Clinical Science and Molecular Medicine (1975) 48,17-26.
Changes in the renin-angiotensin-aldosterone system and in sodium and potassium balance during development of renal hypertension in rats F. H. H. LEENEN, J. W. SCHEEREN, D. OMYLANOWSKI,(1) J. D. ELEMA,(2) B. VAN DER WAL(3) AND W. DE JONG Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, The Netherlands
(Received 29 May 1974)
Summary
4. The initial sodium retention could be a factor in the early rise of blood pressure and could account for the delay in the rise of peripheral plasma renin activity. The subsequent loss of the retained sodium and potassium during the development of severe hypertension could have facilitated the rise in peripheral plasma renin activity, but did not initiate this rise.
1. The relationships between the renin-angiotensin-aldosterone system, sodium and potassium balance and systolic blood pressure were studied during development of moderate (160-180 mmHg; clip i.d. 0·25 mm) and severe (200-230 mmHg; clip i.d, 0·20 mm) renal hypertension in rats with an undisturbed contralateral kidney. 2. In severely hypertensive rats renin activity in the peripheral plasma increased from day 9, by which time the systolic blood pressure was elevated to 160180 mmHg. The rate of total corticosteroid and aldosterone production in vitro increased from day 14 and plasma renin substrate concentration increased from day 24. In moderately hypertensive rats, none of these changes occurred. 3. During the first 10 days after the application of 0·25 and 0'20 mm clips, sodium and potassium retentionjg gain in body weight were higher than in shamoperated controls. During the next 10 days, the positive balance stabilized in animals with a 0·25 mm clip whereas, in animals with a 0'20 mm clip, sodium and potassium balance returned to the level of the sham-operated controls through increased renal losses. Despite these changes the systolic pressure rose further in animals with a 0'20 mm clip.
Key words: renal hypertension, plasma renin activity, aldosterone production, sodium and potassium balance. Introduction In a previous study in rats it was found that;. after application of silver clips to a renal artery, basal peripheral plasma renin activity (PRA) increased when the systolic blood pressure increased above 160-180 mmHg. No changes were found in basal peripheral PRA in animals with moderate hypertension (Leenen, de Jong & de Wied, 1973). In renal hypertensive rats renal sodium excretion increased when the systolic blood pressure exceeded 180 mmHg (Gross, 1971). Gross (1971) suggested that the observed negative sodium balance contributed to the stimulation of the renin-angiotensin-aldosterone system and that renin hypersecretion may be the consequence rather than the cause of the hypertension. A similar relationship was suggested to occur during development of severe renal hypertension in sheep (Blair-West, Coghlan, Denton, Orchard, Scoggins & Wright, 1968). In the present study, the time-course of the changes in the renin-angiotensin system (peripheral PRA and
• (1) Present address: Department of Pharmacology, University of Toronto, Toronto, Canada. (2) !=>epartment of Pathology, University of Groningen, Groningen, The Netherlands. (~) Pr~sent address: Department of Pharmacology, Free University, Amsterdam, The Netherlands. Correspondence: Dr F. H. H. Leenen Rudolf Magnus Institute for Pharmacology, Vondellaan' 6, Utrecht, The Netherlands.
17
F. H. H. Leenen et al.
18
plasma renin substrate concentration) and adrenocortical activity were assessed during the development of two levels of renal hypertension in rats with an undisturbed contralateral kidney. Daily sodium and potassium balances were measured in order to establish the time-relationship between the changes in these balances and in the renin-angiotensin-aldosterone system.
