Renin Reactivity, Renin Activity and Renin Concentration in Patients with Normal and Low Renin Essential Hypertension WALTER J. MCDONALD,1 EDWIN L. COHEN, AND JEROME W. CONN2 Endocrinology Division, Department of Medicine, University of Michigan, Ann Arbor, Michigan, and Research Service, Veterans Administration Hospital, Portland, Oregon in the low renin hypertensives. These findings suggest that plasma renin concentration may be suppressed in most hypertensive subjects. Furthermore, plasma renin activity may be "normalized" in most hypertensive subjects by the effect of circulating modifiers of the renin reaction. While renin reactivity in the plasma of low-renin hypertensive subjects is accelerated to a lesser degree than that of the normal-renin hypertensives, this finding alone does not explain the low plasma renin activity. (J Clin Endocrinol Metab 45: 685, 1977)
ABSTRACT. Renin activity, concentration, substrate and reactivity were determined in normal subjects as well as in hypertensive subjects with suppressed and normal plasma renin activity. Renin substrate measurements were similar in all groups. Renin reactivity, a measure of circulating modifiers of the renin reaction, was significantly increased in both hypertensive groups. Reactivity was significantly greater in the normal renin hypertensive group than the low renin hypertensive group. Renin concentration was significantly suppressed in both hypertensive groups, but to a greater degree
P
LASMA renin activity measurements have been traditionally employed for evaluating the renin system in man and experimental animals. These methods have generally proven satisfactory. Several observations, however, raise questions regarding the applicability of these methods in experimental studies requiring more precise knowledge of circulating renin concentration. First, the kinetic characteristics of the renin-renin substrate reaction remain unclear. A recent review of investigation designed to clarify this point suggests that the renin reaction in vitro does not follow the laws of zero order kinetics with respect to its substrate (1). This observation alone limits the usefulness of plasma renin activity determination. Under these non-zero Received October 26, 1976. Supported in part by United States Public Health Service Grant No. HL-14520, National Heart and Lung Institute, National Institutes of Health; by a Grant (RR-42) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. Send all communications to: Walter J. McDonald, M.P., Veterans Administration Hospital, Portland, Oregon 97207. 1 MRIS 5577. 2 VA Distinguished Physician.
order conditions, plasma renin activity is at least partly dependent upon substrate concentration as well as enzyme concentration. Of equal concern are observations that, independent of substrate concentrations, angiotensin generation per unit of renin in plasma (renin reactivity) is not the same in all plasmas. Patients with essential hypertension (2-4), uremia (2), primary aldosteronism (5), renovascular hypertension (2), and those on oral contraceptives (6) have been demonstrated to have increased renin reactivity in their plasmas. These findings have been interpreted as reflecting the presence of an accelerator (7), the absence of an inhibitor (8), or a molecular alteration of either renin or its substrate (7). From the foregoing, it is apparent that plasma renin activity does not always reflect renin concentration. For these reasons, we have investigated the relationship between plasma renin activity, renin activity, and renin concentration in both normal subjects and patients with essential hypertension. The hypertensive subjects were classified into normal and low-renin categories according to their plasma renin activities. Significantly lower plasma renin concentrations and higher
685
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MCDONALD, COHEN AND CONN
renin reactivities were found in both the low and normal-renin hypertensive groups when compared to normotensives. Renin reactivity and renin concentration were significantly higher in the normal renin hypertensives as compared to the low-renin hypertensives. Materials and Methods Fourteen nonhypertensive subjects (Group A), 10 subjects with essential hypertension and normal plasma renin activity (Group B), and 19 subjects with hyporeninemic essential hypertension (Group C) were studied. All subjects were white and less than 60 years of age. Secondary forms of hypertension were ruled out by iv pyelogram, peripheral renin activity determinations, urinary VMA, aldosterone, and Porter-Silber determinations. Patients were categorized as hypertensive if the blood pressure measurements were consistently greater than 145/95. Subjects were maintained for three days on a diet containing 10 milli-equivalent of sodium per day, followed by three days on one containing 120 milliequivalent of sodium per day. Following the third day of each dietary period, blood was obtained at 0800 h following 2 h of upright posture for determination of plasma renin activity, renin substrate, renin concentration and renin reactivity. If the low sodium diet and