Mechanisms of enhanced renin secretion during CO2 retention in dogs KENNETH
Department
D. KURZ AND JOHN E. ZEHR of Physiology and Biophysics, University
KURZ, KENNETH D., AND JOHN E. ZEHR. Mechanisms of enhanced renin secretion during CO, retention in dogs. Am. J. Physiol. 2346): H573-H581, 1978 or Am. J. Physiol.: Heart Circ. Physiol. 36): H573-H581, 1978. -Arterial renin activity and renal renin secretion were studied during CO, inhalation (constant minute ventilation) in chloralose-anesthetized dogs. In eight dogs with both renal nerves and adrenals intact, successive 30.min periods of 4, 8, and 12% CO, inhalation resulted in a dose-dependent and reversible increase in arterial renin activity. Ten dogs with intact adrenals were studied 3-4 days after denervation of one kidney. Measurement of the simultaneous rate of renin secretion from the denervated kidney and from the contralateral innervated kidney indicated that both increased their rate of renin secretion after 10 min of 12% CO, inhalation. In six adrenalectomized dogs with one kidney denervated, the denervated kidney failed to respond to an identical hypercapnic stimulus while the contralateral innervated kidney doubled its rate of renin secretion. Comparable hypercapnemia was produced in six intact dogs, but concomitant acidemia was prevented by infusion of tris(hydroxymethyl)aminomethane (Tris; 0.5 mM kg+ min-l, iv). Plasma renin activity (PRA) increased significantly despite the maintenance of a relatively constant arterial pH. In contrast, PRA did not change in six dogs when pH was lowered by lactic acid infusion (0.33 mM kg+ min-l iv) but relatively constant (controlled minute pace, was maintained ventilation). The data indicated that acute respiratory acidosis stimulates renin release via adrenal medullary and renal nerve activation. The response is dependent upon elevation of rather than reduction of arterial pH per se. PQO, l
l
angiotensin; acidosis; hypercapnia; pressure; chemoreceptors
renal
l
l
blood flow; arterial
FROMTHISLABORATORY demonstrated that, in the dog, graded respiratory acidosis is accompanied by a dose-dependent and reversible elevation of circulating renin (8, 25). More recently Anderson et al. (1) reported that patients with acute respiratory insufficiency also exhibit elevated levels of circulating renin activity. Since regulation of renin is under complex and redundant influences (4), several potential mechanisms could be invoked to explain the renin response to respiratory acidosis. Both circulating catecholamines (10) and renal nerve pathways (10, 26) have been shown to modify renin secretion. In addition, an intrarenal baroreceptor (4), presumably responding to changes in renal tierent wall tension, has been demonstrated (7). Finally sodium (18), and probably chloride (ll), delivery to the macula PREVIOUSSTUDIES
0363.6135/78/0000-0000$01.25
Copyright
0 1978 the American
Physiological
of Illinois,
Urbana,
Illinois
61801
densa have been shown to modify renin release. These mechanisms participate to various degress in several reflex responses to physiological stimuli. Since hypercapnia results in increased peripheral sympathetic nerve activity (19) and sympathoadrenal stimulation (2l), it was of interest to study the involvement of the renal nerves and adrenal catecholamines in the increase in renin secretion during controlled acute respiratory acidosis. The relative importance of Pace, and arterial pH per se in mediating the renin response was also assessed. METHODS
All studies were conducted on mature mongrel dogs of both sexes. Anesthesia was induced with sodium methohexital (sodium Brevital, 12 mg/kg iv) and supplemented with an initial dose of cu-chloralose (80 mg/ kg iv). Maintenance doses of chloralose were administered at approximately 10 mg/kg every 30 min or as required to maintain a steady plane of anesthesia. The dogs were intubated with a cuffed endotracheal tube and ventilated with a Harvard Instruments model 607 positive-pressure respirator at an end-expiratory pressure of 3-4 cmH,O to minimize atelectasis. Expiratory gases were continuously measured for CO, with a calibrated, rapidly responding (100 ms) Beckman Instruments type LB2 respiratory gas analyzer. Ventilation rate was maintained at 15 breathslmin, and tidal volume was adjusted so that end-expiratory CO, was constant at 5% during control conditions. Positive control of respiration throughout prevented compensatory hyperventilation during CO, inhalation and thus permitted achievement of reproducible levels of arterial blood gases and pH. A femoral arterial catheter (PE240) was advanced into the abdominal aorta to a suprarenal location for purposes of recording arterial blood pressure and for withdrawal of arterial blood samples. A femoral venous catheter was used to administer supplemental anesthetic and to replace fluid losses. The dogs were suspended in a upright position to facilitate a retroperitoneal kidney exposure via flank incision. The renal artery and vein were carefully exposed, so as to prevent damage to nerves or vessels, and a noncannulating electromagnetic flow probe was fitted around the renal artery. Renal blood flow was measured with a Zepeda model SW-3 square-wave electromagnetic flowmeter. Zero flow was determined by momentary occlusion of the renal artery distal to the Society
H573
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 15, 2019.
