AMERICAN JOURNAL OF PHYSIOLOGY Vol. 230, No. 2, February 1976. Printed
in U.S.A.
Renin-angiotensin-aldosterone system in experimental renal hypertension in the rabbit THOMAS E. LOHMEIER Department of Physiology,
AND JAMES 0. DAVIS University of Missouri School of Medicine, Columbia, Missouri 65201
LOHMEIER, THOMAS E., AND JAMES 0. DAVIS.R~CZ-WZ~~otensin-aldosterone system in experimental renal hypertension in the rabbit. Am. J. Physiol. 230(2): 311-318. 1976. -Hypertension was produced in 25 rabbits by constricting the right renal artery and leaving the opposite kidney intact (twokidney hypertension). After 30 days mean arterial pressure and plasma renin activity (PRA) were significantly elevated (p < O.Ol), and arterial pressure was correlated with PRA (r = 0.551, P < 0.01); however, not all hypertensive rabbits had elevated PRA, and in animals in which sodium balance was monitored, only rabbits in negative sodium balance had increased levels of PRA. To investigate the role of angiotensin II (A-II) in the hypertension, [Lsarcosine,%alanine]angiotensin II was infused at 6 ,ug/kg per min for 30 min in anesthetized hypertensive animals (n = 25). For the group, arterial pressure fell significantly (P < O.Ol), but several animals with minimal hypertension failed to give a depressor response. The declines in arterial pressure were highly correlated with PRA (r = 0.853, P < 0.01). Aldosterone secretion in hypertensive animals was correlated with PRA (r = 0.851, P < 0.01). Thus, two-kidney hypertension in the rabbit persists with normal PRA, but during periods of spontaneous sodium depletion, AII plays a role in the maintenance of the hypertension. aldosterone secretion; corticosterone secretion; deoxycorticosterone secretion; plasma renin activity; arterial pressure; twokidney hypertension; [ 1-sarcosine,8-alaninelangiotensin II; mineralocorticoids; sodium balance; angiotensin II
THE ROLE OF THE renin-angiotensin-aldosterone
system in the pathogenesis of renovascular hypertension is poorly understood. There is disagreement in the literature concerning the role of the renin-angiotensin system in the maintenance of two-kidney hypertension in the rabbit (one renal artery constricted and the contralatera1 kidney intact). In the established phase of the hypertension, plasma renin activity (PRA) has been reported to be elevated in some studies (11, 12) but normal in others (14, 18, 24). Moreover, the arterial pressure responses to partial blockade of the reninangiotensin system with renin antibodies (24) or with specific inhibitors of the renin angiotensin system (14, 25) have been inconsistent. Under physiological conditions there is a well-documented reciprocal relationship between PRA and sodium balance. Although sodium balance has not been monitored in rabbits with twokidney hypertension, rats (13, 28) and sheep (2, 20) with this type of renal hypertension have been reported to be in negative sodium balance. Therefore, the different levels of PRA reported in the literature for two-kidney
hypertension in the rabbit may be attributed to variations in sodium balance among the animals. There are few published reports dealing with adrenal steroid secretion rates in two-kidney hypertensive animals and none at all in the rabbit. Since the reninangiotensin system has been shown to be an important physiological regulator of aldosterone secretion in other animals, perturbation of this system could conceivably increase arterial pressure by sodium retention and expansion of body fluid volume via angiotensin II-mediated increases in aldosterone secretion. Hypertension of this etiology might be partially or totally independent of enhanced pressor activity of angiotensin II. This study was undertaken in an attempt to resolve the current controversy regarding the role of the reninangiotensin system in the maintenance of two-kidney hypertension in the rabbit and to evaluate the potential role of mineralocorticoids in the hypertensive process. The data also contribute to our knowledge of the reninangiotensin system in the control of aldosterone secretion in the rabbit. Arterial pressure, PRA, sodium balance, and adrenal steroid secretion rates of aldosterone, deoxycorticosterone (DOC), and corticosterone were measured 30 days after unilateral renal artery constriction in rabbits with the contralateral kidney intact. Subsequently, the arterial pressure and adrenal steroid secretory responses to infusion of the specific angiotensin II antagonist, [1-sarcosine&alanine]angiotensin II ([Sar1,Ala8]angiotensin II), were determined. There are no prior studies which have attempted to evaluate arterial pressure and adrenal steroidogenic responses to blockage of the renin-angiotensin system in hypertensive animals in which all of the above interrelated parameters were determined. METHODS
Male, New Zealand white rabbits, weighing 2.7-3.7 kg, were housed in a room with a constant temperature of20-22°C and maintained on Purina rabbit chow (sodium content, 100-110 meq/kg; potassium content, 510570 meqkg) and water ad libitum except during sodium depletion. Renal hypertensive rabbits Series 1. Renal hypertension was induced in 25 rabbits by applying a silver clip (ID, 0.6 mm) of the type described by Brooks and Muirhead (4) on the right renal artery; the left kidney was undisturbed. The rabbits
311
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 13, 2019.