upright posture failed to stimulate a rise in plasma renin activities to values greater than 3.2 ng/ml/h, the patient was classified as hyporeninemic. This value represents the lowest observed renin activity in a previous study involving twenty non-hypertensive subjects following a similar protocol. Renin Human renal renin was prepared by the method of Lucas et al. (9). This renin preparation was devoid of angiotensinase activity in the concentrations used. Measurement of plasma renin activity, concentration, and substrate The measurement of renin parameters was accomplished in all specimens by radioimmunoassay of angiotensin I by the method of Cohen et al. (10). The complete methods for these measurements have been described in detail elsewhere (11). EDTA and diisopropylfluoro-
JCE & M • 1977 Vol 45 • No 4
phosphate are employed as angiotensinase inhibitors and the incubation is carried out at pH 5.5. It should be emphasized that renin concentration was measured by a modification of the method of Haas (12). This method employs an external renin standard to eliminate the influence of variations of substrate and other undefined factors. A comparison of angiotensin I generation, both before and after the addition of the known quantity of renin, permits the calculation of renin concentration by the following two formulae: 1. Angiotensin I generated/unit exogenous renin = (Angiotensin I generated/unit exogenous + endogenous renin) — endogenous plasma renin activity. 2. Plasma renin concentration = Plasma renin activity/(Angiotensin I generated/unit exogenous renin/ml). The results are expressed in Goldblatt units/ml. Measurement of renin reactivity Renin reactivity (defined as the amount of angiotensin generated/ml/h/1 x 10"4 Goldblatt Units/ml) was measured by a method which employs an external renin standard. Plasma was prepared as for plasma renin activity measurement and separated into 100 /xl aliquots. To the first aliquot, 10 pi of saline were added and to the second, 10 /xl of purified human renin containing 1.1 x 10~5 Goldblatt units of renin. After 30 min of incubation, each sample was assayed in triplicate for angiotensin. Subtraction of the angiotensin generated in the first sample from that generated in the second sample yields the amount of angiotensin generated per 1 x 10~4 Goldblatt units of renin per ml or the renin reactivity. To assure that reaction velocity did not change with substrate utilization, a third aliquot was utilized and 2.2 x 10~5 Goldblatt units of renin were added to this sample prior to incubation. In all cases, angiotensin generation was linear with respect to added renin.
Results
Plasma renin activity and substrate concentration Table 1 lists the mean plasma renin activity and substrate concentration for the
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687
RENIN REACTIVITY IN HUMAN HYPERTENSION normal group during the 10 and 120 milliequivalent sodium diets, and the two hypertensive groups during the higher sodium intake. The mean plasma renin activity during the two sodium intake periods in normal subjects were significantly different at the P < .01 level. The mean plasma renin activities of Group A and B during the higher salt intake did not differ significantly from each other. Plasma renin activity in Group C patients was significantly different from both Group A and Group B at the P < .01 level. There was no significant difference in the mean substrate concentrations between any of the groups. Renin reactivity Figure 1 illustrates the mean renin reactivity in normal subjects (Group A) on the two sodium diets. The mean values are not statistically different. Renin reactivity appears to be independent of the sodium intake. Because of this finding, and prior observations showing renin reactivity to
TABLE 1. Renin activity and substrate concentration in normal and hypertensive subjects
Normal—Group A 120 meq Na+ Diet (N = 14) 10 meq Na+ Diet (N = 10) Hypertensive 120 meq Na+ Diet Normal PRA-Group B (N = 10) Low PRA-Group C (N = 19)
Renin activity (ng/mg/h ± SEM)
Substrate (/ig/ml ± SEM)
5.4 ± 0.7» 10.3 ± 1.1
1.39 + 0.15 1.54 ±0.14
6.0 ± l.lf 1.2 ± 0.3*
1.54 ± 0.16 1.33 ± 0.08
• Significantly lower than all other renin activity values at P < .01. f Significantly lower than renin activity in Group A on the 10 meq diet at P < .01.
be independent of renin concentration (2,11), renin reactivity in the hypertensive population was determined only in samples obtained during the 120 milliequivalent sodium intake. Renin reactivity in all three groups during the higher sodium intake is also depicted in Fig. 1. All three mean values are significantly different from each other at the P < .01 level. The renin reaction is accelerated in both groups of patients with essential hypertension. It is less accelerated, how-
16r
14
12
FiG. 1. Renin reactivity in normal subjects on a 10 and a 120 millequivalent sodium diet and hypertensive subjects on a 120 millequivalent diet. Mean renin reactivities, marked by a dash, are not significantly different in the two normal groups. All reactivities on the 120 millequivalent diet differ from each other at P < .01.
