical outcome. If the resting energy expenditure (REE) correlated with the APACHE II score, the latter, simpler measurement could be used as part of a formula to predict metabolic rate. My studies of REE in patients with ARF showed no correlation with the APACHE II score which should therefore not be used to predict energy expenditure in ARE. Nor was there any association between metabolic rate and the clinical outcome of the patient.