312 were subjected to acute experimentation 30 days after renal artery constriction. Two hours prior to surgery the rabbits were injected intramuscularly with 10 mg of dexamethasone sodium phosphate (Decadron; Merck Sharpe & Dohme) to suppress ACTH secretion. Using local anesthesia (2% lidocaine), a marginal ear vein and a femoral vein and artery were cannulated with polyethylene tubing for infusions and blood pressure recordings. Subsequently, the rabbits were anesthetized with intermittent injections of 2% thiopental and laparotomized. Following ligation of the left renal artery and vein close to the kidney, a catheter (French 5 polyvinyl chloride) was placed in the left renal vein to allow sampling of adrenal venous blood. All collateral veins emptying into the renal vein were ligated. The animals were then heparinized (1,000 U/kg) and left undisturbed for 30 min before taking a 7- to &ml arterial blood sample for determinations of PRA,’ plasma sodium and potassium concentration, hematocrit, and serum urea nitrogen (SUN). Then, a timed adrenal blood sample (45 ml) was collected by retrograde blood flow by applying a clamp on the renal vein between the vena cava and the adrenal gland. Following collection of this control sample, [Sar1,Ala8]angiotensin II was infused in 0.9% saline at 0.1 ml/min into the femoral vein for 30 min before obtaining a second timed adrenal blood sample; the concentration of the antagonist in the infusate was adjusted to achieve a rate of infusion of 6 pg/kg min. One hour after infusion of the antagonist, a recovery adrenal venous blood sample was obtained. A final arterial blood sample was then collected for determinations of hematocrit and plasma electrolytes. During all blood sampling, blood was simultaneously replaced with an equal volume of 6% dextran in 0.9% saline. Throughout the experiment mean arterial pressure was measured continuously by a Statham pressure transducer (P23Db) and a Sanborn recorder (model 7700), and the values presented in the results were those recorded just prior to blood sampling. Body temperature was monitored with a rectal probe (Yellow Springs Instrument Company) and was maintained at 35-37°C with a heat lamp. Twelve of these rabbits were placed in metabolic cages either during the final 5 days of the hypertensive period (n = 6) or for 5 days prior to renal artery constriction and for the entire 30 days thereafter (n = 6). Food consumption, urine volume, and urinary excretion of sodium and potassium were determined daily. In each rabbit daily sodium balance was estimated on the basis of dietary intake and urinary excretion, and in the latter group of six animals feces were also collected at weekly intervals, and the sodium content was determined and included in the calculation of sodium balante. Series 2. Renal hypertension was again produced in seven additional rabbits by constricting the right renal 1 In unpublished studies from our laboratory in the rabbit, PRA was found to be unchanged by the anesthesia and the experimental procedure used here for collection of adrenal vein blood. PRA measured on plasma obtained from conscious rabbits with thoracic caval constriction or controls was 8.2 + SEM 1.0 ng angiotensin II per milliliter with a range of 4.7-16.0 compared with 8.3 + 0.9 ng angiotensin II per milliliter with a range of 4.0-14.8 after anesthesia and the experimental procedure (n = 12).