10
mm
RfAcnvirr Ȥ/ml/ki I x 10" 4 GO 6
Noraol
Momal
10 meq
120
«M
Hyperteisire NOMMI
Hyptrftisiv*
Stppntiti
m
120 mte
120 «M
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MCDONALD, COHEN AND CONN
688
TABLE 2. Mean renin concentrations in normal and hypertensive subjects on 120 mEq sodium intake PRC (GU x 10-7mI) ± SEM Normals (Group A) Essential Hypertension Normal PRA (Group B) Low PRA (Group C)
1.20 ± 0.21 0.47 ± 0.09* 0.21 ± 0.05t
* Significantly different from both other groups at P > .05. f Significantly different from Group A at P < .01.
ever, in patients with hyporeninemic hypertension. Plasma renin concentration The mean plasma renin concentrations for the three groups during the 120 milliequivalent sodium intake are tabulated in Table 2. The mean renin concentration in hypertensive subjects with normal renin activity differs significantly from that of normal subjects despite the fact that renin activity is identical in the two groups. The mean renin concentration in both groups of patients with essential hypertension is significantly suppressed in comparison to normals. Discussion It has been repeatedly demonstrated that between 20 and 40% of patients with essential hypertension display blunted responses to manipulations designed to stimulated renin activity (13). The etiology of this hyporeninemic response is unclear and remains a source of speculation, much of which centers around the possibility of mineralocorticoid excess (13,14). Our finding of suppressed renin concentration in patients with both low and normal renin activity, however, raises another possibility. It is possible that there is a factor common to both hypertensive categories which is acting to suppress renin concentration. What that factor or factors might be is a matter of conjecture. One possibility is that all patients with essential hypertension have derangements of
JCE&M Vol 45
< 1977 No 4
mineralocorticoid metabolism leading to manifestations of mineralocorticoid excess. Studies designed to demonstrate such an abnormality have generally been unsuccessful. Nevertheless, Genest (4), in a recent review, documented several such disturbances in patients with benign essential hypertension. Included in this list of abnormalities were the inability of these patients to suppress aldosterone secretion in response to an oral salt load, and a significant decrease in aldosterone metabolic clearance rate. Additionally, plasma aldosterone has been shown to be elevated in 33% and 18-hydroxy-deoxycorticosterone secretion elevated in 65% of patients with benign essential hypertension (4). Under these abnormal conditions, those patients currently being classified as low-renin hypertensives might merely be those with the more profound disturbances. Another possibility is that renin suppression may simply be a response to elevation of blood pressure. It is clear that such elevations tend to suppress renin values acutely in isolated perfused kidneys (15,16). This suppression is achieved by altering renal proximal arterial perfusion pressure. In support of this possibility, Walker et al. (17) have recently demonstrated a negative correlation between both systolic and diastolic blood pressure and plasma renin activity in hypertensive subjects. A third possibility is that renin suppression among hypertensive subjects may reflect an increased availability of angiotensin II at receptor sites. This latter concept, proposed by Sambhi et al. (7), suggests that increased angiotensin II levels acting directly at the level of the juxtaglomerular apparatus inhibit renin release. Sambhi has suggested that the increased availability of angiotensin may be the result of enhanced renin reactivity in the hypertensive plasmas. These are but three of a number of hypotheses which might explain our finding of suppressed renin concentration in patients with essential hypertension. According to these postulates, those patients with the lowest renin concentration (and also the
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RENIN REACTIVITY IN HUMAN HYPERTENSION lowest renin activity) represent the lower end of the spectrum and not a unique entity. Our findings of a 60% increase in renin reactivity in patients with low renin hypertension and 130% increase in normal renin hypertensives is at variance with many previous reports which have recently been reviewed by Poulson (1). Nevertheless, they compare remarkably well with several recent studies. Kotchen et al. have reported a 25% acceleration (2) and a 50-100% acceleration (8) in patients with essential hypertension. Principle differences between their renin reactivity methodology and ours were their use of dimercaprol and 8-hydroxy-quinoline as angiotensinase inhibitors and a physiologic pH for incubation. In general, they have used exogenous renin concentrations comparable to ours. Nonetheless, even using 100 times greater concentrations of exogenous renin, they detected increased renin reactivity in hypertensive plasma. Sambhi et al. (4) have demonstrated 50-100% increases in renin reactivity in plasma from hypertensive subjects. The major difference in their methodology was the use of larger (0.5 ml) concentrations of exogenous renin. Similar angiotensinase inhibitors and incubation pH were employed in their studies and ours. We have also demonstrated that patients with normal renin hypertension have a somewhat greater acceleration of the renin reaction than do patients with low renin hypertension. It is possible that this difference in reactivity partially explains the low plasma renin activity (relative to the normal renin hypertension) observed in hyporeninemic patients. Nevertheless, while renin reactivity was 27% lower in the low renin group, renin concentration was 56% lower in comparison to the normal renin hypertensive population. Thus, suppression of renin concentration would appear to be at least as important in determining the hyporeninemic state as decreased renin reactivity. This study does not permit further clarification of the cause of the increased renin
