Open-Loop Analysis of the Renin-Angiotensin System in the Dog

Open-Loop Analysis of the Renin-Angiotensin

System in the Dog

By A. W. Cowley, Jr., J. P. Miller, and A. C. Guyton

ABSTRACT

The loop gain and time response of the renin-angiotensin system was determined in dogs in which the cardiovascular control loops of the central nervous system were eliminated by spinal cord destruction and decapitation. Step decreases in renal artery perfusion pressure were introduced for a 30-minute period, and changes were measured in systemic arterial blood pressure, cardiac output, and renin activity. The calculated loop gain of both kidneys was —1.59 ±0.11 (SE) and for a single kidney was -0.92 ± 0.02 between renal perfusion pressures of 65 to 100 mm Hg. This indicates nearly a 65% and 50% compensation in pressure, respectively. Below renal perfusion pressures of 65 mm Hg, the gain decreased. The system exhibits a relatively rapid time course with maximum steady-state systemic pressure elevations occurring in an average of 20 minutes, and returning to control levels in an average of 17.8 minutes for bilateral constrictions and 31.9 minutes for single artery constrictions with contralateral nephrectomy. Cardiac outputs were not significantly changed during the constriction period. The arterial renin activity and renal renin secretion rates were consistently elevated during the constrictions. Systemic pressure changes were inhibited by infusion of angiotensin II before constriction and by the injection of angiotensin II antiserum. These results demonstrate that the systemic pressure changes were the result of the renin-angiotensin vasoconstrictor system. The results indicate that the renin-angiotensin system possesses sufficient time response and gain characteristics to participate significantly in the normal regulation of arterial pressure.

KEY WORDS antiangiotensin II radioimmunoassay of angiotensin I renin secretion rate blood pressure regulation renal artery constriction time response of renin-angiotensin system

• The renin-angiotensin system may play a role in the normal control of arterial pressure. Renin activity increases when man assumes the upright posture (1-3), and it has been shown to increase in hemorrhage (4, 5) and in hypotension without hemorrhage (6). Worcel et al. (7) reported a transitory hypotension following injection of a specific antibody against angiotensin II in normal rats and concluded that angiotensin plays a role in

From the Department of Physiology and Biophys-ics, University Medical Center, 2500 N. State Street, Jackson, Mississippi 39216.

This investigation was supported by research grants-in-aid from the American Heart Association and U. S. Public Health Service.

Received November 30, 1970. Accepted for publication February 25, 1971.

maintaining the blood pressure at a normal level.

On the other hand, recent find'ngs have cast doubt on the significance of renin and angiotensin in the pathogenesis of at least some types of renal hypertension. Macdonald et al. (8) reported that immunizing rabbits against angiotensin II did not prevent the development or reverse previously established renal-clip hypertension. Similar findings were reported by Eide and Aars (9) in perinephric hypertension established by wrapping the kidney in turpentine-soaked silk. Evidence exists, therefore, that the renin-angiotensin system is not necessary for the production or maintenance of two types of long-term renal hypertension. This, however, does not pre-clude a role for renin and angiotensin in other

568 Circulation Research, Vol. XXVIII, May 1971

types of hypertension or in the short-term daily regulation of blood pressure.

Although the exact mechanisms are not clearly understood, the kidney has the ability to sense small decreases in mean arterial pressure which results in renin release (10, 11). However, it is not known whether the release of renin and the activation of angio-tensin initiate a feedback mechanism with sufficient gain and time response characteris-tics to significantly offset rapid pressure changes presented to the system. The purpose of these experiments was to evaluate quantita-tively the ability of the reniii-angiotensin system to alter peripheral blood pressure in response to changes in renal perfusion pres-sure. This was studied by measuring the loop gain and time response of the system over a range of blood pressures. The feedback loop was opened by applying a decreased perfusion pressure to the kidneys and holding it constant while the resulting systemic pressure changes were recorded. Neural and hormonal in-fluences of the central nervous system were eliminated by spinal destruction and decapita-tion.

Methods ANIMAL PREPARATION

Forty-three mongrel dogs weighing 16 to 20 kg were anesthetized with sodium thiamylal (Suri-tal), 30 mg/kg, or sodium pentobarbital, 30 mg/kg, to permit exposure of the kidneys, catheterization of blood vessels, destruction of the spinal cord, and decapitation of the animal. The choice of anesthetics appeared to make no difference in the results of the experiment.

A polyvinyl catheter was placed in a femoral artery to measure arterial blood pressure and another in a femoral vein for intravenous infusion of norepinephrine. The renal artery was exposed retroperitoneally through a flank incision, and an adjustable screw clamp was placed around the arteiy. A polyethylene catheter for monitoring pressure was inserted into the artery through an 18-gauge needle distal to the occluder. Renal blood flow was measured with a Biotronex blood flowmeter (Model 410) with a noncannulating Biotronex flow transducer placed on the proximal side of the clamp. When renal arteriovenous blood samples were desired, an additional catheter was inserted into the abdominal aorta through a carotid artery and another catheter

Circulation Research, Vol. XXVlll, May 1971

threaded into the renal vein from the spermatic or ovarian vein.

The spinal cord was destroyed by injecting 80% ethanol (8 to 12 ml) into the spinal canal until respiration ceased and no carotid sinus pressure reflex could be elicited by carotid artery clamp-ing. Blood pressure was maintained by intrave-nous infusion of norepinephrine and respiration by using a Harvard respirator (Model 607A). The neck was then crushed rapidly in a large steel vise, and the head above the vise was subsequently removed without bleeding from the lower neck. The pressure was maintained con-stant at 100 mm Hg for 1 hour preceding any experimental manipulations by carefully adjusting the level of norepinephrine infusion from a Beckman solution metering pump (Model 746). The preparations generally required increasing amounts of norepinephrine for 20 to 40 minutes following decapitation to maintain arterial pres-sure at 100 mm Hg. Following this initial period of adjustment, the preparations generally re-mained stable for several hours. The norepineph-rine infusion was not altered throughout the entire period of data accumulation and the preparations used for the data presented in this paper showed no deterioration during this period.