T. E. LOHMEIER
AND
J. 0.
DAVIS
artery as described above. Approximately 25 days after renal artery stenosis, the animals were placed in metabolic cages for 5-7 days so that sodium balance could be monitored. Subsequently, the central ear artery was cannulated using local anesthesia, and each rabbit was placed in a rectangular box and left undisturbed. Mean arterial pressure was recorded with the rabbit conscious, and a 6-ml blood sample was taken for determination of PRA, plasma concentrations of sodium and potassium, and hematocrit. After replacing the blood with fresh blood from a donor rabbit, the rabbits were returned to their metabolic cages and given a sodiumdeficient diet (General Biochemicals) containing 10 meq sodium per kilogram and 230 meq potassium per kilogram for 7 days. Each rabbit also received 0.1 ml/kg Mercuhydrin intramuscularly for the first 2 days of the low-sodium diet. At the end of this period of sodium depletion, the central ear artery was again cannulated, mean blood pressure measured, and a blood sample taken for PRA, plasma electrolytes, and hematocrit. The blood was replaced with donor blood, and once again the rabbits were returned to their metabolic cages. The animals were then maintained on the regular laboratory chow for 7-10 days before taking a final measurement of arterial pressure and a final blood sample for PRA, plasma electrolytes, and hematocrit. Normal
rabbits
In seven rabbits food intake and urine volume were measured for at least 3 days prior to subjecting the animals to the acute experimental procedure used for the hypertensive rabbits in series 1. Analytical
methods
The double-isotope derivative assay was used to measure adrenal steroids. Aldosterone, deoxycorticosterone, and corticosterone were determined in 2-3 ml of adrenal vein plasma by the double-isotope derivative procedure of Kliman and Peterson (16) with modifications for the measurement of DOC (30). These methods have been validated previously for analysis of adrenal steroids in rabbit plasma (3). Adrenal secretion rates were calculated from the concentration of each steroid and the adrenal plasma flow. For measurement of PRA, blood was collected in 10% ethylenediaminetetracetate (EDTA) and centrifuged, and the plasma was frozen for later analysis. Plasma samples for renin activity were processed for generation of angiotensin I as described previously (3) and assayed by the pressor response in the pentobarbital-anesthetized, pentolinium-blocked rat. PRA is expressed as nanograms angiotensin generated during 90 min of incubation. Concentrations of sodium and potassium in plasma, food, feces, and urine were determined by flame photometry. Food and feces were digested by a technique described elsewhere (9). Hematocrit was determined by a microhematocrit method, and SUN levels were measured in the Clinical Chemistry Laboratory of the University Hospital. Statistical analysis of data between groups and between pairs was done by the Student t test.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 13, 2019.
RENAL
HYPERTENSION
IN
THE
313
RABBIT
RESULTS
Normal
rabbits
The seven control rabbits had an average arterial pressure of 84 * 2 SEM mmHg with a range from 80 to 92 mmHg. Mean PRA was 4.0 t 0.2 ng angiotensin per milliliter plasma, and the secretion rates for aldosterone, corticosterone, and DOC were 1.7 t 0.6 ng/min, 0.88 * 0.26 pg/min, and 0.08 t 0.03 Fg/min, respectively. Adrenal plasma flow was 0.7 t 0.1 ml/min. During the infusion of [Sar1,Ala8]angiotensin II, mean arterial pressure and aldosterone secretion were not significantly different from their respective control or recovery values, although the values for aldosterone secretion increased in six of seven animals and were as high as 5.7 ng/min during antagonist infusion. Similarly, there were no significant alterations in adrenal plasma flow or the secretion rates of corticosterone and DOC. Control values for sodium concentration, potassium concentration, and hematocrit were 139 t 1 meq/liter, 2.6 2 0.1 meq/liter, and 38 t l%, respectively, and although concentrations of plasma electrolytes remained constant during experimentation, hematocrit fell significantly to 34 -+ 1% (P < 0.01) by the end of the recovery period. Food intake in this group of animals was 188 t 4 SEM g/day; daily urine volume averaged 207 t 16 ml. Hypertensive
rabbits
Series 1. In the 25 rabbits with renal artery constriction, mean arterial pressure ranged from 81 to 129 mmHg and PRA ranged from 1.6 to 32.0 ng angiotensin per milliliter plasma; the corresponding means were 102 2 3 SEM mmHg and 9.5 t 1.8 ng angiotensin per milliliter plasma, and both of these means were significantly greater than those for normal rabbits (P < 0.01). For the entire series there was a significant positive correlation between PRA and arterial pressure (r = 0.551, P < 0.01) (Fig. 1). Adrenal steroid secretion was measured in 16 of the 25 animals. The average adrenal secretion rates for aldosterone (6.0 t 2.3 ng/min) and DOC (0.12 t 0.02 pg/min) and the rate of adrenal plasma flow were unchanged