689
reactivity in hypertensive plasmas. There is disagreement as to whether this increased reactivity reflects the presence of an accelerator, the absence of an inhibitor, or molecular alteration of either renin or its substrate. Sambhi et al. (7) have demonstrated that hypertensive plasma contains an accelerator of the enzyme reaction. Utilizing an isolated system of human renin and homologous renin substrate, they have shown that the addition of small quantities of hypertensive plasma increases both Km and V max of the reaction. This action suggests the presence of an uncompetitive accelerator in the hypertensive plasma. In contrast, Kotchen et al. (8) demonstrated that acetone extraction of normal plasma accelerated the renin reaction while similar treatment did not affect the reaction velocity in hypertensive plasma. Furthermore, addition of the acetone extract obtained from plasmas of normotensive subjects to the hypertensive plasmas caused deceleration of the renin reaction. Kotchen interprets this to mean that an inhibitor, normally present in the non-hypertensive state, is absent from hypertensive plasma. Which, if either, of these interpretations is correct is not known and the answer will require further study. There is reason for caution in interpreting all data, including ours, which deal with renin concentration. In our case, the use of an external renin standard makes the assumption that only a single physiologically important molecular species of renin is present in peripheral plasma. It is not clear that this is the case and at least one report (18) suggests that under some conditions, altered molecular forms of renin may occur in plasma. On the other hand, methods for measuring renin concentration which depend upon the addition of exogenous, homologous or heterologous, substrate may not permit the expression of the effect of circulating modifiers of the renin reaction. As this is the most frequently employed method for measuring renin concentration, this might at least partially account for previous failures to observe low renin concentrations
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in hypertensive plasmas. At present, there are potentially pitfalls in the utilization of either of these methods for measuring renin concentration. In summary, renin reactivity appears to be increased in plasmas of most hypertensive patients, and to a significantly greater degree in patients with normal plasma renin activity. Renin concentration, when assessed by the methods we have utilized, is suppressed in all plasma samples of hypertensive patients and to a greater degree in those from patients with low renin hypertension. References 1. Poulson, K., Kinetics of the renin system, Scan J Clin Lab Med (Suppl 132) 31: 11, 1973. 2. Kotchen, T. A., T. W. Rice, and D. W. Walters, Renin reactivity in normal, hypertensive, and uremic plasma, J Clin Endocrinol Metab 34: 928, 1972. 3. McDonald, W. J., E. L. Cohen, C. P. Lucas, and J. W. Conn, Renin reactivity in plasma of patients with essential hypertension and suppressed PRA (abstract), Clin Res 21: 284, 1973. 4. Sambhi, M. P., M. G. Crane, and J. Genest, Essential hypertension: New concepts about mechanisms, Ann Intern Med 79: 411, 1973. 5. McDonald, W. J., J. W. Conn, E. L. Cohen, and C. P. Lucas, Increased generation velocity of angiotensin I in plasma of patients with primary aldosteronism, Clin Res 19: 652, 1971 (Abstract). 6. Rieger, D., J. C. Romero, J. Lazar, and S. W. Hoobler, Definition and use of renin reaction velocity in the study of human hypertension, J Lab Clin Med 80: 342, 1972. 7. Sambhi, M. P., P. Eggena, J. D. Barrett, M. Tuck and C. E. Wiedeman, A circulating renin activator in essential hypertension, Circ Res (Suppl 1) 36-37: 28, 1975.
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8. Kotchen, T. A., R. T. Talwalker, 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. 9. Lucas, C. P., S. Fukuchi, J. W. Conn, F. G. Berlinger, W. Waldhausl, E. L. Cohen, and D. A. Rovner, Purification of human renin, J Lab Clin Med 76: 689, 1970. 10. Cohen, E. L., C. E. Grim, J. W. Conn, W. N. Blough, R. B. Guyer, D. C. Kern, and C. P. Lucas, Accurate and rapid measurement of plasma renin activity by radioimmunoassay, / Lab Clin Med 77: 1025, 1971. 11. Cohen, E. L., J. W. Conn, C. P. Lucas, W. J. McDonald, C. E. Grim, G. H. Meyer, S. E. Saltman, and J. M. Caldwell, In Genest, J., and E. Koiw (eds.) Hypertension, Springer-Verlag, Berlin, 1972, p. 569. 12. Haas, E., A. Gould, and H. Goklblatt, Estimation of endogenous renin in human blood, Lancet 1: 657, 1968. 13. Crane, M. G., J. J. Harris, V. J. Johns, Hyporeninemic hypertension, Am J Med 52: 457, 1972. 14. Spark, R. F., J. C. Melby, Hypertension and low plasma renin activity; presumptive evidence for mineralocorticoid excess, Ann Intern Med 75: 831, 1971. 15. Hofbauer, K. G., H. Zschiedrich, E. Hackenthal and F. Gross, Function of the renin-angiotensin system in the isolated perfused rat kidney, Circ Res (Suppl 1) 34-35: 193, 1974. 16. Kolyanides, G. J., R. D. Bastron, and G. F. DiBona, Effect of ureteral clamping and increased renal arterial pressure on renin release, Am J Phijsiol 225: 95, 1973. 17. Walker, W. G., J. S. Horvath, M. A. Moore, P. Whelton and R. P. Russel, Relation between plasma renin activity, angiotensin and aldosterone and blood pressure in mild untreated hypertension, Circ Res 38: 470, 1976. 18. Day, R. P., J. A. Luetscher, and C. M. Gonzales, Occurrence of high renin in human plasma, amniotic fluid and kidney extracts, J Clin Endocrinol Metab 40: 1078, 1975.
