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Measurement of circulating renin activity in the rat is complicated by the need to obtain amounts of blood which are large in comparison with its blood volume. Removal of blood samples of the necessary magnitude required for standard methods (1-3) induces physiological responses which lead to an alteration of the level of peripheral renin.This difficulty has been approached in several ways. Gross and associates (4) have utilized an isovolemic cross circulation technique to demonstrate an alteration in renin concentration in the rats circulation. This approach lacks specificity and is expensive in its requirement of assay rats. Boucher and co-workers (5) have devised a micromethod, employing exogenous rat renin substrate, which enables assay of renin activity in the plasma of the unanesthetized rat. The small size of the sample taken does not cause any disturbance of the circulating levels of peripheral renin activity. They have established in a quantitative fashion the levels found in a variety of states (6).We have utilized dialysis of the serum sample against disodium EDTA to inactivate angiotensinase. This dialysis is accomplished without volume alterations when 1 ml of serum is used. Employing bilateral nephrec-* Supported by PHS Grant FR-5360-06, the John A. Hartford Foundation Inc., and the Georgetown Kidney Research Fund. J. M. H. was a Fellow of the National Kidney Foundation during the period in which this work was performed.tomy immediately before collection of a large blood sample we clearly separated the renin activity results occurring in several differing states.Methods. Experimental animals. Sprague-Dawley rats, weighing 250-350 g, of both sexes were used. Blood samples were collected from the right carotid artery after bilateral nephrectomy had been performed under sodium pentobarbital anesthesia, 40 mg/kg ip. The interval between nephrectomy and sacrifice by exsanguination was 15 min. The serum was stored frozen until dialyzed.Groups of rats were subjected to the following treatments: bilateral adrenalectomy without replacement therapy for 7 days; hemorrhage with the kidneys in situ; deoxycorticosterone (DOCA) in peanut oil im, 5 mg administered on alternate days for a total dose of 95 mg with 0.9% saline as drinking water; and 5% dextrose, 10 ml/kg im on one occasion.PeripheraJ renin activity determinution ( P R A ) . One-ml aliquots of sera were dialyzed in 8J32-in. cellophane tubing sacs against 0.003 M disodium EDTA in 0.9% sodium chloride solution for 16 hr in the cold. Two-tenths-ml of serum was added to a siliconized tube containing 0.2 ml of rat renin substrate; 0.1 ml of 0.001 M phenylmercuric acetate; 0.1 ml of 0.5 M sodium phosphate buffer, pH 5.5; 0.01 ml of 5% diisopropyfluorophosphate in isopropyl alcohol; and 0.4 ml of 0.9% sodium chloride solution. Incuat GEORGETOWN UNIV MED CTR on July 22, 2015 ebm.sagepub.com Downloaded from

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