Simultaneous bilateral partial renal artery constrictions were carried out on 12 dogs using two identical renal artery clamps individually controlled while recording renal artery pressure from each distal renal artery. In 33 additional dogs one kidney was first removed, arterial pressure was stabilized for 1 hour, and the artery of the single remaining kidney was constricted.

Additional catheters were placed into the right atrium and aorta of six dogs through the jugular vein or carotid artery to determine cardiac output by dye dilution. Cardio-Green dye,1 a Gilford Model 1031R cuvette densitometer, and a Gilford Model 105A constant flow system were used. A maximum of 10 ml of blood was removed from the animal, and this generally resulted in a pressure drop of 5 mm Hg in the areflexic dog. All dilution curves were analyzed with a curve tracer connected to a digital computing system with analog-to-digital conversion capabilities, previous-ly described (12). A Model 7 Grass Poprevious-lygraph was used to record renal blood flow, and all blood pressures were measured using Statham P23 strain gauge transducers.

EXPERIMENTAL PROTOCOL

Following the 1-hour stabilization period with arterial pressure at 100 mm Hg, a step-function Supplied by Hynson, Westcott, and Dunning, Baltimore, Md.

decrease in renal artery pressure ranging from 5 to 55 mm Hg was introduced while the response of the aortic blood pressure was recorded and cardiac output was measured intermittently. The renal artery clamp was readjusted as required to maintain the decreased perfusion pressure of the kidney constant over a 30-minute occlusion period, after which the clamp was released and the off-transient responses were recorded. Since the preparation retained good stability for only 3 to 4 hours, an average of only two step-function responses were obtained in each animal. Results were used only if postocclusion blood pressure returned to the control value, indicating no drift from control levels of vascular tone and blood volume. A 1-hour restabilization period followed each occlusion before the procedure was repeat-ed, with the exception of six experiments in which the effects of consecutive step decreases in pressure were studied.

For the experiments in which cardiac outputs were measured, two dye dilution curves were obtained during the 5 minutes preceding the occlusion, and two curves during the resulting plateau period of the maximum aortic pressure rise.

The loop gain of the system was calculated in accordance with the principles of linear control systems by dividing the increase in aortic blood pressure above control values by the pressure drop from control in the renal artery. The immediate slight pressure increase resulting from the increased peripheral resistance at the time of occlusion (about 2 mm Hg) was subtracted from the aortic pressure increase before making the calculation. The pressures used for calculations of the gains were steady-state values recorded during the plateau of the response.

Three procedures were used to make certain that the pressure responses were a result of renin release from the kidney and the activation of angiotensin II.

Radioimmunoassay of Renin Activity.—Arterial

and venous renal blood samples (3 ml) were simultaneously withdrawn immediately before constriction of the renal artery, during the plateau of the pressure response, and after the pressure had returned to preocclusion levels. Zero flow base-line occlusions for the renal artery flowmeter were obtained immediately after each blood sample was withdrawn and required less than 5 seconds.

All blood samples were drawn into ice-cold siliconized syringes and immediately injected into Vacutainer tubes containing 6 mg sodium EDTA. These tubes were kept on ice and centrifuged within 30 minutes at 4°C to obtain 1 ml of plasma for analysis. The protocol used to measure renin activity was based on that described by

Haber et al. (2). Angiotensin I was formed during a 3-hour plasma incubation period under conditions which prevented its conversion to angiotensin II, and at the end of this time its quantity was measured. EDTA, dimercaprol, and 8-hydroxyquinoline were used to inhibit convert-ing enzyme and angiotensinases. Duplicate sam-ples were run for all standards and all plasma assays. Each sample had its own control unincubated sample kept at 4°C so that possible nonspecific effects of plasma or cross reactions with renin substrate were subtracted and correct-ed for in the assay. Measurements made on standards yielded the known quantities of angiotensin I present, and a standard curve was run with each group of incubated samples. A Nuclear-Chicago Corporation (Model 130 B) solid crystal scintillation counter was used for radioactive counting procedures. Renin release rates were calculated by multiplying the arterio-venous renin activity difference times renal plasma flow. The angiotensin I antiserum, I-angiotensin I and I-angiotensin I standard solution were obtained from Schwartz BioResearch (Mountain View Avenue, Orangeburg, New York).

Angiotensin Infusion.—In six dogs angiotensin

II (CIBA) was administered before the renal artery occlusion to inhibit the release of renin from the kidney. In four of these dogs an infusion of angiotensin, 0.6 ^.g/kg/min, was initiated 1 hour before the renal artery occlusion and adjusted before the occlusion so the arterial pressure stabilized at some constant level between 100 and 120 mm Hg. In another two animals the angiotensin infusion was stopped before the occlusion, and norepinephrine was used to set the pressure to 100 mm Hg before the occlusion. The renal arteries of these two animals had also been occluded prior to the angiotensin infusion and had demonstrated a normal pressor response.

Angiotensin II Antiserum.—Enough antiserum

specific against angiotensin II,2 generated in rabbits, was obtained to immunize one 15-kg dog to determine whether the pressure effects of renal artery constriction could be reversed and prevent-ed thereafter. The antiserum was first bioassayprevent-ed using nephrectomized rats given pentolinium tartrate for ganglionic blockade. It was found that 0.2 ml/kg antiserum (the dosage also used in the dog experiment) consistently reduced die pressor effects of a test injection of 2.0 ng angiotensin II (CIBA) from 18 mm Hg to 4 mm Hg. For the dog experiment, a unilaterally nephrectomized areflexic dog was used as previously described. The renal artery pressure was decreased and held 2Courtesy of Dr. Norman Fleischer, Baylor College of Medicine, Houston, Texas.

Circulation Research, Vol. XXVlll, May 1971

Right Renal Artery j * Pressure (mmHg) Constriction FIGURE 1

Effect on systemic arterial blood pressure of decreasing renal artery perfusion pressures bilaterally from 100 to 75 mm Hg.

at 70 mm Hg while the systemic pressure rose. During the plateau of the maximum pressure response, 3 ml of nonimmunized rabbit plasma was first injected through a carotid artery catheter. This was followed in 3 minutes by a 3-ml injection of the rabbit antiserum. The response was followed for another 10 minutes, and the occlusion was then released. At the end of 1 hour the renal artery was again occluded, and the response was recorded once more. Standard test injections of 250 ng angiotensin II were given every 20 minutes after administration of the antiserum to follow the change in sensitivity to this standard dose.