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ABSTRACT. Renin activity, concentration, substrate and reactivity were determined in normal subjects as well as in hypertensive subjects with suppressed and normal plasma renin activity. Renin substrate measurements were similar in all groups. Renin reactivity, a measure of circulating modifiers of the renin reaction, was significantly increased in both hypertensive groups. Reactivity was significantly greater in the normal renin hypertensive group than the low renin hypertensive group. Renin concentration was significantly suppressed in both hypertensive groups, but to a greater degree
P
LASMA renin activity measurements have been traditionally employed for evaluating the renin system in man and experimental animals. These methods have generally proven satisfactory. Several observations, however, raise questions regarding the applicability of these methods in experimental studies requiring more precise knowledge of circulating renin concentration. First, the kinetic characteristics of the renin-renin substrate reaction remain unclear. A recent review of investigation designed to clarify this point suggests that the renin reaction in vitro does not follow the laws of zero order kinetics with respect to its substrate (1). This observation alone limits the usefulness of plasma renin activity determination. Under these non-zero Received October 26, 1976. Supported in part by United States Public Health Service Grant No. HL-14520, National Heart and Lung Institute, National Institutes of Health; by a Grant (RR-42) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. Send all communications to: Walter J. McDonald, M.P., Veterans Administration Hospital, Portland, Oregon 97207. 1 MRIS 5577. 2 VA Distinguished Physician.
order conditions, plasma renin activity is at least partly dependent upon substrate concentration as well as enzyme concentration. Of equal concern are observations that, independent of substrate concentrations, angiotensin generation per unit of renin in plasma (renin reactivity) is not the same in all plasmas. Patients with essential hypertension (2-4), uremia (2), primary aldosteronism (5), renovascular hypertension (2), and those on oral contraceptives (6) have been demonstrated to have increased renin reactivity in their plasmas. These findings have been interpreted as reflecting the presence of an accelerator (7), the absence of an inhibitor (8), or a molecular alteration of either renin or its substrate (7). From the foregoing, it is apparent that plasma renin activity does not always reflect renin concentration. For these reasons, we have investigated the relationship between plasma renin activity, renin activity, and renin concentration in both normal subjects and patients with essential hypertension. The hypertensive subjects were classified into normal and low-renin categories according to their plasma renin activities. Significantly lower plasma renin concentrations and higher
685
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MCDONALD, COHEN AND CONN
renin reactivities were found in both the low and normal-renin hypertensive groups when compared to normotensives. Renin reactivity and renin concentration were significantly higher in the normal renin hypertensives as compared to the low-renin hypertensives. Materials and Methods Fourteen nonhypertensive subjects (Group A), 10 subjects with essential hypertension and normal plasma renin activity (Group B), and 19 subjects with hyporeninemic essential hypertension (Group C) were studied. All subjects were white and less than 60 years of age. Secondary forms of hypertension were ruled out by iv pyelogram, peripheral renin activity determinations, urinary VMA, aldosterone, and Porter-Silber determinations. Patients were categorized as hypertensive if the blood pressure measurements were consistently greater than 145/95. Subjects were maintained for three days on a diet containing 10 milli-equivalent of sodium per day, followed by three days on one containing 120 milliequivalent of sodium per day. Following the third day of each dietary period, blood was obtained at 0800 h following 2 h of upright posture for determination of plasma renin activity, renin substrate, renin concentration and renin reactivity. If the low sodium diet and upright posture failed to stimulate a rise in plasma renin activities to values greater than 3.2 ng/ml/h, the patient was classified as hyporeninemic. This value represents the lowest observed renin activity in a previous study involving twenty non-hypertensive subjects following a similar protocol. Renin Human renal renin was prepared by the method of Lucas et al. (9). This renin preparation was devoid of angiotensinase activity in the concentrations used. Measurement of plasma renin activity, concentration, and substrate The measurement of renin parameters was accomplished in all specimens by radioimmunoassay of angiotensin I by the method of Cohen et al. (10). The complete methods for these measurements have been described in detail elsewhere (11). EDTA and diisopropylfluoro-
JCE & M • 1977 Vol 45 • No 4
phosphate are employed as angiotensinase inhibitors and the incubation is carried out at pH 5.5. It should be emphasized that renin concentration was measured by a modification of the method of Haas (12). This method employs an external renin standard to eliminate the influence of variations of substrate and other undefined factors. A comparison of angiotensin I generation, both before and after the addition of the known quantity of renin, permits the calculation of renin concentration by the following two formulae: 1. Angiotensin I generated/unit exogenous renin = (Angiotensin I generated/unit exogenous + endogenous renin) — endogenous plasma renin activity. 2. Plasma renin concentration = Plasma renin activity/(Angiotensin I generated/unit exogenous renin/ml). The results are expressed in Goldblatt units/ml. Measurement of renin reactivity Renin reactivity (defined as the amount of angiotensin generated/ml/h/1 x 10"4 Goldblatt Units/ml) was measured by a method which employs an external renin standard. Plasma was prepared as for plasma renin activity measurement and separated into 100 /xl aliquots. To the first aliquot, 10 pi of saline were added and to the second, 10 /xl of purified human renin containing 1.1 x 10~5 Goldblatt units of renin. After 30 min of incubation, each sample was assayed in triplicate for angiotensin. Subtraction of the angiotensin generated in the first sample from that generated in the second sample yields the amount of angiotensin generated per 1 x 10~4 Goldblatt units of renin per ml or the renin reactivity. To assure that reaction velocity did not change with substrate utilization, a third aliquot was utilized and 2.2 x 10~5 Goldblatt units of renin were added to this sample prior to incubation. In all cases, angiotensin generation was linear with respect to added renin.
