Renin Reactivity in Plasma of Patients with Normal Renin and Low Renin Essential Hypertension CHARLES S. BROOKS, RAMESH T. TALWALKER, AND THEODORE A. KOTCHEN Department of Medicine, University of Kentucky College of Medicine, Lexington, Kentucky 40506 tension, PRR was suppressed (P < 0.005) during 30, 60, and 180 min incubations in the low renin patients. Overall, in the hypertensive patients, there was a significant positive correlation (r = +0.58; P < 0.01) between PRR and the PRA response to furosemide. PGA in patients with low renin hypertension (0.86 ng/ml ± 0.06 SE) was less (P < 0.05) than that in patients with normal renin hypertension (1.10 ng/ml ± 0.07 SE) and control subjects (1.18 ng/ml ± 0.10 SE); PGA of normal renin patients and control subjects did not differ (P > 0.1). These results suggest that an alteration of the kinetics of the renin reaction may contribute to the apparent renin suppression in patients with low renin hypertension. Hypertensive patients with suppressed PRA also have low PGA. (J Clin Endocrinol Metab 44: 322, 1977)
ABSTRACT. Plasma renin reactivity (PRR) is the rate of angiotensin generation in vitro after addition of exogenous renin to plasma. To evaluate the hypothesis that suppressed plasma renin activity (PRA) in patients with low renin essential hypertension may be related to an alteration of the kinetics of the in vitro renin reaction, PRR was compared in plasma of patients with low renin and normal renin essential hypertension. Prostaglandin A (PGA) inhibits renin, and PGA was also measured to determine if suppressed PRA may be related to increased PGA. Low renin and normal renin hypertension were defined by comparing PRA responses of 30 hypertensive patients and 16 matched control subjects to upright posture and furosemide (80 mg p.o.). Nine of 30 patients had low PRA. Compared to that in plasma of patients with normal renin hyper-
P
LASMA renin activity (PRA) is suppressed in approximately 25% of patients with essential hypertension (1). Although increased production of a specific mineralocorticoid other than aldosterone has been demonstrated in a relatively small per cent of these patients (2-4), in the majority of patients with low renin essential hypertension, the mechanism for the apparent renin suppression has not been defined (1). Because PRA is a measure of the in vitro rate of angiotensin production, the measurement of PRA may be influenced by renin enzyme concentration, renin substrate concentration, and by additional circulating factors in plasma that may modify the reninrenin substrate reaction (5). It is possible that suppressed PRA in patients with low renin hypertension may reflect an alteration Received July 23, 1976. Supported by Grant HL15528 and NephrologyUrology Training Program AM05701, both from the National Institutes of Health, and by a grant from the Eagles' Max Baer Heart Fund. Address correspondence to: Theodore A. Kotchen, M.D., Department of Medicine, University of Kentucky College of Medicine, Lexington, Kentucky 40506.
of the kinetics of the in vitro renin reaction, rather than decreased renin enzyme concentration. We have previously used the term plasma renin reactivity (PRR) to refer to the in vitro rate of angiotensin generation after addition of exogenous renin to plasma (6). Differences of endogenous renin are minimized by adding relatively high concentrations of exogenous enzyme. Compared to that in plasma of normotensive control subjects, PRR is increased in plasma of patients with essential hypertension (6-8) and to a greater extent in plasma of patients with renal insufficiency (6). Increased PRR in hypertensive and uremic plasma is not related to differences of renin substrate or angiotensinase activity (6), and may reflect the deficiency of a normally occurring acetone soluble renin inhibiting factor (9). The purpose of the present study is to compare measurements of PRR in patients with normal renin and low renin essential hypertension. In addition, we have recently shown that high concentrations of a renomedullary prostaglandin, prostaglandin A (PGA), inhibit the in vitro renin reaction (10). In the pres-
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RENIN REACTIVITY IN HYPERTENSION
ent study, measurements of PGA are also compared in patients with normal renin and low renin hypertension to determine if suppressed PRA may be related to increased circulating PGA. Materials and Methods To identify patients with low renin essential hypertension, PRA was measured after an overnight fast, before and 4 h after the combined stimulus of 80 mg furosemide, given orally, and the upright posture in 30 patients with essential hypertension and in 16 matched normotensive control subjects. Informed consent was obtained from all hypertensive patients and control subjects, and the use of this protocol was approved by the Human Investigations Committee at the University of Kentucky. Hypertensive patients and control subjects had not received any medications for at least two weeks; women on estrogen therapy were excluded from study. Essential hypertension was diagnosed on the basis of complete history and physical examination, urinalysis, measurement of creatinine clearance and serum Na+, K+, Cl~, and CO2 concentrations, rapid sequence iv pyelogram, and in selected patients measurement of 24 h urine VMA and/or catecholamine excretion and renal angiography. Mean arterial blood pressures of the hypertensive patients and normotensive control subjects were 124 mmHg ± 12 SD and 93 mmHg ± 8 SD, respectively; respective mean ages of the two groups were 48 years ± 11 SD and 42 years ± 12 SD. Sixty per cent of the hypertensive patients and 37% of the control subjects were black; 23% of the patients and 25% of control subjects were male. In addition to PRA, plasma aldosterone and PGA concentrations were measured before and 4 h after furosemide; serum creatinine concentration was measured before furosemide. Urine excreted during the 4 h period following furosemide was collected and analyzed for Na+, K+, and aldosterone concentrations. PRR and plasma renin substrate (PRS) were measured in plasma of hypertensive patients before furosemide. PRR is not affected by sodium deprivation (unpublished observations) and consequently was not measured again after furosemide. Urine Na+ and K+ concentrations were measured with a flame photometer. Serum creatinine concentrations were measured by the method of
323
Kennedy etal. (11). Plasma and urine aldosterone concentrations were measured in quadruplicate by the radioimmunoassay procedure described by Ito et al. (12), except that bound and free aldosterone were separated with dextran coated charcoal rather than Florisil. Plasma renin activity was measured in quadruplicate by the radioimmunoassay procedure of Haber et al. (13), previously validated in our laboratory by bioassay (14). For the measurement of both PRS and PRR, renin was extracted from human kidneys by an ammonium sulfate precipitation procedure of Haas (15). The concentration of renin in this preparation was determined by comparing the rate of angiotensin I generated, in the presence of excess renin substrate, with a human renin standard obtained from the Medical Research Council (Division of Biological Standards, National Institutes for Medical Research, London, England). Plasma renin substrate concentration was measured, as previously described (6), by adding a high concentration of exogenous human renin to plasma (1.5 x 10~2 GU/0.5 ml plasma) and measuring the concentration of angiotensin I generated after exogenous substrate had been completely consumed. For the measurement of plasma renin reactivity, a lower concentration of exogenous human renin was added to plasma (7.3 x 10~5 GU/0.5 ml plasma), and the concentrations of angiotensin I generated were measured after 30, 60 and 180 min incubation periods at 37 C and pH 7.4. At the time of venepuncture, blood was anticoagulated with sodium EDTA to inhibit calcium dependent angiotensinases. Dimercaprol (1.6 DIM) and 8-hydroxyquinoline (3.4 mM) were added to in vitro incubations to inhibit converting enzyme activity. Tris buffer (100 ul/ml), pH 7.4, was added to maintain a constant pH during the incubation, and neomycin sulfate (50 ul/0.5 ml of a 10% solution) was added to inhibit bacterial growth. To adjust for relatively small differences of angiotensin generation due to endogenous renin activity, angiotensin production was measured in the absence of exogenous enzyme in a control aliquot of each plasma sample at each incubation period. For the reported PRR values, concentrations of angiotensin generated by endogenous renin, which were invariably less than 3% of the total, were subtracted from the amount of angiotensin measured after addition of exogenous enzyme. Production of rabbit anti-PGA antibodies, ex-
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statistical significance of differences was computed with analysis of variance. Student's t test was used to determine the significance of differences between two groups.