compared with control animals. Corticosterone secretion was significantly higher (P < 0.05) in the hypertensive animals (2.3 * 0.3 pg/min) than in the control rabbits (0.89 t .26 pglmin). The secretion rate of aldosterone was extremely variable (0.3-37 ng/min) but was highly correlated with PRA (r = 0.851, P < 0.01) (Fig. 2). Adrenal venous plasma was discarded in four rabbits with very low adrenal blood flows, and in five other animals no adrenal venous blood could be obtained because the adrenal vein emptied directly into the vena cava. In the 25 rabbits with renal artery stenosis, mean plasma sodium and potassium concentrations were similar to control, although hematocrit (43 t 1%) was significantly elevated (P < O.Ol>, particularly in the eight animals (46 t 1%) with the highest PRA. Serum urea nitrogen averaged 24 t 1 mg/lOO ml, a value similar to that reported by Johnson et al. (14) for control rabbits. Mean food consumption (n = 12) during the last 5 days of the hypertension was 204 2 4 g/day, which is slightly greater (P < 0.01) than control, whereas the average urine volume for the respective 5-day period (424 -+ 19 ml/day) was markedly elevated (P < 0.01). In the six animals in this series where daily food consumption and urine volume were monitored prior to and following renal artery constriction, the average food intake for the final 5 days of the hypertensive period was essentially the same as the preconstriction level, while mean urine volume had increased significantly from 261 t 20 to 391 -+ 27 ml/day (P < 0.01). In these six rabbits in which extensive sodium balance measurements were made, sodium recovery (urinary sodium + fecal sodium)/(dietary sodium) during the control period averaged 90 * 3 SEM % with a range of 80-90%. For the first 3 wk after renal artery constriction, there were no significant alterations in mean sodium recovery or any consistent trends in sodium balance in the individual animals. Similarly, during the final week of the hypertensive period, five of the six
130
9E 120 5. E
I IO
ii K
100 d E P CK 90 a
80
0
5
IO PLASMA
FIG.
activity
1. Relationship in 25 rabbits
I5 RENIN
20 ACTIVITY
25
30
35
(rig/ml)
between arterial pressure and plasma with chronic 2-kidney hypertension.
renin
FIG.
renin
2. Relationship between acti vity in 16 rabbits with
aldosterone secretion and plasma chronic 2-ki dney hypertension.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 13, 2019.
314
T. E. LOHMEIER
animals apparently maintained their sodium status. In contrast to these five animals, one rabbit was in negative sodium balance (16 meq sodium) after the final 5 day period of the hypertension (Table 1). PRA was elevated in this rabbit (32.0 ng angiotensin/ml plasma) but normal in the other five animals. Table 1 shows the relationship between PRA and sodium balance during the final 5 days of the 30-day hypertensive period for 12 of the rabbits in this series. Elevated levels of PRA were not found in all hypertensive animals, but PRA was highest in those animals that were in negative sodium balance. Further, hypertension was severe in three of the four rabbits with the highest values for PRA. Since fecal sodium content was not determined in all 12 rabbits, it was not possible to correlate PRA with the absolute state of sodium balance; however, it is clear from urinary and dietary sodium data alone that the four animals with the highest PRA were in negative sodium balance during the 5 day period. During infusion of [Sar1,Ala8]angiotensin II, mean arterial pressure fell from 102 t 3 to 91 t 3 mmHg (P < 0.01; n = 24) and remained depressed during the recovery period. The depressor response to analogue infusion ranged from 0 to 35 mmHg and was well correlated with the preexisting level of PRA (r = 0.853, P < 0.01) (Fig. 3). Similarly, there was considerable variation in the aldosterone secretory response to administration of the antagonist. Changes in aldosterone secretion during infusion of analogue were highly correlated (r = - 0.945, P < 0.01) (Fig. 4) with preinfusion secretion rates of aldosterone. During infusion of the angiotensin antagonist, aldosterone secretion declined when initially elevated and fell a maximum of 17 nglmin in the animal with the highest preinfusion secretion rate of aldosterone (37 nglmin). As in the normal rabbits, aldosterone secretion often appeared to increase slightly (3 ng/min or less) in the hypertensive animals with normal secretion rates of mineralocorticoid. Overall, the decline in aldosterone secretion to a mean value of 4.2 st 1.2 from 6.0 t 2.3 ng/min was not statistically significant. 1. Relationship between plasma renin activity and sodium balance in rabbits with two-kidney Goldblatt hypertension (series 1) ---
AND
J. 0.
DAVIS
0
-5 r=-0 853 35 I
(p=
in U.S.A.