H574
K.
probe at least 30 min before the initial experimental period and again after completion of the final observation. To avoid the renin secretion that results from renal artery occlusion, occlusive flow zeros were not determined during the experimental procedures; however, electronic zeros were checked periodically. Although in our experience the Zepeda SW-3 is subject to minimal base-line drift, if uncertainty with regard to flow-zero was evident at the end of the experiment, the data were discarded as unreliable. Flow probes were calibrated in vivo on a femoral artery with step flow controlled by a Harvard infusion-withdrawal syringe pump. Arterial blood pressure was measured with a Statham P23Db strain gauge transducer. The pulsatile arterial pressure signal served as the input to a Beckman Instruments type 9857-B cardiotachometer for a moment-to-moment determination of the heart rate. Pulsatile pressure, electronically averaged arterial pressure and renal blood flow, heart rate, and endexpiratory CO, were continuously recorded on a Beckman Instruments type RM six-channel ink-writing Dynograph. Arterial blood samples for determination of pH, Pa,,, and Pa,, were drawn into heparinized 3-ml plastic syringes and immediately measured with an Instrumentation Laboratory model IL-213 digital blood gas analyzer. To assure reliability of blood gas determinations, special care was taken to avoid air contamination and all samples were analyzed within l-2 min after withdrawal. Experimental
Protocol
After completion of the experimental preparatory procedures, a minimum of 30 min equilibration was observed. In every instance during this period, ventilatory rate was held at 15 breaths/min, and tidal volume was adjusted to achieve an end-expiratory CO, level of 5%. Under these conditions, control blood gases and pH were within normal ranges (6) for dogs. The criteria were established that renal blood flow and mean arterial pressure were to remain stable for a minimum of 30 min before initiation of any experimental procedure. The experiment was aborted if renal blood flow or arterial pressure was not stabilized within 60 min or if mean blood pressure fell below 90 mmHg during this period. This precaution was taken because the cardiac depression usually observed during controlled hypercapnic ventilation oRen leads to reduction of systemic arterial pressure (15). To avoid activation of potential intrarenal baroreceptor and tubular mechanisms, it was desirable that serious compromise of blood pressure during experimental procedures be avoided. In order to suppress compensatory respiratory movement and resultant motion artifacts during COz inhalation, the animals were paralyzed throughout with succinylcholine (Anectine, iv). This procedure also results in a greater degree of reproducibility with regard to blood gases and cardiovascular responses to chemoreceptor stimulation than would be possible with uncontrolled ventilation. Since it is difficult to monitor the anesthetic plane in neuromuscularly blocked animals, a rigorous schedule of 10 mg/kg of alpha-chloralose was adminis-
D. KUM
AND
J. E. ZEHR
tered at 30-m in intervals immediately after a sampling recorded variables period. Data for all continuously were analyzed over the final 2 min of the respective control or experimenta 1 period. Arterial samples were drawn for blood gases, PH, ami renin during the final 1 min. The general protocol outlined above was followed during each of the following series of studies. Se&s 1: analysis of &relationship between acute respiratory acid&is and circulating r&in. In this series, a group of eight dogs was used to test the basic hypothesis that acute respiratory acidosis is capable of eliciting short-term reflex adjustments in the circulating levels of renin. A protocol was followed which permitted observations after three different consecutive doses of CO2 in the same animal. After completion of the preparatory surgery, a 30.min equilibration period was followed by successive 30.min periods of ventilation with room air (control), 4% CO, in air (4% COe), 8% CO* in air (8% CO*), 12% COz in air (12% CO&, and room air to recovery. Analysis of variance combined with a Tukey comparison among means was used to determine which doses of CO, resulted in a significant increase in the level of circulating renin or a significant change in the measured cardiovascular parameters. Minimum criteria for rejection of a null hypothesis was the 95% confidence level. Because the results of this series indicated that the rate of renin release was elevated by acute respiratory acidosis, additional experiments were designed to characterize the pathways responsible for the response. Series 2: role of renal nerves in the presence of intact adrenals. This