Results

Time Response.—Figure 1 is representative

of a bilateral renal artery occlusion experiment in which a step-function pressure decrease of 25 mm Hg was simultaneously introduced to each kidney. The systemic pressure rose from 100 to 120 mm Hg within the first 5 minutes of occlusion and then climbed more slowly to a maximum of 145 mm Hg by 20 minutes. The manual adjustments of the renal artery occluders to offset the aortic pressure eleva-tion resulted in some renal artery pressure fluctuations in the record, but these were generally held to within ±2.5 mm Hg of the desired pressure value. Release of the occlud-ers resulted in return of the systemic arterial pressure to the control level in 20 minutes.

The average time course of the peripheral pressure responses is presented in Figure 2. To average the pressure responses at the

indi-Circulalion Research, Vol. XXVIII, May 1971

cated time intervals, only those single and double artery occlusions were used in which the renal artery pressure was decreased to 75 to 80 mm Hg. Within 1 minute after the start of the occlusion, there was a pressure rise which increased progressively to its maximum response in 18.8 ± 1.5 ( SE ) minutes for the experiments with single artery occlusion and 21.3 ± 2.0 minutes for bilateral artery occlu-sions. After this time, the pressure plateaued and stabilized until the occlusion was released at the end of 30 minutes. Following release of

5O 4 0 I3O I2O no bilateral Bilateral

A

*

Time course of average systemic arterial pressure changes resulting from unilateral renal artery con-striction (N = 12) with contralateral nephrectomy and simultaneous bilateral renal artery constriction (N = 12). Renal perfusion pressures during constriction were maintained at 75 to 80 mm Hg. Standard errors of the means are indicated.

85-90 75-80 65-70 55-60 45-50 Renol Artery Blood Pressure tmmHg)

FIGURE 3

Relation between renal artery perfusion pressure and the resulting systemic pressure response. Hatched portion represents single renal artery constriction; open portion, double renal artery constriction. Data were obtained from 41 separate occlusions on 26 dogs. Standard errors of the means are indicated.

the occlusion, the peripheral pressure returned to control levels in 31.9 ± 4.4 (SE) minutes for the single artery occlusions and 17.8 ± 3.1 minutes for the bilateral artery occlusions.

Because of the limited stability of the decapitated preparation, the maximum dura-tion of the sustained pressure response could not be determined, but in three exceptionally stable preparations the occlusion was pro-longed to 1 hour. In these experiments, the peripheral pressure response did not decline, and pressures returned to control levels following release of the occlusions. In most animals, the pressure response became pro-gressively weaker following more than two 30-minute occlusions separated by a 1-hour stabilization period.

Pressure Responses.—Figure 3 relates the

average steady-state renal artery perfusion pressures to the resulting systemic arterial pressure values. When the perfusion pressures to both kidneys were simultaneously lowered from 100 mm Hg to any pressure between 65 and 100 mm Hg, the systemic arterial pressure rose an amount equal to 160% of the renal artery decrease. Single renal artery

constric-tion following contralateral nephrectomy re-sulted in a systemic arterial pressure rise equal to 100% of the renal artery pressure decrease. A maximum peripheral pressure response up to a level of 145 to 150 mm Hg was reached when the renal perfusion pressures were bilaterally set at 65 to 70 mm Hg. When the renal artery perfusion pressure was lowered below 60 mm Hg, the resulting systemic pressure rose less than 45% of the decrease in renal artery pressure.

Uninterrupted Step Changes in Renal Per-fusion Pressure.—In six experiments using a

single kidney (the other removed), consecu-tive step decreases in renal perfusion of 10 to 15 mm Hg were introduced and maintained until the elevations of systemic arterial pres-sure reached a steady state. This approach differed from the preceding experiments in that the constrictor was not released at the end of each step decrease, but was instead directly lowered to the next perfusion pres-sure.

100 90 80 70 60 50 Renal Artery Blood Pressure (mmHg)

FIGURE 4

Effect on systemic arterial blood pressure of con-secutive step decreases in renal perfusion pressure to one kidney with contralateral nephrectomy in three areflexic dogs. Each point represents the steady-state pressure elevation at the indicated renal perfusion pressure. The solid line indicates pressure elevations during constriction and the broken line the return of the systemic arterial pressures following release of the constriction.

Circulation Research, Vol. XXVIU, May 1971

573 2.0-1.8 .S 1.6 5 1.4 I.2-i.o

as

FIGURE 5

Calculated loop gains at various renal artery perfusion pressures. Hatched portions of the bars represent single renal artery constriction with contralateral nephrectomy and open portions simultaneous bilateral renal artery constriction.

—*— (2) (12) id31 (2) = RT7T1 ( 3 ) 1(6)1

Figure 4 shows three of these experiments in which renal perfusion pressures were varied between 100 and 55 mm Hg. The systemic arterial pressures responded by increasing an amount equal to nearly 100% of the decrease in renal artery pressure (solid line) between renal perfusion pressures of 65 to 100 mm Hg. The arterial pressure was not elevated much further at renal artery pressures below this range.

The experiment in the lower part of this figure shows the effect of releasing the occluder in consecutive steps, in contrast to the data in the upper part of the figure where the constriction was released in one step. The responses are generally similar, with the exception that the systemic pressures were slightly lower at all stages of the return curve in the animal in which the constriction was released in steps.