Results
PRE POST HYPERTENSIVES (n.JOl
PRE
POST NORMALS
FIG. 1. Mean (±SE) PRA before and after furosemide.
traction of prostaglandins with ethylacetate, separation of PGA on silicic acid columns, and radioimmunoassay for PGA were carried out according to the procedures described by Zusman et al. (16). Compared to PGA2, cross-reactivity of our antibody was 43% with PGAj, 19% with PGEl5 0.4% with PGE2, and there was no detectable cross-reactivity with PGF. Prior to assay, PGE was separated from PGA by silica gel column chromatography. PGB is not separated from PGA in this system. However, the lack of prostaglandin isomerase in human plasma suggests that contamination with PGB is of little consequence (17). For assay, the standard curve was derived using PGA2 standard, (obtained from Upjohn Co., Kalamazoo, Michigan) ranging from 10 pg/ml to 1,000 pg/ml, and tritiated PGA tracer (obtained from New England Nuclear, Corp, Boston, Mass.). The final dilution of antibody, for 50% displacement of tracer PGA, was 1:750. To validate further the assay, peripheral venous PGA was measured in 8 control rabbits and 8 indomethacin treated rabbits (10 mg/kgm body weight given SC 20 h, 9 h and again at 2 h prior to bleeding). Control animals received SC injections of the sodium carbonate vehicle. Mean PGA concentration of indomethacin treated animals (0.24 ng/ml ± 0.06 SE) was significantly lower (P < 0.05) than that of control animals (0.39 ng/ml ± .04). All PGA measurements were done in duplicate. In instances where differences among more than two groups were compared simultaneously,
Before furosemide, mean PRA in normotensive control subjects (0.9 ng/ml/h ± 0.2 SE) and patients with essential hypertension (0.9 ng/ml/h ± 0.2) did not differ (Fig. 1). After furosemide, PRA increased significantly in both control subjects and hypertensive patients (P < 0.001); however, mean PRA of patients with essential hypertension (1.9 ng/ml/h ± 0.3 SE) was significantly (P < 0.05) lower than that of normotensive control subjects (3.1 ng/ml/h ± 0.5 SE). PRA was undetectable both before and after furosemide in one 65-year-old black normotensive man. Excluding this subject, the lowest PRA after furosemide in normotensive subjects was 0.8 ng/ml/h. Nine of the 30 patients (30%) with essential hypertension had stimulated PRA lower than this value, and these patients were designated as having low renin essential hypertension. Comparing patients with low renin and normal renin hypertension, mean arterial pressure (126 mmHg ± 4 SE vs. 123 mmHg ± 3 SE) and age (45 yrs. ± 2 SE vs. 49 yrs. ± 2) were similar (P > 0.3) in both groups. Of the low renin patients, 89% were black; 48% of normal renin patients were black (P < 0.05). Twenty-two per cent of low renin and 24% of normal renin patients were male. Mean serum creatinine concentrations of normotensive subjects, patients with low renin hypertension, and patients with normal renin hypertension did not differ (P > 0.05) significantly (Table 1). During the 4 h after furosemide, sodium and potassium excretion of hypertensive patients did not differ from respective values in control subjects (P > 0.5), suggesting a comparable furosemide induced stimulus for renin secretion. Furthermore, comparing patients with low renin and normal renin hypertension, sodium and potassium excre-
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RENIN REACTIVITY IN HYPERTENSION tion did not differ (P > 0.8). Mean serum sodium and potassium concentrations of patients with low renin and normal renin hypertension did not differ (P > 0.7). Four hour aldosterone excretion of hypertensive patients following furosemide did not differ from that of control subjects (P > 0.6). Despite suppressed PRA, aldosterone excretion of patients with low renin hypertension did not differ from that of patients with normal renin hypertension or control subjects (P > 0.7). Similarly, before furosemide, plasma aldosterone (Fig. 2) of hypertensive patients and control subjects did not differ (P > 0.1). Plasma aldosterone increased significantly in response to furosemide in both groups (P < 0.02), and after furosemide, plasma aldosterone in hypertensive patients and controls did not differ (P > 0.05). Both before and after furosemide, plasma aldosterone concentrations of low renin patients and normal renin patients did not differ (P > 0.7). Measurements of PRR were compared in plasma of patients with low renin and normal renin hypertension during 30, 60, and 180 min incubation periods (Fig. 3); significantly less angiotensin was generated in plasma of the low renin patients (P < 0.005). Overall, in the entire group of hypertensive TABLE 1. Mean (±SE) serum creatinine, serum and urine electrolytes, and urine aldosterone in control subjects, all patients with essential hypertension (EH), and in hypertensive patients separated into normal renin (NREH) and low renin essential hypertension (LREH)
Serum Creatinine (mg/dl) Serum Na (mEq/1) Serum K+ (niEq/1) UN> + V (inEq/4 hr) (mEq/4 hr)
uAldDv
(MK/4 hr)
Norniotensive Controls
All EH
NREH
LREH
1.2 ± 0.1
1.0 ± 0.1
1.1 ±0.1
1.1 ±0.1
_
141.1 ± 0.5
140.9 ± 0.6
141.3 ± 0.9
_
4.2 ± 0.1
4.2 ± 0.1
4.2 ± 0.2
167 ± 13
157 ± 11
158 + 15
156 ± 12
28±3
28±3
29 ± 3
28±4
4.4 ± 0.4
4.7 ± 0.4
4.6 ± 0.5
4.9 ± 0.8
60 -
50
t 40
30
20
PRE POST HYPERTENSIVES (r.^24)
PRE
POST NORMALS (n-15)
FIG. 2. Mean (±SE) plasma aldosterone before and after furosemide.