series was conducted in 10 dogs with intact adrenals and was designed to determine renal sympathetic input to the renin response during hypercapnia. In each dog of this series, one kidney was denervated several days prior to the experiment by cutting all visible renal nerves, stripping fascia from the artery and vein, and painting with a 10% phenol in ethanol solution. Simultaneous determinations of actual renin secretion from the denervated kidney and that from the contralateral innervated kidney allowed a direct examination of the role of the renal . nerves during CO, inhalation. Bilateral renal venous blood samples were collected from a Silastic collection tube attached to a 22.gauge needle that was inserted into each renal vein. It was expected that if sympathetic reflex factors were of major involvement, the response should be relatively rapid in onset. Therefore, a lO-min period of 12% CO2 inhalation was used as the experimental stimulus. Two lO-min control periods were followed by 10 min of 12% CO, inhalation and 30 min of ventilation with room air to recovery. In addition to analysis of variance, a paired Student t-test was used to compare the renin release from the innervated and denervated kidneys. Series 3: role of rqzal nerves in the absence of adrenal influences. Since the data collected in the previous study indicated that with intact adrenals the renal nerves played a minor, if any, role in the renin response, it appeared likely that adrenal catecholamine activation may have beena dom inant factor. Therefore,
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 15, 2019.
RENIN
RELEASE
DURING
RESPIRATORY
ACIDOSIS
this series was designed to test the hypothesis that adrenal activation may have mediated the renin response. Studies in this group were conducted in six adrenalectomized dogs with one kidney denervated. As in the previous series these ablative procedures were carried out 3-4 days prior to the experimental study. Daily mineralo- and glucocorticoid replacement therapy with desoxycorticosterone acetate (Percorten acetate, Ciba, 5 mg im) and dexamethasone (Azium, Schering, 1 mg im) was provided for each dog. The experimental protocol in this group was identical in every respect with that of series 2. Series 4: separate effects of arterial pH and Pace,. Since it was clear that controlled CO, retention elicited a short-term renin response, it was of interest to determine whether the response was a result of elevated Pace per se or secondary to the accompanying reduction in pfl. To differentiate between these possibilities, three groups of six dogs each were studied. The dogs were first tested for their individual response to 10 min of 12% COz. During this trial, an intravenous Tyrode solution was administered at a rate to match the rate at which tris(hydroxymethyl)aminomethane (Tris) or lactic acid were later infused in the same dog. Buffering with Tris, rather than sodium bicarbonate, was chosen to avoid potential renin responses to sodium. The purpose of the Tyrode infusion was to equalize any volume effects between the two conditions. Arterial pH and Pace, were monitored at 1-min intervals. The animals were allowed to reequilibrate for at least 30 min and then retested. In the first group, arterial pH was maintained during the second period of CO, inhalation by infusion of 0.6 M Tris (0.5 mM*kg+ l rein+, iv). There are several reports of the use of Tris buffer to restore pH during CO, accumulation (13, 16). Tris will also remove CO, from blood by the reaction RNH,
+ CO, + H,O G RNH,+
+ HCO,-
Therefore, preliminary testing was conducted which indicated that during Tris infusion an inhalation mixture of approximately 21% CO, in air was required to achieve Pace, levels similar to those achieved during 12% CO, inhalation without ‘KS. The second group of dogs was studied in converse experiments during which Pace, was held constant while pH was depressed by administration of lactic acid (0.33 mM min-l kg-l, iv). Each dog was first tested, as before, for its response to 12% COz inhalation and then retested during lactic acid infusion at levels which resulted in arterial pH values approximating those during its first trial. With a fixed minute ventilation, acid infusion results in hypercapnemia; therefore, to maintain Pea, relatively constant, minute ventilation was regulated as required to control end-expired CO2 near control levels. The availability of the rapidly responding LB2 respiratory gas analyzer permitted moment-to-moment monitoring and control of expired CO, and by inference Pace,. Finally, a third group of dogs was studied to test whether the increase inrenin during Tris infusion might be a result of the direct effects of Tris rather than Pace, related. An infusion of Tris (0.5 mM . kg-l min-l) identical to the first group was given, l