Renin-angiotensin-aldosterone system in experimental renal hypertension in the rabbit THOMAS E. LOHMEIER Department of Physiology,
AND JAMES 0. DAVIS University of Missouri School of Medicine, Columbia, Missouri 65201
LOHMEIER, THOMAS E., AND JAMES 0. DAVIS.R~CZ-WZ~~otensin-aldosterone system in experimental renal hypertension in the rabbit. Am. J. Physiol. 230(2): 311-318. 1976. -Hypertension was produced in 25 rabbits by constricting the right renal artery and leaving the opposite kidney intact (twokidney hypertension). After 30 days mean arterial pressure and plasma renin activity (PRA) were significantly elevated (p < O.Ol), and arterial pressure was correlated with PRA (r = 0.551, P < 0.01); however, not all hypertensive rabbits had elevated PRA, and in animals in which sodium balance was monitored, only rabbits in negative sodium balance had increased levels of PRA. To investigate the role of angiotensin II (A-II) in the hypertension, [Lsarcosine,%alanine]angiotensin II was infused at 6 ,ug/kg per min for 30 min in anesthetized hypertensive animals (n = 25). For the group, arterial pressure fell significantly (P < O.Ol), but several animals with minimal hypertension failed to give a depressor response. The declines in arterial pressure were highly correlated with PRA (r = 0.853, P < 0.01). Aldosterone secretion in hypertensive animals was correlated with PRA (r = 0.851, P < 0.01). Thus, two-kidney hypertension in the rabbit persists with normal PRA, but during periods of spontaneous sodium depletion, AII plays a role in the maintenance of the hypertension. aldosterone secretion; corticosterone secretion; deoxycorticosterone secretion; plasma renin activity; arterial pressure; twokidney hypertension; [ 1-sarcosine,8-alaninelangiotensin II; mineralocorticoids; sodium balance; angiotensin II
THE ROLE OF THE renin-angiotensin-aldosterone
system in the pathogenesis of renovascular hypertension is poorly understood. There is disagreement in the literature concerning the role of the renin-angiotensin system in the maintenance of two-kidney hypertension in the rabbit (one renal artery constricted and the contralatera1 kidney intact). In the established phase of the hypertension, plasma renin activity (PRA) has been reported to be elevated in some studies (11, 12) but normal in others (14, 18, 24). Moreover, the arterial pressure responses to partial blockade of the reninangiotensin system with renin antibodies (24) or with specific inhibitors of the renin angiotensin system (14, 25) have been inconsistent. Under physiological conditions there is a well-documented reciprocal relationship between PRA and sodium balance. Although sodium balance has not been monitored in rabbits with twokidney hypertension, rats (13, 28) and sheep (2, 20) with this type of renal hypertension have been reported to be in negative sodium balance. Therefore, the different levels of PRA reported in the literature for two-kidney
hypertension in the rabbit may be attributed to variations in sodium balance among the animals. There are few published reports dealing with adrenal steroid secretion rates in two-kidney hypertensive animals and none at all in the rabbit. Since the reninangiotensin system has been shown to be an important physiological regulator of aldosterone secretion in other animals, perturbation of this system could conceivably increase arterial pressure by sodium retention and expansion of body fluid volume via angiotensin II-mediated increases in aldosterone secretion. Hypertension of this etiology might be partially or totally independent of enhanced pressor activity of angiotensin II. This study was undertaken in an attempt to resolve the current controversy regarding the role of the reninangiotensin system in the maintenance of two-kidney hypertension in the rabbit and to evaluate the potential role of mineralocorticoids in the hypertensive process. The data also contribute to our knowledge of the reninangiotensin system in the control of aldosterone secretion in the rabbit. Arterial pressure, PRA, sodium balance, and adrenal steroid secretion rates of aldosterone, deoxycorticosterone (DOC), and corticosterone were measured 30 days after unilateral renal artery constriction in rabbits with the contralateral kidney intact. Subsequently, the arterial pressure and adrenal steroid secretory responses to infusion of the specific angiotensin II antagonist, [1-sarcosine&alanine]angiotensin II ([Sar1,Ala8]angiotensin II), were determined. There are no prior studies which have attempted to evaluate arterial pressure and adrenal steroidogenic responses to blockage of the renin-angiotensin system in hypertensive animals in which all of the above interrelated parameters were determined. METHODS
Male, New Zealand white rabbits, weighing 2.7-3.7 kg, were housed in a room with a constant temperature of20-22°C and maintained on Purina rabbit chow (sodium content, 100-110 meq/kg; potassium content, 510570 meqkg) and water ad libitum except during sodium depletion. Renal hypertensive rabbits Series 1. Renal hypertension was induced in 25 rabbits by applying a silver clip (ID, 0.6 mm) of the type described by Brooks and Muirhead (4) on the right renal artery; the left kidney was undisturbed. The rabbits
311
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 13, 2019.