Calculated Loop Gains.-The average

cal-culated loop gains of the single and bilateral occlusions are illustrated in Figure 5. It can be seen that lowering the renal artery perfusion pressure from 100 mm Hg to any pressure down to 65 mm Hg resulted in an average loop gain of —0.92 ± 0.02 (SE) for single renal artery occlusions and 1.59 ±0.11 for bilateral occlusions. When the perfusion pressure was

Circulation Research, Vol. XXVIII, May 1971

lowered below 65 mm Hg, the gain sharply declined. In other words, the system appears to operate at maximum pressure feedback gain only between renal perfusion pressures of 65 to 100 mm Hg. Several animals responded with maximum gains down to renal perfusion pressures of 55 to 60 mm Hg, but this was uncommon.

Cardiac Output vs. Total Peripheral Resis-tance during Renal Artery

Constriction.—Ta-ble 1 presents the cardiac output values obtained immediately prior to occlusions and during the plateaus of the maximum pressure response. Cardiac outputs were not significant-ly altered (P>0.1) but, if anything, were decreased rather than increased, while the systemic pressure rose an average of 24 mm Hg in response to a renal artery pressure drop of 30 mm Hg. This represents a significant increase of the calculated total peripheral resistance from 0.095 to 0.125 mm Hg/ml/min ( P < 0.001).

Influence of Norepinephrine on Loop Gain.—

Different rates of infusion of norepinephrine were required to set the control levels of arterial pressures at 100 mm Hg. The effects of these varying rates of infusion on the maxi-mum calculated gain for each animal are shown in Figure 6. The infusion rates were not

TABLE 1

Average Hemodynamic Changes during Single Renal Artery Constriction

Control Constriction Mean arterial blood pressure (mm Hg)

Renal artery perfusion pressure (mm Hg) Cardiac output (liters/min)

Total peripheral resistance (mm Hg/ml/min)

d = mean difference; Sd = standard error of the mean difference; N = number of observations on 7 animals. *P < 0.001; fP > 0.1. 100 100 1.16 0.095 124 70 1.06 0.125 +24* - 3 0 * - o . i o t +0.030* ±9.0 ±6.2 ±0.24 * ±0.010 . 0 10 10 10 0.6 0.4 0.2-N =30 P>0.2 0.1 0.2 0.3 Q4 05 0.6 0.7 Norepinephrine (pg/kg/min.)

Relation between the rate of norepinephrine in-fusion required to stabilize arterial pressure at 100 mm Hg before and during artery constriction and the resulting calculated loop gain. Renal perfusion pres-sures used in this graph were between 65 and 90 mm

Hg. This graph demonstrates the lack of significant

correlation between the two parameters.

altered during the experimental procedures. It can be seen that there was no correlation between the quantity of norepinephrine in-fused and the resulting gain of the system, indicating that the gain of the system was not influenced by plasma catecholamine levels that were used in these experiments (0 to 0.8 ng/kg/min).

Effect of Prior Infusion of Angiotensin II on Peripheral Pressure Response.—The combined

results of three types of experiments indicated that the systemic pressure elevations during the renal artery constriction resulted from activation of the renin-angiotensin system.

First, the peripheral pressure response was nearly abolished in all experiments in which angiotensin II was infused for 1 hour before renal artery constriction, as seen in Figure 7. The renal artery perfusion pressure was lowered to 75 to 80 mm Hg in all experiments represented in this bar graph. Control values were obtained from 12 dogs in which norepinephrine was used to stabilize the blood pressure before and during the renal artery constrictions. The data for the angiotensin infusion experiments is the average of the six dogs described in Methods. Although two of these animals were maintained on angiotensin throughout the constriction period, it appears

- 25.0 20.0-| m .9 10.0-5.0 [112)1 No Angiotensin Angiotensin

Inhibition of the systemic arterial pressure response caused by angiotensin infusion prior to decreasing the renal perfusion pressure to 75 to 80 mm Hg. Standard errors of the mean are indicated.

Circulation Research, Vol. XXVHI, May 1971

575 (10) (10)

m

Control Constriction Post-constriction

The tower graph illustrates the average arterial (hatched portion) and renal venous (open portion) renin activity before, during, and after renal artery constriction. Renin activity expressed as nanograms of angiotensin 1 generated during incubation per milli-liter of plasma per hour. The middle graph represents the net secretion rate of renin by the kidney (using the same units) as calculated from A-V difference times renal plasma flow per gram of kidney. The top graph represents the systemic arterial pressure changes during experiments in which renal perfusion pressure was decreased to 75 to 80 mm Hg. Standard errors of the mean are indicated.

that a high infusion rate of angiotensin before the occlusion will also block the response.

Renin Responses Resulting from Renal Artery Constrictions.—Figure 8 relates the

average systemic arterial pressure increase to the net secretion rate of renin and to the average changes in arterial and renal venous renin activities immediately before, during, and after release of renal artery constriction. The renin activity is expressed as nanograms

Circulation Research, Vol. XXVlll, May 1971

of angiotensin I per milliliter of plasma generated per hour of incubation. Only the occlusions in which a stable base line for renal blood flow was obtained were analyzed for the net release of renin. A significant ele-vation in plasma renin activity occurred dur-ing the constriction period in both renal venous (P < 0.005) and peripheral arterial

(P < 0.025) plasma, and these increases were

accompanied by widening of the mean arteriovenous concentration difference. More important, an increase in the net secretion of renin by the kidney occurred during every occlusion period in which the systemic arterial blood pressure became elevated. One hour following release of the renal artery constric-tion, the plasma renin values had returned to control levels. The calculated increase in the net renin secretion rate was a result of the increased arteriovenous difference in renin activities, since renal blood flow did not change measurably during the con-striction periods, presumably because of autoregulation. The average renal blood flow of 3.5 ±0.5 (SE) ml/min/g of kidney be-fore, and 3.5 ±0.7 ml/min/g during, the plateau of the maximum pressure response.