l
l
but no CO, stress was imposed. A Student paired t-test was used to compare the experimental response with its matched control. Renin
Assay
Previously described techniques for radioimmunoassay of angiotensin I were used to estimate plasma renin activity (PBA) and renin secretion rates from individual kidneys (26). Benin secretion was calculated as the product of renal plasma flow and the difference between renal venous and arterial renin activities. Renal plasma flow was calculated from the renal blood flow and hematocrit. Since the enzymatic activity of renin is pH dependent and since blood pH is decreased during respiratory acidosis, all plasma samples used for renin determinations were buffered to pH 7.4 to provide uniform conditions for generation of angiotensin I. To assure that the renin-substrate reaction was not substrate limiting, an aliquot of partially purified homologous renin substrate, extracted from the plasma of 48-h nephrectomized dogs (9), was added to all samples. This quantity of substrate when generated to completion with excess dog renin yielded approxi .mately 200 ng angiotensin I. In the absence of added renin no detectable angiotensin Iw as generated from this material. While it is recognized that in the presence of excess substrate, renin may be expressed as units of concentration, this paper will define circulating renin as activity. Benin secretion is expressed in nanograms per minute. RESULTS
Series 1: analysis of the relation ship between acute respiratory acidosis and circulating renin. The purpose of this group was to provide basic information regarding the dose-response relationship between acute respiratory acidosis and circulating renin. The data presented in Table 1 show the effects of 30 min of graded CO, inhalation at a constant minute ventilation on cardiovascular parameters and arterial blood gases and pH for the eight dogs tested. The mean control values observed for arterial Pacol, Paog9 and pH are in close agreement with the previously reported values in unanesthetized dogs (6). None of the dogs became hypoxemit at any time during the course of-the experiments. Data summarized in Fig. 1 shows arterial renin activity was significantly elevated after 8% (P < 0.05) and aRer 12% CO, (P < 0.01) inhalation. It was clear from these data that graded CO2 inhalation was capable of inducing a dose-dependent but reversible increase in circulating renin. Although underlying mechanisms were obscure, it was hypothesized that acute activation of sympathetic and adrenal medullary pathways may have been involved, because of the complex action of CO, on peripheral and central chemoreceptors as well as its action on adrenal medullary function (17, 21). Therefore, two additional series of experiments were conducted to study renal sympathetic and adrenal medullary influences. Series 2: role of renal nerves in the presence of intact adrenak. Ten dogs with unilateral renal denervation were ventilated with 12% CO, for 10 min to assess the
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 15, 2019.
K. D. KURZ AND J. E. ZEHR
H576 TABLE 1. Effects of graded CO2 inhalation (constant minute ventilation) on cardiovascular variables, blood gases, and pH (n = 8) Control
Heart rate, beats/min Mean arterial pressure, mmHg Renal blood flow, ml/min Pk02, mmHg Pa,, mmHg PH
4%
108
8%
12%
Recovery
84t
113
125 210.1 130
k6.2
81t 24.1
k5.3
k5.4
131
112”
116t
132
25.6
25.3
k7.8
26.2
k6.7
137t 222.2 89.8$ 22.5
161
173
162
220.0 34.2
d7.2 45.2$
220.2 70.7$
21.0
+1.6 112.9
22.9 105.4
27.8 7.27$
k3.1 7.13$ 20.02
82.4 22.4 7.37
92.5 23.6 7.03$
168
+21.9 34.8 21.5 81.7 24.0 7.36 kO.02
creased from the innervated side (P < 0.05), whereas there was no change in rate of release from the denervated side. Thus it seems apparent that catecholamines, originating from either the adrenals or from renal sympathetic nerve terminals, were required for the acute (10 min) renin response. Series 4: separate effects of arterial pH and PacO .
This series was designed to determine if the renl?n response to CO, retention was dependent on the elevated P&o, per se or the resultant reduced arterial pH. Three groups of six intact dogs each were tested. In the first group the renin response of each dog to 10 min of *
+O.Ol 20.01 to.01 Values are means +, SE for each variable. Analysis of variance was applied to test null hypothesis that mean values for each variable were equal across the experimental treatments. A Tukey test for comparison among means was applied to identify individual differences for each variable. P value indicates the confidence level that the given variable is different from its respective control value. * P -c 0.05. t P < 0.01. $ P c 0.001.