Results
Plasma renin activity and substrate concentration Table 1 lists the mean plasma renin activity and substrate concentration for the
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687
RENIN REACTIVITY IN HUMAN HYPERTENSION normal group during the 10 and 120 milliequivalent sodium diets, and the two hypertensive groups during the higher sodium intake. The mean plasma renin activity during the two sodium intake periods in normal subjects were significantly different at the P < .01 level. The mean plasma renin activities of Group A and B during the higher salt intake did not differ significantly from each other. Plasma renin activity in Group C patients was significantly different from both Group A and Group B at the P < .01 level. There was no significant difference in the mean substrate concentrations between any of the groups. Renin reactivity Figure 1 illustrates the mean renin reactivity in normal subjects (Group A) on the two sodium diets. The mean values are not statistically different. Renin reactivity appears to be independent of the sodium intake. Because of this finding, and prior observations showing renin reactivity to
TABLE 1. Renin activity and substrate concentration in normal and hypertensive subjects
Normal—Group A 120 meq Na+ Diet (N = 14) 10 meq Na+ Diet (N = 10) Hypertensive 120 meq Na+ Diet Normal PRA-Group B (N = 10) Low PRA-Group C (N = 19)
Renin activity (ng/mg/h ± SEM)
Substrate (/ig/ml ± SEM)
5.4 ± 0.7» 10.3 ± 1.1
1.39 + 0.15 1.54 ±0.14
6.0 ± l.lf 1.2 ± 0.3*
1.54 ± 0.16 1.33 ± 0.08
• Significantly lower than all other renin activity values at P < .01. f Significantly lower than renin activity in Group A on the 10 meq diet at P < .01.
be independent of renin concentration (2,11), renin reactivity in the hypertensive population was determined only in samples obtained during the 120 milliequivalent sodium intake. Renin reactivity in all three groups during the higher sodium intake is also depicted in Fig. 1. All three mean values are significantly different from each other at the P < .01 level. The renin reaction is accelerated in both groups of patients with essential hypertension. It is less accelerated, how-
16r
14
12
FiG. 1. Renin reactivity in normal subjects on a 10 and a 120 millequivalent sodium diet and hypertensive subjects on a 120 millequivalent diet. Mean renin reactivities, marked by a dash, are not significantly different in the two normal groups. All reactivities on the 120 millequivalent diet differ from each other at P < .01.
10
mm
RfAcnvirr Ȥ/ml/ki I x 10" 4 GO 6
Noraol
Momal
10 meq
120
«M
Hyperteisire NOMMI
Hyptrftisiv*
Stppntiti
m
120 mte
120 «M
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MCDONALD, COHEN AND CONN
688
TABLE 2. Mean renin concentrations in normal and hypertensive subjects on 120 mEq sodium intake PRC (GU x 10-7mI) ± SEM Normals (Group A) Essential Hypertension Normal PRA (Group B) Low PRA (Group C)
1.20 ± 0.21 0.47 ± 0.09* 0.21 ± 0.05t
* Significantly different from both other groups at P > .05. f Significantly different from Group A at P < .01.
