The urinary tract is lined by a mitotically-quiescent, but highly regenerative epithelium, the urothelium. The mechanisms regulating urothelial regeneration are incompletely understood although autocrine stimulation of the Epidermal Growth Factor Receptor (EGFR) signalling pathway has been implicated. The hypothesis developed in this thesis is that urothelial homeostasis is regulated through resolution of interactive signal transduction networks downstream of local environmental cues, such as cell:cell contact. Here, canonical Wnt signalling was examined as a candidate key pathway due to the pivotal role of β-catenin in both nuclear transcription and intercellular adherens junctions. Normal human urothelial (NHU) cells isolated from surgical biopsies were grown as finite cell lines in monolayer culture. mRNA analysis from proliferating cultures inferred all components for a functional autocrine-activated canonical Wnt cascade were present. In proliferating cells, β-catenin was nuclear and Axin2 expression provided an objective hallmark of β-catenin/TCF transcription factor activity. This endogenous activity was not mediated by Wnt receptor activation, as Wnt ligand was produced in inactive (non-palmitylated) form in serum-free culture, but instead, β-catenin activation was driven via EGFR-mediated phosphorylation of GSK3β and inhibition of the β-catenin destruction complex. In quiescent, contact–inhibited cultures, β-catenin was seen to re-localise to the adherens junctions and GSK3β activity was re-established. Knock-down of β-catenin using RNA interference led to significant changes in p-ERK and p-AKT activity as well as an increase in E-cadherin protein expression. The results presented in this thesis identifies β-catenin as a central component of a bi-directional feedback loop between growth factor-mediated cell signalling and cell:cell contact and provides preliminary evidence that de-regulation of the mechanisms that control β-catenin regulation and EGFR signalling are important in neoplastic growth.

Arginine vasopressin (AVP) is also known as anti-diuretic hormone (ADH). The two major effects of this peptide, that of increasing blood pressure by vasoconstriction and reducing water loss by promoting water re-absorption in the kidney, are described as its primary functions. But other effects of AVP have been demonstrated. For example, in the adult mammal AVP has a role in platelet aggregation, hepatic glycogenloysis, and memory consolidation, and the purpose of the course of study described in this thesis was to examination a putative alternative function for AVP. In certain rat myogenic cell lines introduction of vasopressin results in promotion of fusion and up-regulation of muscle specific gene expression. This effect has been described as being mediated by the V1a-vascular receptor. In addition, Data were published suggesting there was a significant amount of AVP immunoreactivity (ir-AVP) in human foetal skeletal muscle. ir-AVP was described at concentrations that could not be explained by plasma concentration, and described in relation to gestation age. Taken together these results suggest a significant role for vasopressin in skeletal muscle for development, and point to an additional alternative site for the synthesis of biologically active AVP. An extraction method was developed which employed solid phase extraction (SPE) followed by radioimmunoassay. The physical recovery of the SPE stage was reproducibly better than 70% when extracting AVP from homogenised muscle tissues. The radioimmunoassay had a cross reactivity of less than 0.01% with both oxytocin and arginine vasotocin. This extraction method was developed as a response to the demonstration that the direct assay of acidified extracts did not supply an accurate measure of the amount of vasopressin in extracted muscle. The reported ir-AVP was shown to probably be the result of acid inference in the assay. Levels of ir-AVP from foetal muscle samples extracted using SPE were not significant. This was in contrast to levels found in several positive control tissues - human foetal adrenal and pituitary glands, and rat adult adrenal glands - that were in close agreement with previously published data.

Renal tubulointerstitial fibrosis is a major predictor of progressive glomerular disease. It occurs as a result of persistent inflammation and is characterised by excessive deposition of extracellular matrix (ECM) proteins, including accumulation of type I collagen, the most abundant protein of the ECM. Type I collagen is encoded by two genes, COL1A1 and COL1A2, that are tightly regulated at a transcriptional level. A key aim of this study was to use the previously identified COL1A2 promoter and enhancer sequences in order to identify novel regulatory cis-acting elements and the relevant transcription factors that regulate collagen transcription in cells derived from healthy or diseased kidneys. Moreover, the effects of hypoxia and transforming growth factor beta (TGFβ), which are both profibrotic stimuli, on collagen transcription were studied. TGFβ is known to activate CDP/cux transcription factors which can suppress gene activation; based on this finding the role of CDP/cux in COL1A2 transcriptional regulation was assessed. In conclusion,the work presented in this thesis provides an insight into the complex control mechanisms that regulate collagen transcription in both physiological and pathological conditions.