patients, there was a significant positive linear correlation between PRA responses to furosemide and the concentrations of angiotensin generated during both a 30 minute (r = +0.46; P < 0.05) and a 60 min (r = +0.58; P < 0.01) incubation after addition of exogenous renin to plasma (Fig. 4). Mean PRS in patients with low renin (1,071 ng/ml ± 96 SE) and normal renin (1,126 ng/ml ± 64 SE) hypertension did not differ (P > 0.6). In addition, during each of the three incubation times (30, 60, 180 min), the measurement of PRR in all hypertensive patients did not correlate with PRS (P > 0.2), indicating that differences of PRS within normal physiologic ranges is not a major factor contributing to differences of PRR. Compared to that in buffer controls, recoveries of standard angiotensin I added to plasma of low renin and normal renin patients did not differ (P < 0.001) after 30, 60, and 180 min incubations. Consequently, different reactivities of renin in plasma of low renin and normal renin patients cannot be attributed to angiotensinase activity. To determine if increased reactivity of renin in hypertensive plasma might be re-
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BROOKS, TALWALKER AND KOTCHEN
centration of exogenous renin, differences of endogenous renin are minimized, and the kinetics of the in vitro renin reaction can be studied at this greater reaction velocity. We 150 have previously demonstrated that PRR is < E increased in plasma of patients with esUJ xi sential hypertension compared to that in 100 plasma of normotensive control subjects (6,9). In the present study, PRR of patients Si with low renin essential hypertension was 50 less than that of patients with normal renin hypertension. This difference cannot be explained by differences of endogenous renin activity, renin substrate concentration, 30 60 180 or angiotensinase activity. INCUBATION TIME(min) Low renin hypertension was defined by comparing PRA responses of patients with FIG. 3. Mean (±SE) PRR in low renin essential hypertension (LREH) and normal renin essential hyperessential hypertension and matched control tension (NREH). subjects to the combined stimulus of furosemide and upright posture. Although mean lated to a lower concentration of PGA, PGA serum creatinine concentration and sodium was measured in normal and hypertensive excretion during the 4 h after furosemide of plasma, both before and after furosemide control subjects and hypertensive patients (Table 2). Before furosemide mean PGA did not differ, as a group, hypertensive paconcentrations in plasma of hypertensive tients did have a reduced PRA response to patients and normotensive control subjects furosemide. Similarly, Padfield et al. have did not differ (P > 0.1), and in neither group recently reported that the renin response did PGA concentration change significantly to iv furosemide is suppressed in patients in response to furosemide (P > 0.3). After with essential hypertension (18). Similar furosemide, mean PGA concentrations in plasma of hypertensive patients and normotensive subjects also did not differ (P > 0.2). However, comparing the two groups of hypertensive patients, both before and after 100 furosemide, mean PGA concentrations of patients with low renin hypertension were less (P < 0.05) than PGA concentrations of patients with normal renin hypertension. Before furosemide, PGA of low renin pa60 tients was also less than that of normotensive control subjects (P < 0.05). 40 200
Discussion The measurement of PRR provides an opportunity to evaluate the possibility that circulating factors in addition to endogenous enzyme and substrate concentrations may modify the in vitro rate of angiotensin generation. By adding a relatively high con-
20
1.0
2.0
3.0
4.0
5.0
6.0
PRA 4 F T M (ng/ml/hr)
FIG. 4. Correlation between PRA after furosemide and PRR in essential hypertension.
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RENIN REACTIVITY IN HYPERTENSION to other reports (1), we found that both aldosterone excretion and plasma aldosterone were normal in hypertensive patients with suppressed PRA. The demonstration of a significant positive correlation between PRR and the PRA response to furosemide in hypertensive patients suggests that an alteration in the kinetics of the renin reaction, as opposed to a decreased concentration of renin, may contribute to the suppression of PRA in patients with low renin hypertension. Similar to these results, McDonald et al. (8) have also reported in abstract form that the reactivity of renin in plasma of patients with low renin essential hypertension was less than that in plasma of patients with normal renin hypertension and suggested that the slower velocity of the enzymatic reaction may contribute to the low renin state. In two models of experimental hypertension, there is also evidence for increased renin inhibition associated with suppressed renin activity (19,20). A number of specific renin inhibitors, generally lipids, have previously been identified (21-25), and we have recently demonstrated that PGA competitively inhibits the in vitro renin reaction (10). In the present experiment, PGA was measured to determine if different reactivities of renin in plasma of low renin and normal renin patients might reflect different concentrations of circulating PGA. PGA was actually decreased in plasma of low renin patients, indicating that low PRR in these patients cannot be attributed to elevated PGA. Zusman et al. (26) and Lee et al. (27) reported that PGA is decreased in plasma of patients with essential hypertension. We found that PGA concentrations in plasma of hypertensive patients and normotensive control subjects did not differ significantly. However, PGA was somewhat lower in our hypertensive patients, and we cannot exclude the possibility that a subtle difference might have become apparent in a more carefully controlled metabolic study. Furthermore, PGA was significantly depressed
TABLE 2. Mean (±SE) PGA (ng/ml), before and after furosemide (f), in control subjects, all patients with essential hypertension (EH), and in hypertensive patients separated into normal renin (NREH) and low renin (LREH) essential hypertension Normotensive Controls
All EH
NREH
LREH
Before f
1.18 ±0.10
1.01 ±0.06
1.10 ±0.07
0.86 ±0.06
After f
1.20 ±0.27
0.94 ±0.06
1.05 ±0.08
0.78 ±0.05
in the group of hypertensive patients who also had suppressed PRA. Infusion of angiotensin II into the renal artery may release renal prostaglandins (28,29), and Krakoff et al. have recently reported that infusion of PGA into hypertensive patients increases renin secretion (30). Renin is decreased by indomethacin, an inhibitor of prostaglandin synthetase (31-33). Whatever relationship may exist between PGA and the renin-angiotensin system, our finding of low PGA concentrations in low renin patients suggests the existence of a delicate balance between these renal vasopressor and vasodepressor mechanisms. Chronic sodium chloride loading is another instance where low PRA is associated with low PGA, and similar to PRA, peripheral PGA concentrations may also increase in response to dietary sodium deprivation (34,35). Indeed, Lee et al. (36) have recently found a significant positive correlation between PGA and PRA as dietary sodium intake was varied in normal subjects. However, our results indicate that PGA does not increase in response to acute furosemide induced diuresis. Patek et al. (31) have also recently reported that furosemide does not affect peripheral venous PGA in normal or hypertensive subjects. In addition, although high concentrations of PGA inhibit the in vitro renin reaction, the present results indicate that low PRR in patients with low renin essential hypertension cannot be attributed to elevated PGA. In summary, although overall PRR is in-
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BROOKS, TALWALKER AND KOTCHEN
creased in plasma of patients with essential hypertension, PRR is suppressed in that group of patients who also have low PRA. The slower velocity of angiotensin generation, even after addition of exogenous renin, suggests that an alteration of the kinetics of the renin-renin substrate reaction may contribute to the apparent renin suppression in patients with low renin essential hypertension. Decreased PRR is not associated with elevated PGA, a previously identified inhibitor of renin. Patients with low renin hypertension also have suppressed PGA, suggesting the existence of a delicate balance between these renal vasopressor and vasodepressor mechanisms. Acknowledgments The authors acknowledge the technical assistance of Mr. William Welch and the secretarial assistance of Ms. Joanne Cecil.