ever, in patients with hyporeninemic hypertension. Plasma renin concentration The mean plasma renin concentrations for the three groups during the 120 milliequivalent sodium intake are tabulated in Table 2. The mean renin concentration in hypertensive subjects with normal renin activity differs significantly from that of normal subjects despite the fact that renin activity is identical in the two groups. The mean renin concentration in both groups of patients with essential hypertension is significantly suppressed in comparison to normals. Discussion It has been repeatedly demonstrated that between 20 and 40% of patients with essential hypertension display blunted responses to manipulations designed to stimulated renin activity (13). The etiology of this hyporeninemic response is unclear and remains a source of speculation, much of which centers around the possibility of mineralocorticoid excess (13,14). Our finding of suppressed renin concentration in patients with both low and normal renin activity, however, raises another possibility. It is possible that there is a factor common to both hypertensive categories which is acting to suppress renin concentration. What that factor or factors might be is a matter of conjecture. One possibility is that all patients with essential hypertension have derangements of
JCE&M Vol 45
< 1977 No 4
mineralocorticoid metabolism leading to manifestations of mineralocorticoid excess. Studies designed to demonstrate such an abnormality have generally been unsuccessful. Nevertheless, Genest (4), in a recent review, documented several such disturbances in patients with benign essential hypertension. Included in this list of abnormalities were the inability of these patients to suppress aldosterone secretion in response to an oral salt load, and a significant decrease in aldosterone metabolic clearance rate. Additionally, plasma aldosterone has been shown to be elevated in 33% and 18-hydroxy-deoxycorticosterone secretion elevated in 65% of patients with benign essential hypertension (4). Under these abnormal conditions, those patients currently being classified as low-renin hypertensives might merely be those with the more profound disturbances. Another possibility is that renin suppression may simply be a response to elevation of blood pressure. It is clear that such elevations tend to suppress renin values acutely in isolated perfused kidneys (15,16). This suppression is achieved by altering renal proximal arterial perfusion pressure. In support of this possibility, Walker et al. (17) have recently demonstrated a negative correlation between both systolic and diastolic blood pressure and plasma renin activity in hypertensive subjects. A third possibility is that renin suppression among hypertensive subjects may reflect an increased availability of angiotensin II at receptor sites. This latter concept, proposed by Sambhi et al. (7), suggests that increased angiotensin II levels acting directly at the level of the juxtaglomerular apparatus inhibit renin release. Sambhi has suggested that the increased availability of angiotensin may be the result of enhanced renin reactivity in the hypertensive plasmas. These are but three of a number of hypotheses which might explain our finding of suppressed renin concentration in patients with essential hypertension. According to these postulates, those patients with the lowest renin concentration (and also the
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RENIN REACTIVITY IN HUMAN HYPERTENSION lowest renin activity) represent the lower end of the spectrum and not a unique entity. Our findings of a 60% increase in renin reactivity in patients with low renin hypertension and 130% increase in normal renin hypertensives is at variance with many previous reports which have recently been reviewed by Poulson (1). Nevertheless, they compare remarkably well with several recent studies. Kotchen et al. have reported a 25% acceleration (2) and a 50-100% acceleration (8) in patients with essential hypertension. Principle differences between their renin reactivity methodology and ours were their use of dimercaprol and 8-hydroxy-quinoline as angiotensinase inhibitors and a physiologic pH for incubation. In general, they have used exogenous renin concentrations comparable to ours. Nonetheless, even using 100 times greater concentrations of exogenous renin, they detected increased renin reactivity in hypertensive plasma. Sambhi et al. (4) have demonstrated 50-100% increases in renin reactivity in plasma from hypertensive subjects. The major difference in their methodology was the use of larger (0.5 ml) concentrations of exogenous renin. Similar angiotensinase inhibitors and incubation pH were employed in their studies and ours. We have also demonstrated that patients with normal renin hypertension have a somewhat greater acceleration of the renin reaction than do patients with low renin hypertension. It is possible that this difference in reactivity partially explains the low plasma renin activity (relative to the normal renin hypertension) observed in hyporeninemic patients. Nevertheless, while renin reactivity was 27% lower in the low renin group, renin concentration was 56% lower in comparison to the normal renin hypertensive population. Thus, suppression of renin concentration would appear to be at least as important in determining the hyporeninemic state as decreased renin reactivity. This study does not permit further clarification of the cause of the increased renin