Effects of Angiotensin II Antiserum on Systemic Pressure Response.—Figure 9 shows

the experimental record of the effects of

25 30 TIME (min.) FIGURE 9

Effects of antiangiotensin U injection on systemic arterial blood pressure. The elevated pressure resulting from the decreased renal perfusion pressure returned rapidly to control levels and remained there in-definitely following release of the constriction.

angiotensin II antibody injection. A typical systemic arterial pressure response was evoked by lowering the renal artery pressure to 70 to 75 mm Hg. The injection of carrier plasma resulted in a slight elevation of arterial blood pressure, which quickly returned to the previous level. Then, immediately after ad-ministration of the antibody, the pressure fell precipitously, initially to slightly below the control level of 100 mm Hg but stabilizing in 5 minutes at 100 mm Hg for the remaining 10 minutes in the occlusion period. The systemic pressure remained at 100 mm Hg for 1 hour after release of the renal constriction. A second occlusion, 1 hour after release of the initial occlusion, resulted in no response when the renal perfusion pressure was decreased to the previous level. Intravenous injections of 250 ng angiotensin II given every 20 minutes for 3 hours following administration of the antibody resulted in a pressure rise of less than 10 mm Hg each time, whereas this same dose raised pressures in nonimmunized are-flexic dogs to 20 to 25 mm Hg.

Decrease in Loop Gain with Intact Nervous System.—To ascertain the effect of the

ner-vous system on the loop gain of the renin-angiotensin system, the head and spinal cord were left intact in five animals in which renal perfusion pressure was lowered to 70 mm Hg. Either no increase or only a slight increase of less than 10 mm Hg occurred in the systemic arterial pressure, resulting in an average calculated gain of —0.16, in contrast to an average gain of —0.92 in the areflexic animal with one kidney removed.

Discussion LOOP GAIN

These experiments show that when operat-ing from a normal pressure of 100 mm Hg, a single kidney in an areflexic dog is capable of elevating the systemic pressure an amount nearly equal to the decrease in renal perfusion pressure in the pressure range of 65 to 100 mm Hg. This corresponds to a loop gain of nearly —1.0 when the feedback circuit was elimi-nated by holding the decreased renal perfu-sion pressure at a constant level while the

systemic blood pressure changed. When the perfusion pressures to both kidneys were simultaneously lowered to between 65 and 100 mm Hg, an open-loop gain of —1.6 was obtained. This means that the systemic pressure was elevated 1.6 times the amount that the renal perfusion pressures were lowered. The maximum short-term systemic pressure response was therefore about 145 to 150 mm Hg when the renal perfusion pressure was lowered from 100 to between 65 and 70 mm Hg.

Since the pressure response following angio-tensin injection exhibits a linear regression with the logarithmic transformation of the dose (13, 14), it comes as no surprise that the gain of a bilateral renal artery constriction is not exactly double that of a single artery constriction.

The calculated gains of -1.0 and -1.6 between renal artery pressures of 65 to 100 mm Hg for the single and double kidney preparations, respectively, indicate that in a closed-loop situation in which systemic pres-sure changes are permitted to act freely on the kidney, the renin-angiotensin mechanism of one kidney could correct a decreased systemic pressure 50% of the way back toward the initial pressure, and both kidneys could correct it nearly 65%. For example, if hemor-rhage resulted in a drop of the systemic arterial pressure (and renal perfusion pressure to both kidneys) to 50 mm Hg, the renin-angiotensin system alone could elevate the systemic arterial pressure to 80 mm Hg. In other words, this system possesses enough gain to have a very significant effect on the final level of systemic blood pressure.

The calculated gain of the system falls off when renal perfusion pressure decreases be-low 65 mm Hg. In the experiments in which renal blood flow was measured, the flows also sharply decreased at renal perfusion pressures below 65 mm Hg, which is consistent with the autoregulatory range reported by others (15, 16). It is possible that renal blood flow was so low at these pressures that the net secretion of renin into the renal vein was compromised.

Circulation Research, Vol. XXVUI, May 1971

The gain measured in decapitated prepara-tions is a result of only the peripheral circulatory and cardiac actions of the renin-angiotensin system. The gain would be expected to be greater in the intact animal with the baroreceptors removed, since the action of angiotensin on the central nervous system has been reported to result in periph-eral vasoconstriction (17-19). This effect, however, was masked in our experiments on intact animals, probably because of action of the baroreceptor mechanism. An increase in the gain of the system could also be expected in situations which stimulate nervous control of renin secretion, which has been shown to occur during hemorrhage even in the absence of changes in mean arterial pressure (20).

There is little information concerning the ability of the system to respond to arterial pressures elevated above normal. Several studies suggest that there is a decrease in renin activity from the normal level with elevated renal perf usion pressures (5, 21), but this decrease could not possibly be great because the rate of renin secretion at normal pressure is already very low. Nevertheless, Worcel et al. (7) concluded that angiotensin plays a role in the maintenance of normal arterial pressure because hypotension oc-curred in normal rats injected with antiangio-tensin II, though hypotension did not occur in rats nephrectomized 24 to 26 hours previously. Fojas and Schmid (22), however, found no change in renin secretion with renal perfusion pressures between 100 and 130 mm Hg, but when pressure decreased below 100 mm Hg, renin secretion rates increased. This could explain our own failure to obtain a systemic pressure change until renal perfusion pres-sures were decreased below 100 mm Hg. Data concerning the normal circulating blood levels of renin and angiotensin are conflicting, so it is difficult to predict the response of the renin-angiotensin feedback system at elevated sys-temic pressures.

The arterial pressures that occur in long standing renal-clip hypertension are greater than those that could result from the gains determined in these studies. The hypertensive

Circulation Research, Vol. XXVlll, May 1971

responses to renal artery clipping appear to occur in two phases. At first, the arterial pressure rises rapidly from a control level of about 100 mm Hg up to about 130 mm Hg and remains near this level for the first 4 days, after which it rises to a maximum at about 20 days (23, 24). With a loop gain of - 1 . 0 for one kidney, a maximum arterial pressure of 130 mm Hg would be expected in an areflexic animal if one kidney was removed and perfusion pressure of the remaining kidney was decreased to 70 mm Hg and maintained at that level. Further reduction in renal pressure would not increase arterial pressure much more because the gain of the system falls greatly at very low renal pressures. The presence of the baroreceptors in an intact animal would reduce the maximum pressure from 130 mm Hg to less than 110 mm Hg for at least the first few days until the reported upward resetting of the baroreceptors oc-curred (25, 26). Only if the effective gain of the renin-angiotensin system increased over prolonged periods could the system account for the high pressure elevation observed in Goldblatt preparations. Such an increased sensitivity has been reported to develop during prolonged infusion of a subpressor dose of angiotensin (17, 27), and this would in effect increase the overall gain of the system and enable it to elevate the pressure to higher levels. It is entirely possible, of course, that the hypertension following renal artery clip-ping is not related to renin or angiotensin, a concept that is supported by the immunization experiments referred to previously (8). TIME RESPONSE OF THE SYSTEM