References 1. Dunn, M. J., and R. L. Tannen, Low-renin hypertension, Kidney Int 5: 317, 1974. 2. Melby, J. C , S. L. Dale, and T. E. Wilson, 18hydroxy-deoxycorticosterone in human hypertension, Circ Res (Suppl 2) 28-29: 143, 1971. 3. Spark, R. F., Low renin hypertension and the adrenal cortex, N Engl J Med 287: 343, 1972. 4. Liddle, G. W., and J. A. Sennett, Steroids and the syndrome of low renin hypertension, In Symposium abstract book, International Congress on Hormonal Steroids, Mexico, September 1974, S-23. 5. Kotchen, T. A., and M. C. Miller, Evidence for the participation of cofactors in the renin reaction, Res Steroids 6: 317, 1974. 6. Kotchen, T. A., T. W. Rice, and D. R. Walters, Renin reactivity in normal, hypertensive, and uremic plasma,/ Clin Endocrinol Metab 34: 928, 1972. 7. Sambhi, M. P., P. Eggena, J. Barrett, and C. E. Wiedeman, In Sambhi, M. P., (ed.). Mechanisms of Hypertension, American Elsevier Publishing Co., New York, 1973, p. 124. 8. McDonald, W. J., E. L. Cohen, C. P. Lucas, and J. W. Conn, Renin reactivity in plasma of patients with essential hyptension and suppressed plasma renin activity, Clin Res 21: 284, 1973 (Abstract). 9. Kotchen, T. A., R. T. Talwalkar, J. M. Kotchen, M. C. Miller, and W. J. Welch, Evidence for the existence of an acetone soluble renin inhibiting factor in normal human plasma, Circ Res (Suppl 1) 36-37: 17, 1975,
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10. Kotchen, T. A., and M. C. Miller, Effect of prostaglandins on renin reactivity, Am J Physiol 226: 314, 1974. 11. Kennedy, T. J., J. G. Hilton, and R. W. Berliner, Comparison of inulin and creatinine clearance in the dog, Am] Physiol 171: 164, 1952. 12. Ito, T., J. Woo, R. Haning, and R. Horton, A radioimmunoassay for aldosterone in human peripheral plasma including a comparison of alternate techniques,; Clin Endocrinol Metab 34: 106, 1972. 13. Haber, F., T. Koerner, L. B. Page, B. Kliman, and A. Purnode, Application of a radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects,/ Clin Endocrinol Metab 29: 1349, 1969. 14. Kotchen, T. A., W. Flamenbaum, W. Cirksena, and W. Gamble, Comparison of a biological and an immunological assay for the measurement of plasma renin activity, / Clin Endocrinol Metab 26: 804, 1973. 15. Haas, E., H. Goldblatt, E. C. Gipson, and L. Lewis, Extraction, purification, and assay of human renin free of angiotensinase, Circ Res 19: 739, 1966. 16. Zusman, R. M., B. V. Caldwell, and L. Speroff, Radioimmunoassay of the A prostaglandins, Prostaglandins 2: 41, 1972. 17. Polet, H., and L. Levine, Serum PGA isomerase, Biochem Biophys Res Commun 45: 1169, 1971. 18. Padfield, P. L., M. E. M. Allison, J. J. Brown, A. F. Lever, R. G. Luke, C. C. Robertson, J. I. Robertson, and M. Tree, Effect of intravenous furosemide on plasma renin concentration: Suppression of response in hypertension, Clin Sci Mol Med 49: 353, 1975. 19. Iwai, J., K. D. Knudsen, and L. K. Dahl, Genetic influence on the renin-angiotensin system: evidence for a renin inhibitor in hypertensionprone r a t s j Exp Med 131: 543, 1970. 20. Zachariah, N. Y., R. R. Smeby, S. Sen, F. M. Bumpus, and C. Singh, Phospholipase A2 in experimental hypertension, Am J Physiol 228:1782,1975. 21. Sen, S., R. R. Smeby, and F. M. Bumpus, Isolation of a phospholipid renin inhibitor from kidney, Biochemistry 6: 1572, 1967. 22. Smeby, R. R., S. Sen, and F. M. Bumpus, A naturally occurring renin inhibitor, Circ Res (Suppl 2) 20-21: 129, 1967. 23. Hiwada, K., T. Kokubu, and Y. Yamamura, Inhibitory effect of rabbit bile on renin angiotensinogen reaction system, Jap Circ J 33: 1231, 1969. 24. Pfeiffer, F. R., S. C. Hoke, C. K. Miao, R. Tedeschi, J. Pasternak, R. Hanh, R. W. Erickson, H. W. Levin, C. A. Burton, and J. A. Weisbach, Lysophosphatidylethanolamine and 2-desoxylysophosphatidylethanolamine derivatives: potential renin inhibitors,/ Med Chem 14: 493, 1971. 25. Turcotte, J. G., and R. E. Boyd, Renin inhibitory
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activity of phospholipid extracted from shark kidney, Pharmacologist 13: 277, 1971. Zusman, R. M., B. V. Caldwell, P. J. Mulrow, and L. Speroff, Sodium homeostasis and blood pressure, Prostaglandins 3: 679, 1973. Lee, J. B., A. Attalah, V. K. Vence, C. Ellwood, and A. Prezyna, Circulating prostaglandin A in human essential hypertension, J Clin Invest 52: 50A, 1973 (Abstract). McGiff, J. C , K. Crowshaw, N. A. Terrango, and A. J. Lonigro, Release of a prostaglandin-like substance into renal venous blood in response to angiotensin II, CircRes(Suppll) 26-27:121,1970. Aiken, J. W., and J. R. Vane, Intrarenal prostaglandin release attenuates the renal vasoconstrictor activity of angiotensin, J Pharmacol Exp Ther 184: 678, 1973. Krakoff, L. R., D. DeGuia, N. Vlachakis, J. Strieker, and M. Goldstein, Effect of sodium balance on arterial blood pressure and renal responses to prostaglandin A in man, Circ Res 33: 539, 1973. Patek, R. V., B. K. Mookerjee, C. J. Bentzel, P. E. Hysert, B. Milos, and J. B. Lee, Antagonism of the
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effects of furosemide by indomethacin in normal and hypertensive man, Prostaglandins 10: 649, 1975. Yun, J. C. H., G. Kelly, F. C. Bartter, and H. C. Smith, Role of prostaglandins in the control of renin secretion, Fed Proc 35: 620, 1976 (Abstract). Romero, J. C., C. G. Strong, C. E. Ott, R. Walker, S. Schryrer, and D. Manahan, The effect of indomethacin on the renin angiotensin system, Clin Res 23: 372, 1975 (Abstract). Lee, J. B., Cardiovascular-renal effects of prostaglandins: the antihypertensive, natriuretic renal "endocrine" function, Arch Int Med 133: 56,1974. Zusman, R. M., D. Spector, B. V. Caldwell, L. Speroff, G. Schneider, and P. J. Mulrow, The effect of chronic sodium loading and sodium restriction on plasma prostaglandin A, E and F concentrations in normal humans,/ Clin Invest 52: 1093, 1973. Lee, J. B., R. V. Patak, B. K. Mookerjee, Renal prostaglandins and the regulation of blood pressure and sodium and water homeostasis, Am J Med 60: 798, 1976.