Materials and methods
General Male white rats of an inbred Wistar strain were used, weighing 140-160 g at the time of application of a renal arterial clip. Animals had free access to food and tap water. Standard rat pellets or finely ground chow (Trouw, Amsterdam, The Netherlands) contained 111 tlmol of sodium and 242 tlmol of potassium/g (mean of four estimations). Under ether anaesthesia, silver clips with internal diameters of 0·25 or 0'20 mm (Leenen & de Jong, 1971) were applied to the left renal artery to achieve moderate or severe constriction. The contralateral kidney re-
50
0·50
mained undisturbed in all rats. Control animals were operated on in the same manner, except for the actual application of a clip. In order to study the development of treatment effects over time, the experiments (five to ten rats per group) were terminated at different time-intervals after operation. Systolic blood pressure was determined in conscious trained rats five to seven times a week by a tail sphygmographic method (Leenen & de Jong, 1971). Approximately 4 ml of blood was collected from the neck within 30 s after decapitation. In order to prevent activation of the hypothalamo-hypophysealadrenal system and non-specific stimulation of renin secretion (Vander, 1967; Hauger-Klevene, Brown & Fleischer, 1969), this was done in a quiet animal-room without disturbing the animals before the decapitation. The data in Fig. 1 indicate that a distinct diurnal variation in PRA and adrenal cortical secretion exists. Because of the possibility that the pattern of changes in these parameters during the development of renal hypertension may differ, depending on the time of measurement, rats were always decapitated at the trough of the endogenous activity of normal rats
I
300. 0",
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e
a. :g
•
V
e
.l!! OJ)
,§
8
"0
«
"5
25
0·25
~
I -----I
~I
150.
I
09.00
I
I
16.00
I
21.00
I
02.00
Time (hours)
FIG. I. Diurnal variation in peripheral plasma renin activity (nmol of angiotensin II equivalents h -1 ml- 1) and total corticosteroid and aldosterone production in vitro of intact rats. Results are mean values (±SEM) for eight rats. Asterisks indicate statistical differences (** P< 0'01; * P< 0'05) as compared with the 09.00 hours group.
Renin and Nar balance in renal hypertension
(between 08.00 and 10.00 hours) to ensure that basal levels were indeed obtained. Organ weights were measured on a torsion balance. Estimation ofsodium and potassium balance
Urine and faeces of twenty individual rats were collected separately in metabolic cages (ACME, Chicago, U.S.A.). Urine was collected in polypropylene cylinders containing two drops of 1 mol/l H 2S0 4 , Demineralized drinking water was offered in calibrated cylinders outside the cages. After an adjustment period of 3 days, the daily food and water intake were measured each morning; spilled food was collected. Urine production was measured and the funnel of the metabolic cage cleaned with demineralized water, diluting the urine to 25 ml. Faeces were dried for 24 h at 90-100°C, weighed and pulverized in a mortar. For the extraction of the electrolytes 1 mI of an acid solution (96% H 2S0 4 - 65% HN0 3 , 1: 1) was added for each gram of dried faeces. After 15 min extraction, each 1 ml of acid was diluted to 25 ml with demineralized water and the sample shaken for 10 min. After filtration the filtrate was diluted 1: 10 and the amounts of electrolytes in the urine and in the faeces extract were measured with an Eppendorff flame photometer against an internal standard. Comparison of the values found after acid extraction of faeces and food or after ashing showed no significant differences. Results were expressed as daily balance (intake-output) and as cumulative balance per gram gain in body weight, because when the increase in body weight is diminished a smaller percentage of the sodium and potassium intake will be retained in the body (Mohring & Mohring, 1972).
19
1973) was estimated by blood pressure bioassay and expressed as nmol of angiotensin II h -1 mI -1 equivalents. Asp I.NH 2 - Val/-anglotensin IT(Hypertensin, CIBA) was used as the standard in all bioassays. Plasma renin substrate was measured by a substrate exhaustion technique (Nasjletti, Matsunaga & Masson, 1969). Partially purified rat kidney renin (0'25 Goldblatt unit/mg dry weight of the freezedried powder) was prepared as reported previously (de Jong, Frankhuyzen & Witter, 1969). Measurement of adrenocortical activity
Adrenal glands were incubated in vitro for 1 h according to Csanky, Van der Wal & de Wied (1968); the total corticosteroid production was assessed spectrophotometrically from a corticosterone standard curve (Van der Vies, Bakker & de Wied, 1960); aldosterone production was assessed by measuring alkaline fluorescence on paper after isolation of the aldosterone by thin-layer chromatography (Csanky et al., 1968). Both measurements were expressed as nmol h -1 g-l. Plasma corticosterone concentration was measured as described by Van der Vies et al. (1960). The width of the zona glomerulosa was determined as described previously (Elema, Hardonk, Koudstaal & Arends, 1968). Statistics
Results are expressed as means ± SEM. Statistical analysis of the data was performed by using Wilcoxon's two sample test.