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reactivity in hypertensive plasmas. There is disagreement as to whether this increased reactivity reflects the presence of an accelerator, the absence of an inhibitor, or molecular alteration of either renin or its substrate. Sambhi et al. (7) have demonstrated that hypertensive plasma contains an accelerator of the enzyme reaction. Utilizing an isolated system of human renin and homologous renin substrate, they have shown that the addition of small quantities of hypertensive plasma increases both Km and V max of the reaction. This action suggests the presence of an uncompetitive accelerator in the hypertensive plasma. In contrast, Kotchen et al. (8) demonstrated that acetone extraction of normal plasma accelerated the renin reaction while similar treatment did not affect the reaction velocity in hypertensive plasma. Furthermore, addition of the acetone extract obtained from plasmas of normotensive subjects to the hypertensive plasmas caused deceleration of the renin reaction. Kotchen interprets this to mean that an inhibitor, normally present in the non-hypertensive state, is absent from hypertensive plasma. Which, if either, of these interpretations is correct is not known and the answer will require further study. There is reason for caution in interpreting all data, including ours, which deal with renin concentration. In our case, the use of an external renin standard makes the assumption that only a single physiologically important molecular species of renin is present in peripheral plasma. It is not clear that this is the case and at least one report (18) suggests that under some conditions, altered molecular forms of renin may occur in plasma. On the other hand, methods for measuring renin concentration which depend upon the addition of exogenous, homologous or heterologous, substrate may not permit the expression of the effect of circulating modifiers of the renin reaction. As this is the most frequently employed method for measuring renin concentration, this might at least partially account for previous failures to observe low renin concentrations
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MCDONALD, COHEN AND CONN
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in hypertensive plasmas. At present, there are potentially pitfalls in the utilization of either of these methods for measuring renin concentration. In summary, renin reactivity appears to be increased in plasmas of most hypertensive patients, and to a significantly greater degree in patients with normal plasma renin activity. Renin concentration, when assessed by the methods we have utilized, is suppressed in all plasma samples of hypertensive patients and to a greater degree in those from patients with low renin hypertension. References 1. Poulson, K., Kinetics of the renin system, Scan J Clin Lab Med (Suppl 132) 31: 11, 1973. 2. Kotchen, T. A., T. W. Rice, and D. W. Walters, Renin reactivity in normal, hypertensive, and uremic plasma, J Clin Endocrinol Metab 34: 928, 1972. 3. McDonald, W. J., E. L. Cohen, C. P. Lucas, and J. W. Conn, Renin reactivity in plasma of patients with essential hypertension and suppressed PRA (abstract), Clin Res 21: 284, 1973. 4. Sambhi, M. P., M. G. Crane, and J. Genest, Essential hypertension: New concepts about mechanisms, Ann Intern Med 79: 411, 1973. 5. McDonald, W. J., J. W. Conn, E. L. Cohen, and C. P. Lucas, Increased generation velocity of angiotensin I in plasma of patients with primary aldosteronism, Clin Res 19: 652, 1971 (Abstract). 6. Rieger, D., J. C. Romero, J. Lazar, and S. W. Hoobler, Definition and use of renin reaction velocity in the study of human hypertension, J Lab Clin Med 80: 342, 1972. 7. Sambhi, M. P., P. Eggena, J. D. Barrett, M. Tuck and C. E. Wiedeman, A circulating renin activator in essential hypertension, Circ Res (Suppl 1) 36-37: 28, 1975.
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8. Kotchen, T. A., R. T. Talwalker, 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. 9. Lucas, C. P., S. Fukuchi, J. W. Conn, F. G. Berlinger, W. Waldhausl, E. L. Cohen, and D. A. Rovner, Purification of human renin, J Lab Clin Med 76: 689, 1970. 10. Cohen, E. L., C. E. Grim, J. W. Conn, W. N. Blough, R. B. Guyer, D. C. Kern, and C. P. Lucas, Accurate and rapid measurement of plasma renin activity by radioimmunoassay, / Lab Clin Med 77: 1025, 1971. 11. Cohen, E. L., J. W. Conn, C. P. Lucas, W. J. McDonald, C. E. Grim, G. H. Meyer, S. E. Saltman, and J. M. Caldwell, In Genest, J., and E. Koiw (eds.) Hypertension, Springer-Verlag, Berlin, 1972, p. 569. 12. Haas, E., A. Gould, and H. Goklblatt, Estimation of endogenous renin in human blood, Lancet 1: 657, 1968. 13. Crane, M. G., J. J. Harris, V. J. Johns, Hyporeninemic hypertension, Am J Med 52: 457, 1972. 14. Spark, R. F., J. C. Melby, Hypertension and low plasma renin activity; presumptive evidence for mineralocorticoid excess, Ann Intern Med 75: 831, 1971. 15. Hofbauer, K. G., H. Zschiedrich, E. Hackenthal and F. Gross, Function of the renin-angiotensin system in the isolated perfused rat kidney, Circ Res (Suppl 1) 34-35: 193, 1974. 16. Kolyanides, G. J., R. D. Bastron, and G. F. DiBona, Effect of ureteral clamping and increased renal arterial pressure on renin release, Am J Phijsiol 225: 95, 1973. 17. Walker, W. G., J. S. Horvath, M. A. Moore, P. Whelton and R. P. Russel, Relation between plasma renin activity, angiotensin and aldosterone and blood pressure in mild untreated hypertension, Circ Res 38: 470, 1976. 18. Day, R. P., J. A. Luetscher, and C. M. Gonzales, Occurrence of high renin in human plasma, amniotic fluid and kidney extracts, J Clin Endocrinol Metab 40: 1078, 1975.
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