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ABSTRACT. Plasma renin reactivity (PRR) is the rate of angiotensin generation in vitro after addition of exogenous renin to plasma. To evaluate the hypothesis that suppressed plasma renin activity (PRA) in patients with low renin essential hypertension may be related to an alteration of the kinetics of the in vitro renin reaction, PRR was compared in plasma of patients with low renin and normal renin essential hypertension. Prostaglandin A (PGA) inhibits renin, and PGA was also measured to determine if suppressed PRA may be related to increased PGA. Low renin and normal renin hypertension were defined by comparing PRA responses of 30 hypertensive patients and 16 matched control subjects to upright posture and furosemide (80 mg p.o.). Nine of 30 patients had low PRA. Compared to that in plasma of patients with normal renin hyper-
P
LASMA renin activity (PRA) is suppressed in approximately 25% of patients with essential hypertension (1). Although increased production of a specific mineralocorticoid other than aldosterone has been demonstrated in a relatively small per cent of these patients (2-4), in the majority of patients with low renin essential hypertension, the mechanism for the apparent renin suppression has not been defined (1). Because PRA is a measure of the in vitro rate of angiotensin production, the measurement of PRA may be influenced by renin enzyme concentration, renin substrate concentration, and by additional circulating factors in plasma that may modify the reninrenin substrate reaction (5). It is possible that suppressed PRA in patients with low renin hypertension may reflect an alteration Received July 23, 1976. Supported by Grant HL15528 and NephrologyUrology Training Program AM05701, both from the National Institutes of Health, and by a grant from the Eagles' Max Baer Heart Fund. Address correspondence to: Theodore A. Kotchen, M.D., Department of Medicine, University of Kentucky College of Medicine, Lexington, Kentucky 40506.
of the kinetics of the in vitro renin reaction, rather than decreased renin enzyme concentration. We have previously used the term plasma renin reactivity (PRR) to refer to the in vitro rate of angiotensin generation after addition of exogenous renin to plasma (6). Differences of endogenous renin are minimized by adding relatively high concentrations of exogenous enzyme. Compared to that in plasma of normotensive control subjects, PRR is increased in plasma of patients with essential hypertension (6-8) and to a greater extent in plasma of patients with renal insufficiency (6). Increased PRR in hypertensive and uremic plasma is not related to differences of renin substrate or angiotensinase activity (6), and may reflect the deficiency of a normally occurring acetone soluble renin inhibiting factor (9). The purpose of the present study is to compare measurements of PRR in patients with normal renin and low renin essential hypertension. In addition, we have recently shown that high concentrations of a renomedullary prostaglandin, prostaglandin A (PGA), inhibit the in vitro renin reaction (10). In the pres-
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RENIN REACTIVITY IN HYPERTENSION
ent study, measurements of PGA are also compared in patients with normal renin and low renin hypertension to determine if suppressed PRA may be related to increased circulating PGA. Materials and Methods To identify patients with low renin essential hypertension, PRA was measured after an overnight fast, before and 4 h after the combined stimulus of 80 mg furosemide, given orally, and the upright posture in 30 patients with essential hypertension and in 16 matched normotensive control subjects. Informed consent was obtained from all hypertensive patients and control subjects, and the use of this protocol was approved by the Human Investigations Committee at the University of Kentucky. Hypertensive patients and control subjects had not received any medications for at least two weeks; women on estrogen therapy were excluded from study. Essential hypertension was diagnosed on the basis of complete history and physical examination, urinalysis, measurement of creatinine clearance and serum Na+, K+, Cl~, and CO2 concentrations, rapid sequence iv pyelogram, and in selected patients measurement of 24 h urine VMA and/or catecholamine excretion and renal angiography. Mean arterial blood pressures of the hypertensive patients and normotensive control subjects were 124 mmHg ± 12 SD and 93 mmHg ± 8 SD, respectively; respective mean ages of the two groups were 48 years ± 11 SD and 42 years ± 12 SD. Sixty per cent of the hypertensive patients and 37% of the control subjects were black; 23% of the patients and 25% of control subjects were male. In addition to PRA, plasma aldosterone and PGA concentrations were measured before and 4 h after furosemide; serum creatinine concentration was measured before furosemide. Urine excreted during the 4 h period following furosemide was collected and analyzed for Na+, K+, and aldosterone concentrations. PRR and plasma renin substrate (PRS) were measured in plasma of hypertensive patients before furosemide. PRR is not affected by sodium deprivation (unpublished observations) and consequently was not measured again after furosemide. Urine Na+ and K+ concentrations were measured with a flame photometer. Serum creatinine concentrations were measured by the method of
323
Kennedy etal. (11). Plasma and urine aldosterone concentrations were measured in quadruplicate by the radioimmunoassay procedure described by Ito et al. (12), except that bound and free aldosterone were separated with dextran coated charcoal rather than Florisil. Plasma renin activity was measured in quadruplicate by the radioimmunoassay procedure of Haber et al. (13), previously validated in our laboratory by bioassay (14). For the measurement of both PRS and PRR, renin was extracted from human kidneys by an ammonium sulfate precipitation procedure of Haas (15). The concentration of renin in this preparation was determined by comparing the rate of angiotensin I generated, in the presence of excess renin substrate, with a human renin standard obtained from the Medical Research Council (Division of Biological Standards, National Institutes for Medical Research, London, England). Plasma renin substrate concentration was measured, as previously described (6), by adding a high concentration of exogenous human renin to plasma (1.5 x 10~2 GU/0.5 ml plasma) and measuring the concentration of angiotensin I generated after exogenous substrate had been completely consumed. For the measurement of plasma renin reactivity, a lower concentration of exogenous human renin was added to plasma (7.3 x 10~5 GU/0.5 ml plasma), and the concentrations of angiotensin I generated were measured after 30, 60 and 180 min incubation periods at 37 C and pH 7.4. At the time of venepuncture, blood was anticoagulated with sodium EDTA to inhibit calcium dependent angiotensinases. Dimercaprol (1.6 DIM) and 8-hydroxyquinoline (3.4 mM) were added to in vitro incubations to inhibit converting enzyme activity. Tris buffer (100 ul/ml), pH 7.4, was added to maintain a constant pH during the incubation, and neomycin sulfate (50 ul/0.5 ml of a 10% solution) was added to inhibit bacterial growth. To adjust for relatively small differences of angiotensin generation due to endogenous renin activity, angiotensin production was measured in the absence of exogenous enzyme in a control aliquot of each plasma sample at each incubation period. For the reported PRR values, concentrations of angiotensin generated by endogenous renin, which were invariably less than 3% of the total, were subtracted from the amount of angiotensin measured after addition of exogenous enzyme. Production of rabbit anti-PGA antibodies, ex-
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statistical significance of differences was computed with analysis of variance. Student's t test was used to determine the significance of differences between two groups.