Estimation of renal function
Results
Plasma and urine urea concentration of eighteen rats were estimated according to Glick (1969). Approximately 0·4 mI of blood was obtained after cutting the tip of the tail. Twenty-four hour urine was collected from the rats in the metabolic cages.
The renin-angiotensin system and adrenocortical activity during development ofexperimental renal hypertension
Measurement ofplasma renin activity andplasma renin substrate
Plasma renin activity was measured according to the method of Pickens, Bumpus, Lloyd, Smeby & Page (1965) with slight modifications (de Jong, 1969). The amount of angiotensin I generated (Leenen et al.,
Application of renal arterial clips with internal diameters of 0'25 or 0'20 mm results in hypertension of 160-180 mmHg within 1-2 weeks and 200-230 mmHg within 2-3 weeks respectively (Leenen & de Jong, 1971). As shown previously (Leenen et al., 1973), during the development of these two levels of hypertension peripheral PRA increases only in animals with the 0'20 mm clip, starting about 1 week after operation. Plasma renin substrate concentration
20
F. H. H. Leenen et aT. 500
1-
400
1/
E 0'10, Table 1). Application of the wider 0·25 mm clip did not increase basal peripheral PRA, neither did it increase aldosterone production or total corticosteroid production in vitro, width of the zona glomerulosa or adrenal weight (Table 1). Sodium and potassium balance during the development of experimental renal hypertension
Sham-operation or application of a renal arterial clip resulted in a decreased food intake during the first day after operation (Fig. 4). This decrease was most pronounced in animals with a 0'20 mm clip; only this group of rats showed a significant (P
Changes in the renin-angiotensin-aldosterone system and in sodium and potassium balance during development of renal hypertension in rats F. H. H. LEENEN, J. W. SCHEEREN, D. OMYLANOWSKI,(1) J. D. ELEMA,(2) B. VAN DER WAL(3) AND W. DE JONG Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, The Netherlands
(Received 29 May 1974)
Summary
4. The initial sodium retention could be a factor in the early rise of blood pressure and could account for the delay in the rise of peripheral plasma renin activity. The subsequent loss of the retained sodium and potassium during the development of severe hypertension could have facilitated the rise in peripheral plasma renin activity, but did not initiate this rise.
1. The relationships between the renin-angiotensin-aldosterone system, sodium and potassium balance and systolic blood pressure were studied during development of moderate (160-180 mmHg; clip i.d. 0·25 mm) and severe (200-230 mmHg; clip i.d, 0·20 mm) renal hypertension in rats with an undisturbed contralateral kidney. 2. In severely hypertensive rats renin activity in the peripheral plasma increased from day 9, by which time the systolic blood pressure was elevated to 160180 mmHg. The rate of total corticosteroid and aldosterone production in vitro increased from day 14 and plasma renin substrate concentration increased from day 24. In moderately hypertensive rats, none of these changes occurred. 3. During the first 10 days after the application of 0·25 and 0'20 mm clips, sodium and potassium retentionjg gain in body weight were higher than in shamoperated controls. During the next 10 days, the positive balance stabilized in animals with a 0·25 mm clip whereas, in animals with a 0'20 mm clip, sodium and potassium balance returned to the level of the sham-operated controls through increased renal losses. Despite these changes the systolic pressure rose further in animals with a 0'20 mm clip.