The mouse renal collecting duct is a heterogeneous segment of the nephron composed of principal cells and three types of intercalated cells Ca, p and non-a non-p intercalated cells). The selective expression of K+ and cr channels in the plasma membranes of these cells plays an important role in K+ and cr handling, maintaining the resting membrane potential and transport function and cell volume regulation in these cell types. The M8 collecting duct cell line is a new model of the collecting duct from embryonic day 18 mice. In the first section of the study we characterized the M8 cells by investing mRNA expression patterns. The mRNA expression favored a principal cell-like cell model, which lacked some key principal cell markers. Stimulating differentiation by increasing temperature had no effect, suggested a fully differentiated cell line. The whole-cell patch-clamp technique was used to investigate the physiological properties of anion and cation currents in the cells. A large cr conductance demonstrated properties similar to that of ICJ(swellh which is found in variety of tissues. Functionally ICI(swell) is involved in the regulation of cell volume. A smaller Ca2+-insensitive inwardly rectifying K+ conductance was assumed to help maintain the resting membrane potential. K+ channels in the cortical collecting duct have been studied for over 20 years and there is much molecular and functional evidence in the literature. A technique to measure the tubule diameter allowed us to measure the response of the cortical collecting duct to a hypotonic shock. Through the application of selective blockers a role was identified for Ca2+-activated K+ channels and Kir channels in volume regulation of the cortical collecting duct. In contrast, a much smaller body of evidence is available on the physiological role of cr channels in the cortical collecting duct. Whole-cell recordings from intercalated cells of the cortical collecting duct demonstrated a CIC-K2-Iike current, consistent wiII previous evidence suggesting CIC-K2 is involved in the cr recycling and reabsorption across the basolateral membrane.

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The aims of the present study were to characterise the location and morphology of ICC in the urethra and bladder and to examine their structural relationships with nerves and smooth muscle. Also, to investigate the physiological role of ICC in the normal bladder by investigating the Ca2+ signalling in smooth muscle cells and ICC, in in situ mucosal preparations of guinea pig bladder, using ca2+~imaging techniques. Rabbit urethras were fixed, labelled with antibodies and examined with confocal microscopy. Staining with Masson's Trichrome showed the arrangement of smooth muscle bundles into distinct layers. Anti-c-kit revealed a population of immuno-positive ICC, which were either elongated with several lateral branches or stellate with branches coming from a central soma. ICC were found throughout the muscularis. c-kit-positive ICC were found on the boundary of the myosin-positive smooth muscle bundles, and also in the spaces between. Anti-c-kit and anti-neurofilament showed frequent points of close contact between the c-kit-positive ICC and nerves some of which were immunopositive for nNos. Spontaneous Ca2+ transients were investigated within the live guinea pig bladder using Oregon green Ca2+ indicator. Spontaneous smooth muscle Ca2+ transients took two forms, uncoordintated single cell Ca2+ events, and coordinated whole bundle flashes. ICC generated Ca2+ transients had longer durations and were significantly less frequent than smooth muscle cell events. There was little evidence of correlation between the spontaneous Ca2+ transients of smooth muscle cells and those of ICC. Both the smooth muscle cell and ICC activity were affected by various store and channel blockers. In conclusion, ICC were located throughout the wall of the rabbit urethra, where they came into close contact with smooth muscle cells and nerves. ICC could have a role in the excitation of smooth muscle cells in the urinary bladder, providing a possible therapeutic target for many urinary tract pathologies.

Previous work in the field has demonstrated two functionally distinct cell types in the rabbit urethra (Sergeant et aI, 2000) but it had never been adequately established that these cells could be clearly distinguished at a molecular level. In this study we demonstrate that urethral SMC and ICC differ at a molecular level. Using a wide range of molecular markers we have demonstrated that cells which were taken to be SMC, by their bright field appearance, did show expression of smooth muscle myosin and did not express any of the other markers. On the other hand, cells that were thought to be ICC expressed c-Kit and vimentin but did not express smooth muscle myosin. ICC exhibit rhythmic Ca2+ oscillations and it is believed that this activity underlies pacemaking in this tissue. Maintenance of this activity depends on influx of calcium through the plasma membrane and on activation of functional RyR and IP3 sensitive stores (Johnston et aI, 2005; Bradley et aI, 2006). We have identified RyR3 as being an essential component for the generation of Ca2+ waves. This statement is based on our finding that RyR3 is expressed in ICC, but is not expressed in SMC, which do not exhibit rhythmic oscillations. A third aim of the present study was to search for a molecular correlate of Ca2+ influx. From current research the most likely candidate appeared to be NCX and, interestingly, we were only able to detect the NCX3 isoform in urethral ICC. This conclusion is supported by the finding, in a recent study (Bradley et aI, 2006), that Ca2+ influx occurs via reverse mode NCX. We have demonstrated that HEK cells transfected so as to express RyR3, became rhythmically active which suggests that this RyR isoform is important in generating intracellular calcium oscillations. We demonstrated that these oscillations had an identical pharmacological profile to those observed in urethral ICC thus making HEK-RyR3 an ideal model for future work.

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No description available.