The time course of the pressure changes resulting from decreased renal perfusion pressure is well documented. Early investiga-tors who completely occluded the renal arteries and followed the pressure rise after the release of the occlusion, observed that the time required to reach maximum pressure was 5 to 15 minutes (28, 29). More recently, it has been reported that the pressure response starts within 5 minutes after reduction of renal perfusion pressures in dogs (30, 31). Injection of renin follows a similar time course with a

gradual rise in pressure following a 15- to 20-second latent period, the height and duration of response being functions of the dose (32). External constriction of the renal artery in conscious dogs with previous contralateral nephrectomy, has been shown to cause an increase in blood pressure after only 10 minutes, with the greatest increase occurring the first 35 to 45 minutes following constric-tion (24). All of these experiments have given results generally similar to the 18- to 20-minute rise to maximum pressure observed in our experiments (Fig. 2).

These relatively rapid elevations of systemic pressure after partial constriction of the renal artery seem to have been obscured as a result of surgical manipulations in studies by some investigators (33), who occluded during a surgical procedure and only later observed slow progressive rise in blood pressure which required about 10 days to reach a plateau.

The time required for the pressure to return to the base line after release of the renal artery constrictor presumably is dependent on the half-life of renin, which has been variously estimated to be from 15 to 45 minutes (32, 34, 35). Since the total pressure response times following complete renal ischemia for more than 10 minutes were reported to average 30 to 40 minutes (28, 29), and 15 to 20 minutes of this time were required to reach peak pressures, one can estimate about 15 to 20 minutes were required for pressures to return to normal. The time course of our double renal artery occlusion experiments, therefore, generally agree with previous observations. It can be seen in Figure 2, however, that there is a difference in the time response of the single and double kidney experiments. With the presence of only one kidney, the time required for the pressure to return to normal following release of the occluder was 31.9 minutes, but in those experiments in which the perfusion pressure to both kidneys was decreased, the pressure returned in only 17.8 minutes despite a higher maximum pressure response. These differences could possibly be accounted for by the suggested role of the kidney in the inactivation of renin (36, 37), which results in

a more rapid destruction of renin in the presence of two kidneys.

The time response of the observed pressure changes is not a result of angiotensin II per se, which when injected at normal doses of 0.25 to 5.0 ng/ml, gives a response averaging only 113 seconds (38).

Due to the limited stability of our prepara-tion it has not yet been possible to determine the maximum duration of the pressure re-sponse.

CAUSE OF BLOOD PRESSURE ELEVATIONS

Since the goal of our experiments was to quantify the open-loop gain of the renin-angiotensin system, it is necessary to discuss the evidence supporting the conclusion that the observed pressure changes were indeed a result of the renin-angiotensin system.

Abundant evidence shows that the rise in arterial pressure following construction of the renal artery is initially associated with an increased plasma renin activity (24, 39, 40). Bianchi et al. (32) showed a linear relation-ship between the increase in blood pressure and plasma renin activity during infusion of exogenous renin into conscious dogs, and they more recently showed a similar relationship with endogenous renin resulting from renal artery constriction (24). In acute experimen-tal situations, the kidney has been shown to be quite sensitive to reduction of arterial perfu-sion pressure, responding with a measurable increase in renin release with reductions of only 10 mm Hg in perfusion pressure (21) and with no detectable change in renal blood flow. The same relationship between renal perfusion pressure and renin release has been demonstrated by other investigators under various conditions such as hemorrhage (34) and aortocaval fistula (41).

Three groups of experiments in our project led to the conclusion that the systemic pressure changes were directly related to the renin-angiotensin system. The first, prolonged infusion of angiotensin preceding renal artery constriction, has been shown by several investigators to inhibit the release of renin induced by decreased renal perfusion pressure in dog and man (42-44). A peripheral

Circulation Research, Vol. XXVIII, May 1971

579

pressure response could not be elicited in our experiments following renal artery constriction in those animals that had received angiotensin infusions for the 1-hour period preceding constriction, or in those animals in which the infusion was maintained during constriction. Failure to respond under these conditions probably was not a result of peripheral tachyphylaxis since 1-hour infusions of similar doses of synthetic angiotensin continue to cause the same pressure responses (45).

A second group of experiments demon-strated that the systemic pressure response is accompanied by elevated plasma renin activ-ity and increased renal secretion rates of renin. Arterial plasma renin activity was elevated during every period of renal artery constric-tion which resulted in elevated systemic blood pressure. Plasma renin activities in samples taken after the systemic pressure had returned to control levels were no longer elevated above the control levels. Since the calculated renin secretion rate was elevated during every renal artery occlusion period, we conclude that the kidney was stimulated to increase renin release during this period.

The third group of experiments, using angiotensin II antibody, gave additional sup-port to the conclusion that the pressure changes resulted from the renin-angiotensin system. A complete reversal of the systemic pressure response was achieved almost imme-diately upon injection of the antibody, and a systemic pressure response could not again be elicited with occlusion of the renal artery. The presence of the antibody did not result in a systemic pressure lower than control levels following release of the occlusion, but the antibody still depressed the pressor response to injected angiotensin.

Taken together, these three groups of experiments support the conclusion that the rapid rise of blood pressure produced by renal artery constriction was mediated through the release of renin from the kidneys, which in turn stimulated the formation of angiotensin II in the plasma. That the actions of angiotensin were peripheral in nature cannot be questioned since complete destruction of

Circulation Research, Vol. XXVIII, May 1971

the spinal cord and decapitation eliminated all possible central nervous system reflex actions as well as any possible hormone or messenger substances from the pituitary gland or else-where in the brain.