P< 0.01
P
Department
D. KURZ AND JOHN E. ZEHR of Physiology and Biophysics, University
KURZ, KENNETH D., AND JOHN E. ZEHR. Mechanisms of enhanced renin secretion during CO, retention in dogs. Am. J. Physiol. 2346): H573-H581, 1978 or Am. J. Physiol.: Heart Circ. Physiol. 36): H573-H581, 1978. -Arterial renin activity and renal renin secretion were studied during CO, inhalation (constant minute ventilation) in chloralose-anesthetized dogs. In eight dogs with both renal nerves and adrenals intact, successive 30.min periods of 4, 8, and 12% CO, inhalation resulted in a dose-dependent and reversible increase in arterial renin activity. Ten dogs with intact adrenals were studied 3-4 days after denervation of one kidney. Measurement of the simultaneous rate of renin secretion from the denervated kidney and from the contralateral innervated kidney indicated that both increased their rate of renin secretion after 10 min of 12% CO, inhalation. In six adrenalectomized dogs with one kidney denervated, the denervated kidney failed to respond to an identical hypercapnic stimulus while the contralateral innervated kidney doubled its rate of renin secretion. Comparable hypercapnemia was produced in six intact dogs, but concomitant acidemia was prevented by infusion of tris(hydroxymethyl)aminomethane (Tris; 0.5 mM kg+ min-l, iv). Plasma renin activity (PRA) increased significantly despite the maintenance of a relatively constant arterial pH. In contrast, PRA did not change in six dogs when pH was lowered by lactic acid infusion (0.33 mM kg+ min-l iv) but relatively constant (controlled minute pace, was maintained ventilation). The data indicated that acute respiratory acidosis stimulates renin release via adrenal medullary and renal nerve activation. The response is dependent upon elevation of rather than reduction of arterial pH per se. PQO, l
l
angiotensin; acidosis; hypercapnia; pressure; chemoreceptors
renal
l
l
blood flow; arterial
FROMTHISLABORATORY demonstrated that, in the dog, graded respiratory acidosis is accompanied by a dose-dependent and reversible elevation of circulating renin (8, 25). More recently Anderson et al. (1) reported that patients with acute respiratory insufficiency also exhibit elevated levels of circulating renin activity. Since regulation of renin is under complex and redundant influences (4), several potential mechanisms could be invoked to explain the renin response to respiratory acidosis. Both circulating catecholamines (10) and renal nerve pathways (10, 26) have been shown to modify renin secretion. In addition, an intrarenal baroreceptor (4), presumably responding to changes in renal tierent wall tension, has been demonstrated (7). Finally sodium (18), and probably chloride (ll), delivery to the macula PREVIOUSSTUDIES
0363.6135/78/0000-0000$01.25
Copyright
0 1978 the American
Physiological
of Illinois,
Urbana,
Illinois
61801
densa have been shown to modify renin release. These mechanisms participate to various degress in several reflex responses to physiological stimuli. Since hypercapnia results in increased peripheral sympathetic nerve activity (19) and sympathoadrenal stimulation (2l), it was of interest to study the involvement of the renal nerves and adrenal catecholamines in the increase in renin secretion during controlled acute respiratory acidosis. The relative importance of Pace, and arterial pH per se in mediating the renin response was also assessed. METHODS
All studies were conducted on mature mongrel dogs of both sexes. Anesthesia was induced with sodium methohexital (sodium Brevital, 12 mg/kg iv) and supplemented with an initial dose of cu-chloralose (80 mg/ kg iv). Maintenance doses of chloralose were administered at approximately 10 mg/kg every 30 min or as required to maintain a steady plane of anesthesia. The dogs were intubated with a cuffed endotracheal tube and ventilated with a Harvard Instruments model 607 positive-pressure respirator at an end-expiratory pressure of 3-4 cmH,O to minimize atelectasis. Expiratory gases were continuously measured for CO, with a calibrated, rapidly responding (100 ms) Beckman Instruments type LB2 respiratory gas analyzer. Ventilation rate was maintained at 15 breathslmin, and tidal volume was adjusted so that end-expiratory CO, was constant at 5% during control conditions. Positive control of respiration throughout prevented compensatory hyperventilation during CO, inhalation and thus permitted achievement of reproducible levels of arterial blood gases and pH. A femoral arterial catheter (PE240) was advanced into the abdominal aorta to a suprarenal location for purposes of recording arterial blood pressure and for withdrawal of arterial blood samples. A femoral venous catheter was used to administer supplemental anesthetic and to replace fluid losses. The dogs were suspended in a upright position to facilitate a retroperitoneal kidney exposure via flank incision. The renal artery and vein were carefully exposed, so as to prevent damage to nerves or vessels, and a noncannulating electromagnetic flow probe was fitted around the renal artery. Renal blood flow was measured with a Zepeda model SW-3 square-wave electromagnetic flowmeter. Zero flow was determined by momentary occlusion of the renal artery distal to the Society
H573
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 15, 2019.
