| Summary: | Many cardiovascular diseases are associated with abnormal function of the endothelium, a condition commonly referred to as endothelial dysfunction (ED). ED is well characterised as the diminished capacity of the endothelium to mediate vasorelaxation that results from a decrease in the availability of nitric oxide (NO). However, additional physiological roles of NO may also be altered. For example, NO plays a vital role in promoting natriuresis/diuresis within the kidney and thus contributes to the maintenance of normal blood pressure. This prompted the hypothesis that diminished availability of NO as a result of ED in the kidney could result in sodium retention and may provide an explanation for the sustained elevation of blood pressure in hypertension. There are several reasons for why NO availability may be decreased during ED. One proposed explanation is altered activity of endothelial nitric oxide synthase (eNOS), the enzyme responsible for generating NO. However, little is known of the mechanisms involved in regulating eNOS activity within the kidney. This thesis describes experiments which aim to further elucidate the cellular and molecular mechanisms involved in the regulation and activation of renal eNOS and discusses how these may be affected during ED. Subcellular localisation to caveolae and interactions with proteins which reside there (e.g. caveolin-1, bradykinin receptor and endothelin type B receptor) are important features of eNOS regulation. The first approach was to determine if interactions between eNOS and regulatory proteins within the kidney were altered in an animal model of ED, the spontaneously hypertensive rat (SHR). Western blot analysis of isolated caveolae revealed no alterations in eNOS or caveolin expression in SHR compared to normotensive WKY rats. Likewise, no change in caveolin expression was observed in whole kidney homogenates, whereas eNOS expression was slightly increased. Although interactions between caveolin-1 and eNOS in isolated endothelial cells were observed, it was not possible to replicate this within tissue, and raises questions concerning the extent to which these proteins interact in vivo. These data imply that alterations in renal caveolin expression are not altered during ED whereas eNOS may be upregulated. Experiments then focused on activation of eNOS by endothelin-1. Within the renal medulla this is mediated by the endothelin type B receptor (ETB) and leads to inhibition of sodium reabsorption. However, the cellular mechanisms remain unknown due to the lack of precise localisation of medullary ETB. Therefore, localisation of ETB was investigated by introducing a LacZ reporter gene into the endothelin type B receptor locus (EDNRB) by homologous recombination in ES cells, thereby generating EDNRB-LacZ mice which expressed LacZ wherever ETB was expressed. These mice, together with another strain of EDNRB-Ea.cZ mice (supplied by Dr. M. Shin), were used to determine renal ETB localisation. Analysis of LacZ expression in homozygote mice revealed intense staining within the medulla, particularly within endothelial cells of the medullary capillaries. In contrast, renal tubules were negative for transgene expression. Since the vasa recta is also a major site of eNOS expression within the medulla, these results suggest that ET-1 activates eNOS by binding to ETB located on vasa recta. These observations enhance the understanding of the basic cellular and molecular mechanisms involved in ETB/eNOS mediated sodium transport and provide a platform for ongoing physiological experiments and investigations into potential pathways which may be associated with renal ED.
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