There is a balance between factors promoting renal salt and water retention (angiotensin, reactive oxygen species) and those favoring excretion (NO, bradykinin). Inappropriate salt retention may lead to hypertension and/or heart failure. This project focuses on one factor favoring salt and water excretion, NO. NO produced by the kidney is an important regulator of renal function. Renal NO production is regulated by a number of factors, including dietary salt intake. The kidney expresses all three NOS isoforms, but the significance of the patterns of expression and the contribution of a single isoform in a single renal structure to the regulation of salt and water excretion have not been studied. The thick ascending limb absorbs 20-30% of the filtered NaCl load, which is essential for proper Na balance. We have reported that NO produced by endothelial nitric oxide synthase (eNOS) in the thick ascending limb acts as an autacoid to inhibit transport. However, it is not known if a high-salt diet enhances the autocrine actions of eNOS, if thick ascending limb eNOS also acts as a paracrine factor and what mediates the effect of a high-salt diet on medullary eNOS expression in general and thick ascending limb eNOS specifically. We hypothesize that NO produced by thick ascending limb eNOS enhances salt and water excretion via autocrine inhibition of thick ascending limb transport, and paracrine inhibition of tubuloglomerular feedback and augmentation of medullary blood flow. A highsalt diet enhances the autocrine and paracrine effects of NO produced by thick ascending limb eNOS, leading to enhanced NaCl excretion. We will address this hypothesis using physiological, pharmacological and molecular techniques. In Aim 1 we will test whether autocrine effects of NO produced by thick ascending limb eNOS on transport are enhanced by a high-salt diet and the mechanism of this inhibition. In Aim 2 we will examine the mechanism whereby a high-salt diet stimulates thick ascending limb eNOS expression. In Aim 3 we will examine whether the second messenger cascade activated by thick ascending limb eNOS is also upregulated by a high-salt diet. In Aim 4 we will test whether NO produced by the thick ascending limb acts as a paracrine factor, diffusing to other tissues and nephron segments and regulating their function, and whether a high-salt diet enhances the paracrine effects of thick ascending limb eNOS. Finally, in Aim 5 we will investigate whether the combined autocrine and paracrine actions of NO produced by thick ascending limb eNOS contribute significantly to renal Na and water excretion in vivo, using newly developed transgenic mice that only express eNOS in the thick ascending limb. These studies are important because they will be the first to investigate the role of a single NOS isoform expressed in a single nephron segment in renal NaCl and water excretion. They will be the first to demonstrate that different amounts of NO produced by thick ascending limb eNOS act via different mechanisms to enhance salt and water excretion. Finally, they will be the first to directly show the paracrine actions of NO produced by the thick ascending limb. The data obtained from this project will significantly advance our understanding of the role of NO in general, and thick ascending limb eNOS in particular, in salt and water excretion during a high-salt diet. Such knowledge is essential to our understanding of the pathogenesis of hypertension, particularly among African Americans in whom hypertension is often salt-sensitive, and may lead to new insights for therapeutic intervention.