312 were subjected to acute experimentation 30 days after renal artery constriction. Two hours prior to surgery the rabbits were injected intramuscularly with 10 mg of dexamethasone sodium phosphate (Decadron; Merck Sharpe & Dohme) to suppress ACTH secretion. Using local anesthesia (2% lidocaine), a marginal ear vein and a femoral vein and artery were cannulated with polyethylene tubing for infusions and blood pressure recordings. Subsequently, the rabbits were anesthetized with intermittent injections of 2% thiopental and laparotomized. Following ligation of the left renal artery and vein close to the kidney, a catheter (French 5 polyvinyl chloride) was placed in the left renal vein to allow sampling of adrenal venous blood. All collateral veins emptying into the renal vein were ligated. The animals were then heparinized (1,000 U/kg) and left undisturbed for 30 min before taking a 7- to &ml arterial blood sample for determinations of PRA,’ plasma sodium and potassium concentration, hematocrit, and serum urea nitrogen (SUN). Then, a timed adrenal blood sample (45 ml) was collected by retrograde blood flow by applying a clamp on the renal vein between the vena cava and the adrenal gland. Following collection of this control sample, [Sar1,Ala8]angiotensin II was infused in 0.9% saline at 0.1 ml/min into the femoral vein for 30 min before obtaining a second timed adrenal blood sample; the concentration of the antagonist in the infusate was adjusted to achieve a rate of infusion of 6 pg/kg min. One hour after infusion of the antagonist, a recovery adrenal venous blood sample was obtained. A final arterial blood sample was then collected for determinations of hematocrit and plasma electrolytes. During all blood sampling, blood was simultaneously replaced with an equal volume of 6% dextran in 0.9% saline. Throughout the experiment mean arterial pressure was measured continuously by a Statham pressure transducer (P23Db) and a Sanborn recorder (model 7700), and the values presented in the results were those recorded just prior to blood sampling. Body temperature was monitored with a rectal probe (Yellow Springs Instrument Company) and was maintained at 35-37°C with a heat lamp. Twelve of these rabbits were placed in metabolic cages either during the final 5 days of the hypertensive period (n = 6) or for 5 days prior to renal artery constriction and for the entire 30 days thereafter (n = 6). Food consumption, urine volume, and urinary excretion of sodium and potassium were determined daily. In each rabbit daily sodium balance was estimated on the basis of dietary intake and urinary excretion, and in the latter group of six animals feces were also collected at weekly intervals, and the sodium content was determined and included in the calculation of sodium balante. Series 2. Renal hypertension was again produced in seven additional rabbits by constricting the right renal 1 In unpublished studies from our laboratory in the rabbit, PRA was found to be unchanged by the anesthesia and the experimental procedure used here for collection of adrenal vein blood. PRA measured on plasma obtained from conscious rabbits with thoracic caval constriction or controls was 8.2 + SEM 1.0 ng angiotensin II per milliliter with a range of 4.7-16.0 compared with 8.3 + 0.9 ng angiotensin II per milliliter with a range of 4.0-14.8 after anesthesia and the experimental procedure (n = 12).