Key words: renal hypertension, plasma renin activity, aldosterone production, sodium and potassium balance. Introduction In a previous study in rats it was found that;. after application of silver clips to a renal artery, basal peripheral plasma renin activity (PRA) increased when the systolic blood pressure increased above 160-180 mmHg. No changes were found in basal peripheral PRA in animals with moderate hypertension (Leenen, de Jong & de Wied, 1973). In renal hypertensive rats renal sodium excretion increased when the systolic blood pressure exceeded 180 mmHg (Gross, 1971). Gross (1971) suggested that the observed negative sodium balance contributed to the stimulation of the renin-angiotensin-aldosterone system and that renin hypersecretion may be the consequence rather than the cause of the hypertension. A similar relationship was suggested to occur during development of severe renal hypertension in sheep (Blair-West, Coghlan, Denton, Orchard, Scoggins & Wright, 1968). In the present study, the time-course of the changes in the renin-angiotensin system (peripheral PRA and
• (1) Present address: Department of Pharmacology, University of Toronto, Toronto, Canada. (2) !=>epartment of Pathology, University of Groningen, Groningen, The Netherlands. (~) Pr~sent address: Department of Pharmacology, Free University, Amsterdam, The Netherlands. Correspondence: Dr F. H. H. Leenen Rudolf Magnus Institute for Pharmacology, Vondellaan' 6, Utrecht, The Netherlands.
17
F. H. H. Leenen et al.
18
plasma renin substrate concentration) and adrenocortical activity were assessed during the development of two levels of renal hypertension in rats with an undisturbed contralateral kidney. Daily sodium and potassium balances were measured in order to establish the time-relationship between the changes in these balances and in the renin-angiotensin-aldosterone system.
Materials and methods
General Male white rats of an inbred Wistar strain were used, weighing 140-160 g at the time of application of a renal arterial clip. Animals had free access to food and tap water. Standard rat pellets or finely ground chow (Trouw, Amsterdam, The Netherlands) contained 111 tlmol of sodium and 242 tlmol of potassium/g (mean of four estimations). Under ether anaesthesia, silver clips with internal diameters of 0·25 or 0'20 mm (Leenen & de Jong, 1971) were applied to the left renal artery to achieve moderate or severe constriction. The contralateral kidney re-
50
0·50
mained undisturbed in all rats. Control animals were operated on in the same manner, except for the actual application of a clip. In order to study the development of treatment effects over time, the experiments (five to ten rats per group) were terminated at different time-intervals after operation. Systolic blood pressure was determined in conscious trained rats five to seven times a week by a tail sphygmographic method (Leenen & de Jong, 1971). Approximately 4 ml of blood was collected from the neck within 30 s after decapitation. In order to prevent activation of the hypothalamo-hypophysealadrenal system and non-specific stimulation of renin secretion (Vander, 1967; Hauger-Klevene, Brown & Fleischer, 1969), this was done in a quiet animal-room without disturbing the animals before the decapitation. The data in Fig. 1 indicate that a distinct diurnal variation in PRA and adrenal cortical secretion exists. Because of the possibility that the pattern of changes in these parameters during the development of renal hypertension may differ, depending on the time of measurement, rats were always decapitated at the trough of the endogenous activity of normal rats
I
300. 0",
0",
1: ~
"0
e
a. :g
•
V
e
.l!! OJ)
,§
8
"0
«
"5
25
0·25
~
I -----I
~I
150.
I
09.00
I
I
16.00
I
21.00
I
02.00
Time (hours)
FIG. I. Diurnal variation in peripheral plasma renin activity (nmol of angiotensin II equivalents h -1 ml- 1) and total corticosteroid and aldosterone production in vitro of intact rats. Results are mean values (±SEM) for eight rats. Asterisks indicate statistical differences (** P< 0'01; * P< 0'05) as compared with the 09.00 hours group.