Since cardiac outputs did not change significantly during the period of renal artery constriction, the increased systemic arterial blood pressures were the result of increased peripheral resistance. Interaction of the renin-angiotensin system with the adrenal cortex would not have had enough time to alter plasma volumes and thereby indirectly in-crease arterial pressure because the renal occlusions lasted only 30 minutes, while approximately 2 hours are required before the effects of aldosterone can be detected (46). Interaction of angiotensin with the adrenal medulla has not been eliminated, and it is possible that angiotensin could be stimulating the release of adrenalin (47). Collins et al. (28) showed, however, that neither adren-alectomy nor splenectomy prevents the pres-sure response following restoration of renal circulation after complete bilateral renal isch-emia.

The failure to demonstrate any relation between the calculated gain and the amount of norepinephrine infused (Fig. 6) indicates that the observed pressor responses were probably not a result of some type of interaction between angiotensin and cate-cholamines.

ROLE OF RENIN-ANGIOTENSIN IN BLOOD PRESSURE REGULATION

The data presented in this paper show that the renin-angiotensin system has sufficient gain and time response characteristics to make a significant contribution in the short-term regulation of blood pressure under conditions of decreased renal perfusion pressure. These characteristics result in a response which is slower but similar in magnitude to those of the baroreceptor system. The static open-loop gain of the carotid sinus pressoregulatory reflex has been reported to be -1.51 and the aortic arch baroreceptor reflex —0.51 resulting in an overall gain of about -2.0 (48-50). These changes are rapid and result in a new

steady-state condition within 2 minutes (48). These values compare with a gain of —1.6 for the renin-angiotensin system and a full response time of about 20 minutes.

The fluid volume feedback mechanisms influencing blood pressure have a slow time response in dogs but with potentially very high gains when the fluid intake exceeds urine output for prolonged periods.

The release of renin from the kidneys and the activation of angiotensin could thus serve as an additional pressure-regulating mecha-nism, bridging the gap between the very rapidly responding reflex mechanisms and the slower fluid volume mechanisms for pressure control. The overlapping of the various parallel negative feedback loops would pro-vide a smooth homeostatic control mechanism to stabilize most of the arterial pressure fluctuations presented to the system.

References

1. CONN, J.W.: Plasma renin activity in primary aldosteronism. J Amer Med Assn 190:213-221,

1964.

2. HABEH, E., KOERNER, T., PAGE, L.B., KLIMAN,

B., AND PURNODE, A.: Application of a radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects. J Clin Edocr 29i1349-1355, 1969.

3. OPARIL, S., VASSAUX, C , SANDERS, C.A., AND

HABEH, E.: Role of renin in acute postural homeostasis. Circulation 41:89-95, 1970.

4. MCKENZIE, J.K., COOK, W.F., AND LEE, M.R.:

Effect of hemorrhage on arterial plasma renin activity in the rabbit. Ore Res 19:269-273, 1966.

5. ZTECLER, M., AND GHOSS, F.: Effect of blood volume changes on renin-like activity in blood. Proc Soc Exp Biol Med 116:774-778, 1964.

6. KANEKO, Y., IKEDA, T., TAKEDA, T., AND UEDA,

H.: Renin release during acute reduction of arterial pressure in normotensive subjects and patients with renovascular hypertension. J Clin Invest 46:705-716, 1967.

7. WORCEL, M., MEYER, P., ANCLES D'AURIAC, C ,

AND MIIXIEZ, P.: Role of angiotensin in normal blood pressure regulation. Pflueger Arch 310:251-263, 1969.

8. MACDONALD, G.J., Louis, W.J., RENZINI, V., BOYD, G.W., AND PEART, W.S.: Renal-clip

hypertension in rabbits immunized against angiotensin II. Circ Res 27:197-211, 1970. 9. EIDE, I., AARS, H.: Renal hypertension in rabbits

immunized with angiotensin. Nature (London) 222:571, 1969.

10. VANDER, A.J.: Control of renin release. Physiol Rev 47:359-382, 1967.

11. PAGE, I.H., MCCUBBIN, J.W.: Renal Hyperten-sion. Chicago, Year Book Medical Publishers, 1968, pp 101-119.

12. COLEMAN, T.G., AND CRIDDLE, F.J., JR.:

Com-puterized analysis of indicator-dilution curves. J Appl Physiol 28:358-360, 1970.

13. ING, T.S., ANDERSON, R.N., PASSOVOY, M., AND

LUCIS, O.J.: Dose-response relationship in angiotensin bioassay. Cardiovasc Res 2:39-42, 1968.

14. PICKENS, P.T., BUMPUS, F.M., LLOYD, A.M., SMEBY, R.R., AND PAGE, I.R.: Measurement of

renin activity in human plasma. Circ Res 17:438-448, 1965.

15. SELKURT, E.E.: Renal circulation. In Handbook of Physiology, Circulation, sec. 2, vol. 2, edited by W. F. Hamilton and P. Dow, Washington, D. C , American Physiological Society 1964, p 1495.

16. BAER, P.G., NAVAR, L.G., AND GUYTON, A.C.:

Renal autoregulation, filtration rate, and elec-trolyte excretion during vasodilatation. Amer J Physiol 219:619-625, 1970.

17. DICKINSON, C.J., AND YU, R.: Mechanisms in-volved in the progressive pressor response to very small amounts of angiotensin in conscious rabbits. Circ Res 21 (suppl I I ) : 11-157-163, 1967.

18. DICKINSON, C.J.: Neurogenic hypertension, with special reference to the central effects of angiotensin. Circ Res 27 (suppl. I I ) : 11-169-176, 1970.

19. UEDA, H., UCHIDA, Y., UEDA, K., GONDATRA, T.,

AND KATAYAMA, S.: Centrally mediated vaso-pressor effect of angiotensin II in man. Jap Heart J 10:243-247, 1969.

20. HODGE, R.L., LOWE, R.D., AND VANE, J.R.:

Effects of alteration of blood volume on the concentration of circulatory angiotensin in anesthetized dogs. J Physiol (London) 185: 613-626, 1966.

