Project 5 examines the role of the angiotensin II in the anatomical loss of microvessels (rarefaction) that occurs in animals fed a high salt diet. He have shown that an elevation in sodium intake can trigger a series of events culminating in a substantial rarefaction throughout the microcirculation in normal rats. In the previous funding period we have demonstrated that rarefaction of the microcirculation can cause an increase in total peripheral resistance, reduced tissue perfusion, decreased oxygen delivery, and impaired organ function. Although the mechanism by which this occurs are not well understood, a number of key observations point to a role for the renin-angiotensin system in this effect. First, maintenance of ANGII at normal levels during periods of HS diet completely eliminates rarefaction. Second, elevated salt intake causes a decrease in microvascular AT/1 receptors which are growth stimulatory and an increase in the microvascular AT/2 receptors which are growth inhibitory. Third, ANGII infused either systematically at sub- pressor levels, or locally into the skeletal muscle interstitium, can induce significant micro-vessel growth. The present study will examine the hypothesis that during period of high slat intake, suppression of either local or circulating ANGII mediate rarefaction. We further hypothesize that actions and augment the rarefaction caused by ANGII suppression. Using a highly sensitive and specific method for the measurement of tissue ANGII that was developed in our laboratories, we have shown that the concentration of ANGII in microvessels of the cremaster muscle is at least 5 times that measured in plasma. To our knowledge this is some of the most direct evidence supporting a role for the local renin-angiotensin system in the microcirculation. The goal of this project is to explore the regulation of the local vascular renin-angiotensin system in the microcirculation and to determine its role in microvascular rarefaction. The role of ANGII in the remodeling of the microcirculation will be assessed in chronically instrumented rats using direct intravenous and local infusions of ANGII to precisely control circulating and local concentrations. Mechanisms of the local control of the renin gene expression will be studied in a unique set of congenic Dahl rat strains in which the R renin gene has been introgressed into the S rat genetic background. We will take advantage of the difference in the renin response in these two unique, genetically matched, strains of rats to test the hypothesis that modulation of the renin-angiotensin system is responsible for the rarefaction due to salt. The application of several exciting and novel techniques will enable examination of this hypothesis. Micro-vessel function will be evaluated in vivo using computer video microscopy. Morphological changes in the microcirculation triggered by salt will be evaluated using quantitative stereological techniques that we have developed and used extensively. Localization of angiotensin receptors in the microcirculation will be carried out using highly specific AT/1 and AT/2 antibodies for immune-histochemistry and immuno-blotting of protein isolated from microvessels dissected from skeletal muscle. The effects of salt intake and circulating levels of ANGII on the distribution of AT/1, and AT/2 mRNA and protein throughout the microcirculation will be determined using competitive RT-PCR, immuno-histochemistry and Western blots to determine if the expression of these receptors is regulated by ANGII. Finally, measurements of circulating and vascular ANGII levels by HPLC combined with local infusion of ANGII and ANGI and local blockade of ACE will determine if change in the locally acting angiotensin system plays a significant role in the remodeling of the microcirculation.
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