T. E. LOHMEIER
AND
J. 0.
DAVIS
artery as described above. Approximately 25 days after renal artery stenosis, the animals were placed in metabolic cages for 5-7 days so that sodium balance could be monitored. Subsequently, the central ear artery was cannulated using local anesthesia, and each rabbit was placed in a rectangular box and left undisturbed. Mean arterial pressure was recorded with the rabbit conscious, and a 6-ml blood sample was taken for determination of PRA, plasma concentrations of sodium and potassium, and hematocrit. After replacing the blood with fresh blood from a donor rabbit, the rabbits were returned to their metabolic cages and given a sodiumdeficient diet (General Biochemicals) containing 10 meq sodium per kilogram and 230 meq potassium per kilogram for 7 days. Each rabbit also received 0.1 ml/kg Mercuhydrin intramuscularly for the first 2 days of the low-sodium diet. At the end of this period of sodium depletion, the central ear artery was again cannulated, mean blood pressure measured, and a blood sample taken for PRA, plasma electrolytes, and hematocrit. The blood was replaced with donor blood, and once again the rabbits were returned to their metabolic cages. The animals were then maintained on the regular laboratory chow for 7-10 days before taking a final measurement of arterial pressure and a final blood sample for PRA, plasma electrolytes, and hematocrit. Normal
rabbits
In seven rabbits food intake and urine volume were measured for at least 3 days prior to subjecting the animals to the acute experimental procedure used for the hypertensive rabbits in series 1. Analytical
methods
The double-isotope derivative assay was used to measure adrenal steroids. Aldosterone, deoxycorticosterone, and corticosterone were determined in 2-3 ml of adrenal vein plasma by the double-isotope derivative procedure of Kliman and Peterson (16) with modifications for the measurement of DOC (30). These methods have been validated previously for analysis of adrenal steroids in rabbit plasma (3). Adrenal secretion rates were calculated from the concentration of each steroid and the adrenal plasma flow. For measurement of PRA, blood was collected in 10% ethylenediaminetetracetate (EDTA) and centrifuged, and the plasma was frozen for later analysis. Plasma samples for renin activity were processed for generation of angiotensin I as described previously (3) and assayed by the pressor response in the pentobarbital-anesthetized, pentolinium-blocked rat. PRA is expressed as nanograms angiotensin generated during 90 min of incubation. Concentrations of sodium and potassium in plasma, food, feces, and urine were determined by flame photometry. Food and feces were digested by a technique described elsewhere (9). Hematocrit was determined by a microhematocrit method, and SUN levels were measured in the Clinical Chemistry Laboratory of the University Hospital. Statistical analysis of data between groups and between pairs was done by the Student t test.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 13, 2019.
RENAL
HYPERTENSION
IN
THE
313
RABBIT
RESULTS
Normal
rabbits
The seven control rabbits had an average arterial pressure of 84 * 2 SEM mmHg with a range from 80 to 92 mmHg. Mean PRA was 4.0 t 0.2 ng angiotensin per milliliter plasma, and the secretion rates for aldosterone, corticosterone, and DOC were 1.7 t 0.6 ng/min, 0.88 * 0.26 pg/min, and 0.08 t 0.03 Fg/min, respectively. Adrenal plasma flow was 0.7 t 0.1 ml/min. During the infusion of [Sar1,Ala8]angiotensin II, mean arterial pressure and aldosterone secretion were not significantly different from their respective control or recovery values, although the values for aldosterone secretion increased in six of seven animals and were as high as 5.7 ng/min during antagonist infusion. Similarly, there were no significant alterations in adrenal plasma flow or the secretion rates of corticosterone and DOC. Control values for sodium concentration, potassium concentration, and hematocrit were 139 t 1 meq/liter, 2.6 2 0.1 meq/liter, and 38 t l%, respectively, and although concentrations of plasma electrolytes remained constant during experimentation, hematocrit fell significantly to 34 -+ 1% (P < 0.01) by the end of the recovery period. Food intake in this group of animals was 188 t 4 SEM g/day; daily urine volume averaged 207 t 16 ml. Hypertensive
rabbits
Series 1. In the 25 rabbits with renal artery constriction, mean arterial pressure ranged from 81 to 129 mmHg and PRA ranged from 1.6 to 32.0 ng angiotensin per milliliter plasma; the corresponding means were 102 2 3 SEM mmHg and 9.5 t 1.8 ng angiotensin per milliliter plasma, and both of these means were significantly greater than those for normal rabbits (P < 0.01). For the entire series there was a significant positive correlation between PRA and arterial pressure (r = 0.551, P < 0.01) (Fig. 1). Adrenal steroid secretion was measured in 16 of the 25 animals. The average adrenal secretion rates for aldosterone (6.0 t 2.3 ng/min) and DOC (0.12 t 0.02 pg/min) and the rate of adrenal plasma flow were unchanged