21. SKINNER, S.L., MCCUBBIN, J. W., AND PAGE, I.H.:

Control of renin secretion. Circ Res 15:64-76, 1964.

22. FOJAS, J.E., AND SCHMID, H.E.: Renin release, renal autoregulation, and sodium excretion in the dog. Amer J Physiol 219:464-468, 1970.

23. LEDINCHAM, J.M., AND PELLINC, D.: Cardiac

output and peripheral resistance in experimen-tal renal hypertension. Circ Res (suppl. II) 21:11-187-199, 1967.

24. BIANCHI, G., TENCONI, L.T., AND LUCCA, R.:

Effect in the conscious dog of constriction of the renal artery to a sole remaining kidney or hemodynamics, sodium balance, body fluid

Circulation Research, Vol. XXVlll, May 1971

volumes, plasma renin concentration, and pressure responsiveness to angiotensin. Clin Sci 38:741-766, 1970.

25. KEZDI, P.: Resetting of the carotid sinus in experimental renal hypertension. In Barorecep-tors and Hypertension, edited by P. Kezdi, London, Pergamon Press, 1967, pp 301-308. 26. KRIEGER, E.M.: Time course of baroreceptor

resetting in acute hypertension. Amer J Physiol 218:486-490, 1970.

27. MCCUBBIN, J.W., DEMOURA, R.S., PAGE, I.H., AND OLMSTED, F.: Arterial hypertension elicit-ed by subpressor amounts of angiotensin. Science 149:1394, 1965.

28. COLLINS, D.A., AND HAMILTON, A.: Pressor response following short, complete renal isch-emia: Characteristics, mechanism, specificity for kidney. Amer J Physiol 130:784-790, 1940.

29. QUINBY, W.C., AND SIMEONE, F.A.: Some observations on acute renal hypertension. Surgery 11:544-556, 1942.

30. SKINNER, S.L., MCCUBBIN, J.W., AND PACE, I.H.: Angiotensin in blood and lymph following reduction in renal arterial perfusion pressure in dogs. CircRes 13:336-345, 1963.

31. HINSHAW, L.B., ARCHER, L.L., PARRY, W.L., AND SHIRES, T.K.: Renal hemodynamics and func-tion in response to renal artery occlusion in canine and primate kidneys. Invest Urol 7:422-432, 1970.

32. BIANCHI, G., BROWN, J.J., LEVER, A.F., ROBERTSON, J.I.S., AND ROTH, W.: Changes in plasma renin concentration during pressor infusions of renin in the conscious dog: Influence of dietary sodium intake. Clin Sci 34:303-314, 1968.

33. OLMSTED, F., AND PAGE, I.H.: Hemodynamic changes in trained dogs during experimental hypertension. Circ Res 16:134-139, 1965. 34. GROSS, F., BRUNNEB, H., AND ZIEGLER, M.:

Renin-angiotensin system, aldosterone and so-dium balance. Recent Progr Hormone Res 21:119-178, 1965.

35. SCHAECHTELIN, G., RECOLI, D., AND GROSS, F.: Quantitative assay and disappearance rate of circulating renin. Amer J Physiol 206:1361-1364, 1964.

36. HOUSSAY, B.A., BRAUN-MENENDEZ, E., AND DEX-TER, L.: Destruction and elimination of renin

in the dog. Ann Intern Med 17:461-473, 1942. 37. FREEDMAN, A.: Response to renin of unanesthe-tized normal and nephrectomized rats. Amer Heart J 20:304-307, 1940.

38. HODGE, R.L., Nc, K.K.F., AND VANE, J.R.: Disappearance of angiotensin from the circula-tion of the dog. Nature (London) 215:138-141, 1967.

39. LEVER, A.F., AND ROBERTSON, J.I.S.: Renin in the plasma of normal and hypertensive rabbits. J Physiol 170:212-218, 1964.

40. BROWN, T.C., DAVIS, J.O., OLICHNEY, M.J., AND JOHNSTON, C.I.: Relation of plasma renin to sodium balance and arterial pressure in experimental renal hypertension. Circ Res 18:475-483, 1966.

41. DAVIS, J.O., URQUHART, J., HIGCINS, J.T., RUBIN, B.C., AND HARTROFT, P.H.: Hyperse-cretion of aldosterone in dogs with a chronic aorric-caval fistula and high output heart failure. Circ Res 14:471-485, 1964.

42. VANDER, A.J., AND CEELHOLD, G.W.: Inhibition of renin secretion by angiotensin II. Proc Soc Exp Biol Med 120:399-403, 1965.

43. BUNAG, R.D., PAGE, I.H., AND MCCUBBIN, J.W.: Inhibition of renin release by vasopressin and angiotensin. Cardiovasc Res 1:67-73, 1967. 44. D E CHAMPLAIN, J., GENEST, J., VEYRATT, R.,

AND BOUCHER, R.: Factors controlling renin in man. Arch Intern Med 117:355-363, 1966. 45. PAGE, I.H., MCCUBBIN, J.W., SCHWARTZ, H., AND

BUMPUS, F.M.: Pharmacologic aspects of synthetic angiotensin. Circ Res 5:552-555, 1957.

46. SHARP, G.W.G., AND LEAF, A.: Mechanism of action of aldosterone. Physiol Rev 46:593-633, 1966.

47. FELDBEBC, W., AND LEWIS, G.P.: Action of peptides on the adrenal medulla: Release of adrenaline by bradykinin and angiotensin. J Physiol (London) 171:98-108, 1964. 48. SAGAWA, K., AND WATANABE, K.: Summation of

bilateral carotid sinus signals in the barostatic reflex. Amer J Physiol 209:1278-1286, 1965. 49. ALLISON, J.L., SACAWA, K., AND KUMADA, M.: An

open-loop analysis of the aortic arch barostatic reflex. Amer J Physiol 217:1576-1584, 1969. 50. SCHER, A.M., AND YOUNC, A.C.: Servoanalysis of

carotid sinus reflex effects on peripheral resistance. Circ Res 12:152-162, 1963.

Circulation Research, Vol. XXVlll, May 1971