Results
PRE POST HYPERTENSIVES (n.JOl
PRE
POST NORMALS
FIG. 1. Mean (±SE) PRA before and after furosemide.
traction of prostaglandins with ethylacetate, separation of PGA on silicic acid columns, and radioimmunoassay for PGA were carried out according to the procedures described by Zusman et al. (16). Compared to PGA2, cross-reactivity of our antibody was 43% with PGAj, 19% with PGEl5 0.4% with PGE2, and there was no detectable cross-reactivity with PGF. Prior to assay, PGE was separated from PGA by silica gel column chromatography. PGB is not separated from PGA in this system. However, the lack of prostaglandin isomerase in human plasma suggests that contamination with PGB is of little consequence (17). For assay, the standard curve was derived using PGA2 standard, (obtained from Upjohn Co., Kalamazoo, Michigan) ranging from 10 pg/ml to 1,000 pg/ml, and tritiated PGA tracer (obtained from New England Nuclear, Corp, Boston, Mass.). The final dilution of antibody, for 50% displacement of tracer PGA, was 1:750. To validate further the assay, peripheral venous PGA was measured in 8 control rabbits and 8 indomethacin treated rabbits (10 mg/kgm body weight given SC 20 h, 9 h and again at 2 h prior to bleeding). Control animals received SC injections of the sodium carbonate vehicle. Mean PGA concentration of indomethacin treated animals (0.24 ng/ml ± 0.06 SE) was significantly lower (P < 0.05) than that of control animals (0.39 ng/ml ± .04). All PGA measurements were done in duplicate. In instances where differences among more than two groups were compared simultaneously,
Before furosemide, mean PRA in normotensive control subjects (0.9 ng/ml/h ± 0.2 SE) and patients with essential hypertension (0.9 ng/ml/h ± 0.2) did not differ (Fig. 1). After furosemide, PRA increased significantly in both control subjects and hypertensive patients (P < 0.001); however, mean PRA of patients with essential hypertension (1.9 ng/ml/h ± 0.3 SE) was significantly (P < 0.05) lower than that of normotensive control subjects (3.1 ng/ml/h ± 0.5 SE). PRA was undetectable both before and after furosemide in one 65-year-old black normotensive man. Excluding this subject, the lowest PRA after furosemide in normotensive subjects was 0.8 ng/ml/h. Nine of the 30 patients (30%) with essential hypertension had stimulated PRA lower than this value, and these patients were designated as having low renin essential hypertension. Comparing patients with low renin and normal renin hypertension, mean arterial pressure (126 mmHg ± 4 SE vs. 123 mmHg ± 3 SE) and age (45 yrs. ± 2 SE vs. 49 yrs. ± 2) were similar (P > 0.3) in both groups. Of the low renin patients, 89% were black; 48% of normal renin patients were black (P < 0.05). Twenty-two per cent of low renin and 24% of normal renin patients were male. Mean serum creatinine concentrations of normotensive subjects, patients with low renin hypertension, and patients with normal renin hypertension did not differ (P > 0.05) significantly (Table 1). During the 4 h after furosemide, sodium and potassium excretion of hypertensive patients did not differ from respective values in control subjects (P > 0.5), suggesting a comparable furosemide induced stimulus for renin secretion. Furthermore, comparing patients with low renin and normal renin hypertension, sodium and potassium excre-
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RENIN REACTIVITY IN HYPERTENSION tion did not differ (P > 0.8). Mean serum sodium and potassium concentrations of patients with low renin and normal renin hypertension did not differ (P > 0.7). Four hour aldosterone excretion of hypertensive patients following furosemide did not differ from that of control subjects (P > 0.6). Despite suppressed PRA, aldosterone excretion of patients with low renin hypertension did not differ from that of patients with normal renin hypertension or control subjects (P > 0.7). Similarly, before furosemide, plasma aldosterone (Fig. 2) of hypertensive patients and control subjects did not differ (P > 0.1). Plasma aldosterone increased significantly in response to furosemide in both groups (P < 0.02), and after furosemide, plasma aldosterone in hypertensive patients and controls did not differ (P > 0.05). Both before and after furosemide, plasma aldosterone concentrations of low renin patients and normal renin patients did not differ (P > 0.7). Measurements of PRR were compared in plasma of patients with low renin and normal renin hypertension during 30, 60, and 180 min incubation periods (Fig. 3); significantly less angiotensin was generated in plasma of the low renin patients (P < 0.005). Overall, in the entire group of hypertensive TABLE 1. Mean (±SE) serum creatinine, serum and urine electrolytes, and urine aldosterone in control subjects, all patients with essential hypertension (EH), and in hypertensive patients separated into normal renin (NREH) and low renin essential hypertension (LREH)
Serum Creatinine (mg/dl) Serum Na (mEq/1) Serum K+ (niEq/1) UN> + V (inEq/4 hr) (mEq/4 hr)
uAldDv
(MK/4 hr)
Norniotensive Controls
All EH
NREH
LREH
1.2 ± 0.1
1.0 ± 0.1
1.1 ±0.1
1.1 ±0.1
_
141.1 ± 0.5
140.9 ± 0.6
141.3 ± 0.9
_
4.2 ± 0.1
4.2 ± 0.1
4.2 ± 0.2
167 ± 13
157 ± 11
158 + 15
156 ± 12
28±3
28±3
29 ± 3
28±4
4.4 ± 0.4
4.7 ± 0.4
4.6 ± 0.5
4.9 ± 0.8
60 -
50
t 40
30
20
PRE POST HYPERTENSIVES (r.^24)
PRE
POST NORMALS (n-15)
FIG. 2. Mean (±SE) plasma aldosterone before and after furosemide.