H574
K.
probe at least 30 min before the initial experimental period and again after completion of the final observation. To avoid the renin secretion that results from renal artery occlusion, occlusive flow zeros were not determined during the experimental procedures; however, electronic zeros were checked periodically. Although in our experience the Zepeda SW-3 is subject to minimal base-line drift, if uncertainty with regard to flow-zero was evident at the end of the experiment, the data were discarded as unreliable. Flow probes were calibrated in vivo on a femoral artery with step flow controlled by a Harvard infusion-withdrawal syringe pump. Arterial blood pressure was measured with a Statham P23Db strain gauge transducer. The pulsatile arterial pressure signal served as the input to a Beckman Instruments type 9857-B cardiotachometer for a moment-to-moment determination of the heart rate. Pulsatile pressure, electronically averaged arterial pressure and renal blood flow, heart rate, and endexpiratory CO, were continuously recorded on a Beckman Instruments type RM six-channel ink-writing Dynograph. Arterial blood samples for determination of pH, Pa,,, and Pa,, were drawn into heparinized 3-ml plastic syringes and immediately measured with an Instrumentation Laboratory model IL-213 digital blood gas analyzer. To assure reliability of blood gas determinations, special care was taken to avoid air contamination and all samples were analyzed within l-2 min after withdrawal. Experimental
Protocol
After completion of the experimental preparatory procedures, a minimum of 30 min equilibration was observed. In every instance during this period, ventilatory rate was held at 15 breaths/min, and tidal volume was adjusted to achieve an end-expiratory CO, level of 5%. Under these conditions, control blood gases and pH were within normal ranges (6) for dogs. The criteria were established that renal blood flow and mean arterial pressure were to remain stable for a minimum of 30 min before initiation of any experimental procedure. The experiment was aborted if renal blood flow or arterial pressure was not stabilized within 60 min or if mean blood pressure fell below 90 mmHg during this period. This precaution was taken because the cardiac depression usually observed during controlled hypercapnic ventilation oRen leads to reduction of systemic arterial pressure (15). To avoid activation of potential intrarenal baroreceptor and tubular mechanisms, it was desirable that serious compromise of blood pressure during experimental procedures be avoided. In order to suppress compensatory respiratory movement and resultant motion artifacts during COz inhalation, the animals were paralyzed throughout with succinylcholine (Anectine, iv). This procedure also results in a greater degree of reproducibility with regard to blood gases and cardiovascular responses to chemoreceptor stimulation than would be possible with uncontrolled ventilation. Since it is difficult to monitor the anesthetic plane in neuromuscularly blocked animals, a rigorous schedule of 10 mg/kg of alpha-chloralose was adminis-
D. KUM
AND
J. E. ZEHR
tered at 30-m in intervals immediately after a sampling recorded variables period. Data for all continuously were analyzed over the final 2 min of the respective control or experimenta 1 period. Arterial samples were drawn for blood gases, PH, ami renin during the final 1 min. The general protocol outlined above was followed during each of the following series of studies. Se&s 1: analysis of &relationship between acute respiratory acid&is and circulating r&in. In this series, a group of eight dogs was used to test the basic hypothesis that acute respiratory acidosis is capable of eliciting short-term reflex adjustments in the circulating levels of renin. A protocol was followed which permitted observations after three different consecutive doses of CO2 in the same animal. After completion of the preparatory surgery, a 30.min equilibration period was followed by successive 30.min periods of ventilation with room air (control), 4% CO, in air (4% COe), 8% CO* in air (8% CO*), 12% COz in air (12% CO&, and room air to recovery. Analysis of variance combined with a Tukey comparison among means was used to determine which doses of CO, resulted in a significant increase in the level of circulating renin or a significant change in the measured cardiovascular parameters. Minimum criteria for rejection of a null hypothesis was the 95% confidence level. Because the results of this series indicated that the rate of renin release was elevated by acute respiratory acidosis, additional experiments were designed to characterize the pathways responsible for the response. Series 2: role of renal nerves in the presence of intact adrenals. This