compared with control animals. Corticosterone secretion was significantly higher (P < 0.05) in the hypertensive animals (2.3 * 0.3 pg/min) than in the control rabbits (0.89 t .26 pglmin). The secretion rate of aldosterone was extremely variable (0.3-37 ng/min) but was highly correlated with PRA (r = 0.851, P < 0.01) (Fig. 2). Adrenal venous plasma was discarded in four rabbits with very low adrenal blood flows, and in five other animals no adrenal venous blood could be obtained because the adrenal vein emptied directly into the vena cava. In the 25 rabbits with renal artery stenosis, mean plasma sodium and potassium concentrations were similar to control, although hematocrit (43 t 1%) was significantly elevated (P < O.Ol>, particularly in the eight animals (46 t 1%) with the highest PRA. Serum urea nitrogen averaged 24 t 1 mg/lOO ml, a value similar to that reported by Johnson et al. (14) for control rabbits. Mean food consumption (n = 12) during the last 5 days of the hypertension was 204 2 4 g/day, which is slightly greater (P < 0.01) than control, whereas the average urine volume for the respective 5-day period (424 -+ 19 ml/day) was markedly elevated (P < 0.01). In the six animals in this series where daily food consumption and urine volume were monitored prior to and following renal artery constriction, the average food intake for the final 5 days of the hypertensive period was essentially the same as the preconstriction level, while mean urine volume had increased significantly from 261 t 20 to 391 -+ 27 ml/day (P < 0.01). In these six rabbits in which extensive sodium balance measurements were made, sodium recovery (urinary sodium + fecal sodium)/(dietary sodium) during the control period averaged 90 * 3 SEM % with a range of 80-90%. For the first 3 wk after renal artery constriction, there were no significant alterations in mean sodium recovery or any consistent trends in sodium balance in the individual animals. Similarly, during the final week of the hypertensive period, five of the six
130
9E 120 5. E
I IO
ii K
100 d E P CK 90 a
80
0
5
IO PLASMA
FIG.
activity
1. Relationship in 25 rabbits
I5 RENIN
20 ACTIVITY
25
30
35
(rig/ml)
between arterial pressure and plasma with chronic 2-kidney hypertension.
renin
FIG.
renin
2. Relationship between acti vity in 16 rabbits with
aldosterone secretion and plasma chronic 2-ki dney hypertension.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 13, 2019.
314
T. E. LOHMEIER
animals apparently maintained their sodium status. In contrast to these five animals, one rabbit was in negative sodium balance (16 meq sodium) after the final 5 day period of the hypertension (Table 1). PRA was elevated in this rabbit (32.0 ng angiotensin/ml plasma) but normal in the other five animals. Table 1 shows the relationship between PRA and sodium balance during the final 5 days of the 30-day hypertensive period for 12 of the rabbits in this series. Elevated levels of PRA were not found in all hypertensive animals, but PRA was highest in those animals that were in negative sodium balance. Further, hypertension was severe in three of the four rabbits with the highest values for PRA. Since fecal sodium content was not determined in all 12 rabbits, it was not possible to correlate PRA with the absolute state of sodium balance; however, it is clear from urinary and dietary sodium data alone that the four animals with the highest PRA were in negative sodium balance during the 5 day period. During infusion of [Sar1,Ala8]angiotensin II, mean arterial pressure fell from 102 t 3 to 91 t 3 mmHg (P < 0.01; n = 24) and remained depressed during the recovery period. The depressor response to analogue infusion ranged from 0 to 35 mmHg and was well correlated with the preexisting level of PRA (r = 0.853, P < 0.01) (Fig. 3). Similarly, there was considerable variation in the aldosterone secretory response to administration of the antagonist. Changes in aldosterone secretion during infusion of analogue were highly correlated (r = - 0.945, P < 0.01) (Fig. 4) with preinfusion secretion rates of aldosterone. During infusion of the angiotensin antagonist, aldosterone secretion declined when initially elevated and fell a maximum of 17 nglmin in the animal with the highest preinfusion secretion rate of aldosterone (37 nglmin). As in the normal rabbits, aldosterone secretion often appeared to increase slightly (3 ng/min or less) in the hypertensive animals with normal secretion rates of mineralocorticoid. Overall, the decline in aldosterone secretion to a mean value of 4.2 st 1.2 from 6.0 t 2.3 ng/min was not statistically significant. 1. Relationship between plasma renin activity and sodium balance in rabbits with two-kidney Goldblatt hypertension (series 1) ---
AND
J. 0.
DAVIS
0
-5 r=-0 853 35 I
(p=
