P2X7 is a unique member of the P2X family of membrane receptors for extracellular ATP. As well as being a non-selective cation channel, this membrane receptor is implicated in several biological functions, including cell death and proliferation. P2X7 expression, initially thought restricted to immune cells, also occurs in epithelial and other cell types. I hypothesised that P2X7 is functionally expressed in the renal tract, particularly in situations in which cell turnover is prominent. I have demonstrated expression of P2X7 in both mouse and human kidney. During mouse nephrogenesis P2X7 was detected in the condensing mesenchyme in early metanephrogenesis and was subsequently restricted to derivatives of the ureteric bud i.e. collecting duct and ureter. P2X7 was immunolocalised in regions of cell turnover, consistent with a role for the nucleotide receptor in nephrogenesis. P2X7 was also detected in cystic collecting ducts of both the cpk/cpk mouse model of autosomal recessive polycystic kidney disease (ARPKD) and human ARPKD. Next I investigated the potential function of P2X7 in cystogenesis using a 3D suspension culture model from the cpk/cpk mouse. Exposure to agonists of the P2X7 receptor caused a significant reduction in numbers of cysts forming in vitro. This was inhibited by P2X7 antagonists, and was greater than the response to other nucleotides, supporting a specific mediation by the P2X7 receptor. My study did not demonstrate a significant effect on markers of cell proliferation, apoptosis or necrosis correlating with P2X7-mediated reduction in cyst numbers, suggesting an alternative function for the receptor in cyst formation. To further understand the molecular mechanisms by which P2X7 mediates its functions, particularly apoptosis, I have developed an in vitro expression system. Stable transfection of a chicken lymphocytes with rat P2X7 enabled characterisation of the receptor's membrane properties, both ion fluxes and pore potential, and established a novel tool with which to further examine the mechanisms by which P2X7 mediates cell death. Further understanding of the molecular mechanisms of this unique nucleotide receptor, and its functional roles in the kidney, particularly in the setting of polycystic kidney disease may in the future lead to a novel therapeutic target for the manipulation of progression of renal injury, via its apoptotic pathways, or other as yet undefined pathways.

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Active uptake of the organic cation, tetraethylammonium (TEA), was shown in freshly isolated human and rat PT cells. This uptake was both inhibitable and temperature dependent. Several other drug substrates including ipratropium, procainamide, amantadine and pindolol were also actively taken up by fresh HPT cells. The human organic cation transporter, hOCT2, is the principal uptake transporter in the human proximal tubule. HEK cells transfected with hOCT2 were used to screen several drugs as possible substrates for this transporter. Substrates identified included TEA, amantadine, quinine, quinidine, procainamide, pindolol, oxytropium, ipratropium, and imipramine. Kinetic parameters for five of these were used to calculate their intrinsic clearances, which were correlated with renal clearance values in vivo. This correlation was significant (R2=0.814), suggesting that the HEK-hOCT2 cell line can be used to make predictions of the renal clearance expected in man for compounds that are hOCT2 substrates. The second-messenger pathways involved in the regulation of organic cation transport in freshly isolated HPT and RPT cells and HEK-hOCT2 cells were elucidated. Several pathways, most notably involving ALP, PKC and calmodulin, are involved in the regulation of organic cation transport in all these systems. Quantitative RT-PCR and immunoblotting were used to detect changes in expression of organic ion transporters in primary cultures of PT cells compared with those in vivo. Expression of apical ion transporters was maintained in these culture systems, while expression of those located basolaterally was down-regulated. As well as successfully predicting in vivo clearance data from cellular uptake rates in vitro, this is the first comprehensive study correlating functional organic cation transport mechanisms with a number of signalling pathways in human renal proximal tubules.

VEGF plays an important role during nephrogenesis and glomeulogenesis. In most adult tissues VEGF is downregulated but it remains constitutively expressed in capillaries with fenestrated endothelium such as in the glomeruli. In the kidney, VEGF is expressed and secreted by podocytes and its receptors are expressed on endothelial cells. The role of constitutive VEGF expression in the adult glomerulus is only partially understood. The aim of this study was to generate an in vivo system whereby VEGF action could be inhibited specifically in the kidney and selectively in the adult fully developed glomeruli. Our working hypothesis was that VEGF could be responsible for the maintenance of endothelial fenestration in glomerular capillaries and possibly playing a role in the filtration or water and solutes. Using a transgenic approach, we over-expressed, with an inducible system, specifically in the glomeruli the soluble truncated VEGF receptor (sFlt-I). By over-expressing sFlt-l in our system, we obtained some preliminary results that suggest a critical role ofVEGF in the functionality of the glomerular filtration barrier.

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There is increasing evidence that extracellular nucleotides (such as ATP, ADP, UTP and UDP), as well as the nucleoside adenosine, behave as autocrine or paracrine agents in most tissues including the kidney, acting on a group of receptors known as purinoceptors. Previous studies have shown that activation of these receptors by exogenous nucleotides can influence a variety of renal vascular and tubular functions. Purinoceptors of various subtypes are present on basolateral and apical membranes of renal tubules. However, the extent to which apical receptors are stimulated by endogenous nucleotides is unknown. Using micropuncture, the first part of this study quantified endogenous ATP in the lumen of proximal and distal tubules of the rat in vivo, both under control conditions and during pathophysiological manoeuvres. The results showed that ATP levels were sufficiently high to activate some purinoceptor subtypes. To assess whether the intraluminal ATP was being secreted or merely filtered at the glomerulus, the ATP content of fluid from Bowman's space (in Munich-Wistar rats) was compared with that in proximal tubules. The conclusion was that tubular epithelial cells secrete ATP. Using a proximal tubular epithelial cell line, the mechanism of ATP release was examined. Intracellular stores of ATP were visualised using a marker compound (quinacrine), and the fate of these stores was monitored following hypotonic stimulation of ATP release. The findings suggested that ATP is stored within the cytoplasm, possibly in vesicles, and is released by exocytosis. In the final part of the investigation, using immunohistochemistry, the distribution of five nucleotide-hydrolysing ectonucleotidases, namely NTPDases 1-3, NPP3 and ecto-5'- nucleotidase, was examined along the rat nephron. These enzymes (which differ in their hydrolysis pathways) were found to be differentially expressed along the major segments of the nephron, suggesting that they may be strategically located to influence the activity of the different purinoceptor subtypes.