patients, there was a significant positive linear correlation between PRA responses to furosemide and the concentrations of angiotensin generated during both a 30 minute (r = +0.46; P < 0.05) and a 60 min (r = +0.58; P < 0.01) incubation after addition of exogenous renin to plasma (Fig. 4). Mean PRS in patients with low renin (1,071 ng/ml ± 96 SE) and normal renin (1,126 ng/ml ± 64 SE) hypertension did not differ (P > 0.6). In addition, during each of the three incubation times (30, 60, 180 min), the measurement of PRR in all hypertensive patients did not correlate with PRS (P > 0.2), indicating that differences of PRS within normal physiologic ranges is not a major factor contributing to differences of PRR. Compared to that in buffer controls, recoveries of standard angiotensin I added to plasma of low renin and normal renin patients did not differ (P < 0.001) after 30, 60, and 180 min incubations. Consequently, different reactivities of renin in plasma of low renin and normal renin patients cannot be attributed to angiotensinase activity. To determine if increased reactivity of renin in hypertensive plasma might be re-
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BROOKS, TALWALKER AND KOTCHEN
centration of exogenous renin, differences of endogenous renin are minimized, and the kinetics of the in vitro renin reaction can be studied at this greater reaction velocity. We 150 have previously demonstrated that PRR is < E increased in plasma of patients with esUJ xi sential hypertension compared to that in 100 plasma of normotensive control subjects (6,9). In the present study, PRR of patients Si with low renin essential hypertension was 50 less than that of patients with normal renin hypertension. This difference cannot be explained by differences of endogenous renin activity, renin substrate concentration, 30 60 180 or angiotensinase activity. INCUBATION TIME(min) Low renin hypertension was defined by comparing PRA responses of patients with FIG. 3. Mean (±SE) PRR in low renin essential hypertension (LREH) and normal renin essential hyperessential hypertension and matched control tension (NREH). subjects to the combined stimulus of furosemide and upright posture. Although mean lated to a lower concentration of PGA, PGA serum creatinine concentration and sodium was measured in normal and hypertensive excretion during the 4 h after furosemide of plasma, both before and after furosemide control subjects and hypertensive patients (Table 2). Before furosemide mean PGA did not differ, as a group, hypertensive paconcentrations in plasma of hypertensive tients did have a reduced PRA response to patients and normotensive control subjects furosemide. Similarly, Padfield et al. have did not differ (P > 0.1), and in neither group recently reported that the renin response did PGA concentration change significantly to iv furosemide is suppressed in patients in response to furosemide (P > 0.3). After with essential hypertension (18). Similar furosemide, mean PGA concentrations in plasma of hypertensive patients and normotensive subjects also did not differ (P > 0.2). However, comparing the two groups of hypertensive patients, both before and after 100 furosemide, mean PGA concentrations of patients with low renin hypertension were less (P < 0.05) than PGA concentrations of patients with normal renin hypertension. Before furosemide, PGA of low renin pa60 tients was also less than that of normotensive control subjects (P < 0.05). 40 200
Discussion The measurement of PRR provides an opportunity to evaluate the possibility that circulating factors in addition to endogenous enzyme and substrate concentrations may modify the in vitro rate of angiotensin generation. By adding a relatively high con-
20
1.0
2.0
3.0
4.0
5.0
6.0
PRA 4 F T M (ng/ml/hr)
FIG. 4. Correlation between PRA after furosemide and PRR in essential hypertension.
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RENIN REACTIVITY IN HYPERTENSION to other reports (1), we found that both aldosterone excretion and plasma aldosterone were normal in hypertensive patients with suppressed PRA. The demonstration of a significant positive correlation between PRR and the PRA response to furosemide in hypertensive patients suggests that an alteration in the kinetics of the renin reaction, as opposed to a decreased concentration of renin, may contribute to the suppression of PRA in patients with low renin hypertension. Similar to these results, McDonald et al. (8) have also reported in abstract form that the reactivity of renin in plasma of patients with low renin essential hypertension was less than that in plasma of patients with normal renin hypertension and suggested that the slower velocity of the enzymatic reaction may contribute to the low renin state. In two models of experimental hypertension, there is also evidence for increased renin inhibition associated with suppressed renin activity (19,20). A number of specific renin inhibitors, generally lipids, have previously been identified (21-25), and we have recently demonstrated that PGA competitively inhibits the in vitro renin reaction (10). In the present experiment, PGA was measured to determine if different reactivities of renin in plasma of low renin and normal renin patients might reflect different concentrations of circulating PGA. PGA was actually decreased in plasma of low renin patients, indicating that low PRR in these patients cannot be attributed to elevated PGA. Zusman et al. (26) and Lee et al. (27) reported that PGA is decreased in plasma of patients with essential hypertension. We found that PGA concentrations in plasma of hypertensive patients and normotensive control subjects did not differ significantly. However, PGA was somewhat lower in our hypertensive patients, and we cannot exclude the possibility that a subtle difference might have become apparent in a more carefully controlled metabolic study. Furthermore, PGA was significantly depressed
TABLE 2. Mean (±SE) PGA (ng/ml), before and after furosemide (f), in control subjects, all patients with essential hypertension (EH), and in hypertensive patients separated into normal renin (NREH) and low renin (LREH) essential hypertension Normotensive Controls
All EH
NREH
LREH
Before f
1.18 ±0.10
1.01 ±0.06
1.10 ±0.07
0.86 ±0.06
After f
1.20 ±0.27
0.94 ±0.06
1.05 ±0.08
0.78 ±0.05
in the group of hypertensive patients who also had suppressed PRA. Infusion of angiotensin II into the renal artery may release renal prostaglandins (28,29), and Krakoff et al. have recently reported that infusion of PGA into hypertensive patients increases renin secretion (30). Renin is decreased by indomethacin, an inhibitor of prostaglandin synthetase (31-33). Whatever relationship may exist between PGA and the renin-angiotensin system, our finding of low PGA concentrations in low renin patients suggests the existence of a delicate balance between these renal vasopressor and vasodepressor mechanisms. Chronic sodium chloride loading is another instance where low PRA is associated with low PGA, and similar to PRA, peripheral PGA concentrations may also increase in response to dietary sodium deprivation (34,35). Indeed, Lee et al. (36) have recently found a significant positive correlation between PGA and PRA as dietary sodium intake was varied in normal subjects. However, our results indicate that PGA does not increase in response to acute furosemide induced diuresis. Patek et al. (31) have also recently reported that furosemide does not affect peripheral venous PGA in normal or hypertensive subjects. In addition, although high concentrations of PGA inhibit the in vitro renin reaction, the present results indicate that low PRR in patients with low renin essential hypertension cannot be attributed to elevated PGA. In summary, although overall PRR is in-
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BROOKS, TALWALKER AND KOTCHEN
creased in plasma of patients with essential hypertension, PRR is suppressed in that group of patients who also have low PRA. The slower velocity of angiotensin generation, even after addition of exogenous renin, suggests that an alteration of the kinetics of the renin-renin substrate reaction may contribute to the apparent renin suppression in patients with low renin essential hypertension. Decreased PRR is not associated with elevated PGA, a previously identified inhibitor of renin. Patients with low renin hypertension also have suppressed PGA, suggesting the existence of a delicate balance between these renal vasopressor and vasodepressor mechanisms. Acknowledgments The authors acknowledge the technical assistance of Mr. William Welch and the secretarial assistance of Ms. Joanne Cecil.
