This is a proposal to study the role of nitric oxide (NO) derived from different isoforms of nitric oxide synthase (NOS) in integrating the tubular and vascular functions in the kidney during changes in salt intake. An antibody to a constitutive NOS from rat brain (B-NOS) localizes strongly to rat macula densa (MD), whereas an antibody to rat induced vascular smooth muscle cells (VSM-NOS). Using reverse transcription polymerase chain reactions (RT-PCR), cDNA's corresponding to B-NOS, MAC-NOS and endothelial cell NOS (EC-NOS) have been amplified from normal rat kidney. The hypothesis that will be tested is that an increase in dietary salt, leading to increased NaCI delivery and reabsorption by the MD segment, activates B-NOS in this segment; the ensuing increase in NO vasodilates the afferent arteriole and blunts the vasoconstrictor signals generated by tubuloglomerular feedback (TGF). In contrast, a reduction in dietary salt increases the generation of angiotensin II (Ang II) which, over the long term, reduces the expression of MAC-NOS in the afferent arteriole; the ensuing reduction in NO promotes pre-glomerular vasoconstriction and TGF responses. Therefore, the juxtaglomerular NOS system acts homeostatically to adapt glomerular filtration to changes in dietary salt. The first two aims propose to use these antibodies, primers and probes to study the distribution, ultrastructural localization and level of expression of different isoforms of NOS in the JGA of normal rats and those given lipopolysaccharide (LPS) to induce NOS. Thereafter, studies will combine immunocytochemistry, quantitative RT-PCR of microdissected afferent and efferent arterioles, glomeruli and MD-containing tubule segments with functional studies based on nephron micropuncture and microperfusion. These techniques will be used to investigate how dietary salt intake alters the expression and function of JGA NOS isoforms. The preliminary results indicate that the expression of one pool of NOS in the endothelium and the afferent arteriole is regulated by Ang II and sets the operating level of TGF while the activity of a second pool of NOS in the MD is set by the level of NaCI delivery and reabsorption at this segment, and mediates a novel vasodilator arm of TGF. Specific inhibitors of NOS, or donors of NO, will be perfused into the renal artery and MD to study the functions of the L-arginine NO pathway in the JGA. Delivery and reabsorption of NaCI at the MD will be varied with dietary salt and diuretics. Ang II receptor activation will be varied with prolonged Ang II infusion or losartan. These protocols will dissociate the effects of dietary salt, Ang II and MD NaCI delivery and reabsorption. This research is devoted to micropuncture physiology, immunocytochemistry and molecular biology.
It aims to define the homeostatic role of NOS in adapting the kidney to changes in salt intake and thereby to provide a framework for future studies of salt-sensitive hypertension.
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