Renin and Nar balance in renal hypertension
(between 08.00 and 10.00 hours) to ensure that basal levels were indeed obtained. Organ weights were measured on a torsion balance. Estimation ofsodium and potassium balance
Urine and faeces of twenty individual rats were collected separately in metabolic cages (ACME, Chicago, U.S.A.). Urine was collected in polypropylene cylinders containing two drops of 1 mol/l H 2S0 4 , Demineralized drinking water was offered in calibrated cylinders outside the cages. After an adjustment period of 3 days, the daily food and water intake were measured each morning; spilled food was collected. Urine production was measured and the funnel of the metabolic cage cleaned with demineralized water, diluting the urine to 25 ml. Faeces were dried for 24 h at 90-100°C, weighed and pulverized in a mortar. For the extraction of the electrolytes 1 mI of an acid solution (96% H 2S0 4 - 65% HN0 3 , 1: 1) was added for each gram of dried faeces. After 15 min extraction, each 1 ml of acid was diluted to 25 ml with demineralized water and the sample shaken for 10 min. After filtration the filtrate was diluted 1: 10 and the amounts of electrolytes in the urine and in the faeces extract were measured with an Eppendorff flame photometer against an internal standard. Comparison of the values found after acid extraction of faeces and food or after ashing showed no significant differences. Results were expressed as daily balance (intake-output) and as cumulative balance per gram gain in body weight, because when the increase in body weight is diminished a smaller percentage of the sodium and potassium intake will be retained in the body (Mohring & Mohring, 1972).
19
1973) was estimated by blood pressure bioassay and expressed as nmol of angiotensin II h -1 mI -1 equivalents. Asp I.NH 2 - Val/-anglotensin IT(Hypertensin, CIBA) was used as the standard in all bioassays. Plasma renin substrate was measured by a substrate exhaustion technique (Nasjletti, Matsunaga & Masson, 1969). Partially purified rat kidney renin (0'25 Goldblatt unit/mg dry weight of the freezedried powder) was prepared as reported previously (de Jong, Frankhuyzen & Witter, 1969). Measurement of adrenocortical activity
Adrenal glands were incubated in vitro for 1 h according to Csanky, Van der Wal & de Wied (1968); the total corticosteroid production was assessed spectrophotometrically from a corticosterone standard curve (Van der Vies, Bakker & de Wied, 1960); aldosterone production was assessed by measuring alkaline fluorescence on paper after isolation of the aldosterone by thin-layer chromatography (Csanky et al., 1968). Both measurements were expressed as nmol h -1 g-l. Plasma corticosterone concentration was measured as described by Van der Vies et al. (1960). The width of the zona glomerulosa was determined as described previously (Elema, Hardonk, Koudstaal & Arends, 1968). Statistics
Results are expressed as means ± SEM. Statistical analysis of the data was performed by using Wilcoxon's two sample test.
Estimation of renal function
Results
Plasma and urine urea concentration of eighteen rats were estimated according to Glick (1969). Approximately 0·4 mI of blood was obtained after cutting the tip of the tail. Twenty-four hour urine was collected from the rats in the metabolic cages.
The renin-angiotensin system and adrenocortical activity during development ofexperimental renal hypertension
Measurement ofplasma renin activity andplasma renin substrate
Plasma renin activity was measured according to the method of Pickens, Bumpus, Lloyd, Smeby & Page (1965) with slight modifications (de Jong, 1969). The amount of angiotensin I generated (Leenen et al.,
Application of renal arterial clips with internal diameters of 0'25 or 0'20 mm results in hypertension of 160-180 mmHg within 1-2 weeks and 200-230 mmHg within 2-3 weeks respectively (Leenen & de Jong, 1971). As shown previously (Leenen et al., 1973), during the development of these two levels of hypertension peripheral PRA increases only in animals with the 0'20 mm clip, starting about 1 week after operation. Plasma renin substrate concentration
20
F. H. H. Leenen et aT. 500
1-
400
1/
E 0'10, Table 1). Application of the wider 0·25 mm clip did not increase basal peripheral PRA, neither did it increase aldosterone production or total corticosteroid production in vitro, width of the zona glomerulosa or adrenal weight (Table 1). Sodium and potassium balance during the development of experimental renal hypertension
Sham-operation or application of a renal arterial clip resulted in a decreased food intake during the first day after operation (Fig. 4). This decrease was most pronounced in animals with a 0'20 mm clip; only this group of rats showed a significant (P