series was conducted in 10 dogs with intact adrenals and was designed to determine renal sympathetic input to the renin response during hypercapnia. In each dog of this series, one kidney was denervated several days prior to the experiment by cutting all visible renal nerves, stripping fascia from the artery and vein, and painting with a 10% phenol in ethanol solution. Simultaneous determinations of actual renin secretion from the denervated kidney and that from the contralateral innervated kidney allowed a direct examination of the role of the renal . nerves during CO, inhalation. Bilateral renal venous blood samples were collected from a Silastic collection tube attached to a 22.gauge needle that was inserted into each renal vein. It was expected that if sympathetic reflex factors were of major involvement, the response should be relatively rapid in onset. Therefore, a lO-min period of 12% CO2 inhalation was used as the experimental stimulus. Two lO-min control periods were followed by 10 min of 12% CO, inhalation and 30 min of ventilation with room air to recovery. In addition to analysis of variance, a paired Student t-test was used to compare the renin release from the innervated and denervated kidneys. Series 3: role of rqzal nerves in the absence of adrenal influences. Since the data collected in the previous study indicated that with intact adrenals the renal nerves played a minor, if any, role in the renin response, it appeared likely that adrenal catecholamine activation may have beena dom inant factor. Therefore,
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 15, 2019.
RENIN
RELEASE
DURING
RESPIRATORY
ACIDOSIS
this series was designed to test the hypothesis that adrenal activation may have mediated the renin response. Studies in this group were conducted in six adrenalectomized dogs with one kidney denervated. As in the previous series these ablative procedures were carried out 3-4 days prior to the experimental study. Daily mineralo- and glucocorticoid replacement therapy with desoxycorticosterone acetate (Percorten acetate, Ciba, 5 mg im) and dexamethasone (Azium, Schering, 1 mg im) was provided for each dog. The experimental protocol in this group was identical in every respect with that of series 2. Series 4: separate effects of arterial pH and Pace,. Since it was clear that controlled CO, retention elicited a short-term renin response, it was of interest to determine whether the response was a result of elevated Pace per se or secondary to the accompanying reduction in pfl. To differentiate between these possibilities, three groups of six dogs each were studied. The dogs were first tested for their individual response to 10 min of 12% COz. During this trial, an intravenous Tyrode solution was administered at a rate to match the rate at which tris(hydroxymethyl)aminomethane (Tris) or lactic acid were later infused in the same dog. Buffering with Tris, rather than sodium bicarbonate, was chosen to avoid potential renin responses to sodium. The purpose of the Tyrode infusion was to equalize any volume effects between the two conditions. Arterial pH and Pace, were monitored at 1-min intervals. The animals were allowed to reequilibrate for at least 30 min and then retested. In the first group, arterial pH was maintained during the second period of CO, inhalation by infusion of 0.6 M Tris (0.5 mM*kg+ l rein+, iv). There are several reports of the use of Tris buffer to restore pH during CO, accumulation (13, 16). Tris will also remove CO, from blood by the reaction RNH,
+ CO, + H,O G RNH,+
+ HCO,-
Therefore, preliminary testing was conducted which indicated that during Tris infusion an inhalation mixture of approximately 21% CO, in air was required to achieve Pace, levels similar to those achieved during 12% CO, inhalation without ‘KS. The second group of dogs was studied in converse experiments during which Pace, was held constant while pH was depressed by administration of lactic acid (0.33 mM min-l kg-l, iv). Each dog was first tested, as before, for its response to 12% COz inhalation and then retested during lactic acid infusion at levels which resulted in arterial pH values approximating those during its first trial. With a fixed minute ventilation, acid infusion results in hypercapnemia; therefore, to maintain Pea, relatively constant, minute ventilation was regulated as required to control end-expired CO2 near control levels. The availability of the rapidly responding LB2 respiratory gas analyzer permitted moment-to-moment monitoring and control of expired CO, and by inference Pace,. Finally, a third group of dogs was studied to test whether the increase inrenin during Tris infusion might be a result of the direct effects of Tris rather than Pace, related. An infusion of Tris (0.5 mM . kg-l min-l) identical to the first group was given, l