References 1. Dunn, M. J., and R. L. Tannen, Low-renin hypertension, Kidney Int 5: 317, 1974. 2. Melby, J. C , S. L. Dale, and T. E. Wilson, 18hydroxy-deoxycorticosterone in human hypertension, Circ Res (Suppl 2) 28-29: 143, 1971. 3. Spark, R. F., Low renin hypertension and the adrenal cortex, N Engl J Med 287: 343, 1972. 4. Liddle, G. W., and J. A. Sennett, Steroids and the syndrome of low renin hypertension, In Symposium abstract book, International Congress on Hormonal Steroids, Mexico, September 1974, S-23. 5. Kotchen, T. A., and M. C. Miller, Evidence for the participation of cofactors in the renin reaction, Res Steroids 6: 317, 1974. 6. Kotchen, T. A., T. W. Rice, and D. R. Walters, Renin reactivity in normal, hypertensive, and uremic plasma,/ Clin Endocrinol Metab 34: 928, 1972. 7. Sambhi, M. P., P. Eggena, J. Barrett, and C. E. Wiedeman, In Sambhi, M. P., (ed.). Mechanisms of Hypertension, American Elsevier Publishing Co., New York, 1973, p. 124. 8. McDonald, W. J., E. L. Cohen, C. P. Lucas, and J. W. Conn, Renin reactivity in plasma of patients with essential hyptension and suppressed plasma renin activity, Clin Res 21: 284, 1973 (Abstract). 9. Kotchen, T. A., R. T. Talwalkar, J. M. Kotchen, M. C. Miller, and W. J. Welch, Evidence for the existence of an acetone soluble renin inhibiting factor in normal human plasma, Circ Res (Suppl 1) 36-37: 17, 1975,
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10. Kotchen, T. A., and M. C. Miller, Effect of prostaglandins on renin reactivity, Am J Physiol 226: 314, 1974. 11. Kennedy, T. J., J. G. Hilton, and R. W. Berliner, Comparison of inulin and creatinine clearance in the dog, Am] Physiol 171: 164, 1952. 12. Ito, T., J. Woo, R. Haning, and R. Horton, A radioimmunoassay for aldosterone in human peripheral plasma including a comparison of alternate techniques,; Clin Endocrinol Metab 34: 106, 1972. 13. Haber, F., T. Koerner, L. B. Page, B. Kliman, and A. Purnode, Application of a radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects,/ Clin Endocrinol Metab 29: 1349, 1969. 14. Kotchen, T. A., W. Flamenbaum, W. Cirksena, and W. Gamble, Comparison of a biological and an immunological assay for the measurement of plasma renin activity, / Clin Endocrinol Metab 26: 804, 1973. 15. Haas, E., H. Goldblatt, E. C. Gipson, and L. Lewis, Extraction, purification, and assay of human renin free of angiotensinase, Circ Res 19: 739, 1966. 16. Zusman, R. M., B. V. Caldwell, and L. Speroff, Radioimmunoassay of the A prostaglandins, Prostaglandins 2: 41, 1972. 17. Polet, H., and L. Levine, Serum PGA isomerase, Biochem Biophys Res Commun 45: 1169, 1971. 18. Padfield, P. L., M. E. M. Allison, J. J. Brown, A. F. Lever, R. G. Luke, C. C. Robertson, J. I. Robertson, and M. Tree, Effect of intravenous furosemide on plasma renin concentration: Suppression of response in hypertension, Clin Sci Mol Med 49: 353, 1975. 19. Iwai, J., K. D. Knudsen, and L. K. Dahl, Genetic influence on the renin-angiotensin system: evidence for a renin inhibitor in hypertensionprone r a t s j Exp Med 131: 543, 1970. 20. Zachariah, N. Y., R. R. Smeby, S. Sen, F. M. Bumpus, and C. Singh, Phospholipase A2 in experimental hypertension, Am J Physiol 228:1782,1975. 21. Sen, S., R. R. Smeby, and F. M. Bumpus, Isolation of a phospholipid renin inhibitor from kidney, Biochemistry 6: 1572, 1967. 22. Smeby, R. R., S. Sen, and F. M. Bumpus, A naturally occurring renin inhibitor, Circ Res (Suppl 2) 20-21: 129, 1967. 23. Hiwada, K., T. Kokubu, and Y. Yamamura, Inhibitory effect of rabbit bile on renin angiotensinogen reaction system, Jap Circ J 33: 1231, 1969. 24. Pfeiffer, F. R., S. C. Hoke, C. K. Miao, R. Tedeschi, J. Pasternak, R. Hanh, R. W. Erickson, H. W. Levin, C. A. Burton, and J. A. Weisbach, Lysophosphatidylethanolamine and 2-desoxylysophosphatidylethanolamine derivatives: potential renin inhibitors,/ Med Chem 14: 493, 1971. 25. Turcotte, J. G., and R. E. Boyd, Renin inhibitory
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effects of furosemide by indomethacin in normal and hypertensive man, Prostaglandins 10: 649, 1975. Yun, J. C. H., G. Kelly, F. C. Bartter, and H. C. Smith, Role of prostaglandins in the control of renin secretion, Fed Proc 35: 620, 1976 (Abstract). Romero, J. C., C. G. Strong, C. E. Ott, R. Walker, S. Schryrer, and D. Manahan, The effect of indomethacin on the renin angiotensin system, Clin Res 23: 372, 1975 (Abstract). Lee, J. B., Cardiovascular-renal effects of prostaglandins: the antihypertensive, natriuretic renal "endocrine" function, Arch Int Med 133: 56,1974. Zusman, R. M., D. Spector, B. V. Caldwell, L. Speroff, G. Schneider, and P. J. Mulrow, The effect of chronic sodium loading and sodium restriction on plasma prostaglandin A, E and F concentrations in normal humans,/ Clin Invest 52: 1093, 1973. Lee, J. B., R. V. Patak, B. K. Mookerjee, Renal prostaglandins and the regulation of blood pressure and sodium and water homeostasis, Am J Med 60: 798, 1976.
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