l
l
but no CO, stress was imposed. A Student paired t-test was used to compare the experimental response with its matched control. Renin
Assay
Previously described techniques for radioimmunoassay of angiotensin I were used to estimate plasma renin activity (PBA) and renin secretion rates from individual kidneys (26). Benin secretion was calculated as the product of renal plasma flow and the difference between renal venous and arterial renin activities. Renal plasma flow was calculated from the renal blood flow and hematocrit. Since the enzymatic activity of renin is pH dependent and since blood pH is decreased during respiratory acidosis, all plasma samples used for renin determinations were buffered to pH 7.4 to provide uniform conditions for generation of angiotensin I. To assure that the renin-substrate reaction was not substrate limiting, an aliquot of partially purified homologous renin substrate, extracted from the plasma of 48-h nephrectomized dogs (9), was added to all samples. This quantity of substrate when generated to completion with excess dog renin yielded approxi .mately 200 ng angiotensin I. In the absence of added renin no detectable angiotensin Iw as generated from this material. While it is recognized that in the presence of excess substrate, renin may be expressed as units of concentration, this paper will define circulating renin as activity. Benin secretion is expressed in nanograms per minute. RESULTS
Series 1: analysis of the relation ship between acute respiratory acidosis and circulating renin. The purpose of this group was to provide basic information regarding the dose-response relationship between acute respiratory acidosis and circulating renin. The data presented in Table 1 show the effects of 30 min of graded CO, inhalation at a constant minute ventilation on cardiovascular parameters and arterial blood gases and pH for the eight dogs tested. The mean control values observed for arterial Pacol, Paog9 and pH are in close agreement with the previously reported values in unanesthetized dogs (6). None of the dogs became hypoxemit at any time during the course of-the experiments. Data summarized in Fig. 1 shows arterial renin activity was significantly elevated after 8% (P < 0.05) and aRer 12% CO, (P < 0.01) inhalation. It was clear from these data that graded CO2 inhalation was capable of inducing a dose-dependent but reversible increase in circulating renin. Although underlying mechanisms were obscure, it was hypothesized that acute activation of sympathetic and adrenal medullary pathways may have been involved, because of the complex action of CO, on peripheral and central chemoreceptors as well as its action on adrenal medullary function (17, 21). Therefore, two additional series of experiments were conducted to study renal sympathetic and adrenal medullary influences. Series 2: role of renal nerves in the presence of intact adrenak. Ten dogs with unilateral renal denervation were ventilated with 12% CO, for 10 min to assess the
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K. D. KURZ AND J. E. ZEHR
H576 TABLE 1. Effects of graded CO2 inhalation (constant minute ventilation) on cardiovascular variables, blood gases, and pH (n = 8) Control
Heart rate, beats/min Mean arterial pressure, mmHg Renal blood flow, ml/min Pk02, mmHg Pa,, mmHg PH
4%
108
8%
12%
Recovery
84t
113
125 210.1 130
k6.2
81t 24.1
k5.3
k5.4
131
112”
116t
132
25.6
25.3
k7.8
26.2
k6.7
137t 222.2 89.8$ 22.5
161
173
162
220.0 34.2
d7.2 45.2$
220.2 70.7$
21.0
+1.6 112.9
22.9 105.4
27.8 7.27$
k3.1 7.13$ 20.02
82.4 22.4 7.37
92.5 23.6 7.03$
168
+21.9 34.8 21.5 81.7 24.0 7.36 kO.02
creased from the innervated side (P < 0.05), whereas there was no change in rate of release from the denervated side. Thus it seems apparent that catecholamines, originating from either the adrenals or from renal sympathetic nerve terminals, were required for the acute (10 min) renin response. Series 4: separate effects of arterial pH and PacO .
This series was designed to determine if the renl?n response to CO, retention was dependent on the elevated P&o, per se or the resultant reduced arterial pH. Three groups of six intact dogs each were tested. In the first group the renin response of each dog to 10 min of *
+O.Ol 20.01 to.01 Values are means +, SE for each variable. Analysis of variance was applied to test null hypothesis that mean values for each variable were equal across the experimental treatments. A Tukey test for comparison among means was applied to identify individual differences for each variable. P value indicates the confidence level that the given variable is different from its respective control value. * P -c 0.05. t P < 0.01. $ P c 0.001.
P< 0.01
P
