Hypertension Induced by Chronic Renal Adrenergic Stimulation Is Angiotensin Dependent
Abstract We designed these studies to assess the role of the renin-angiotensin system in mediating the hypertensive and renal functional effects of chronic renal adrenergic stimulation. Norepinephrine was infused at 0.1 μg/kg per minute for 7 days directly into the renal artery of uninephrectomized dogs under control conditions (n=5) or after plasma angiotensin II (Ang II) concentration was fixed at control levels (n=5) by chronic intravenous infusion of captopril (14 μg/kg per minute) and Ang II (0.58±0.04 ng/kg per minute). During the first 60 minutes of norepinephrine infusion in control dogs, mean arterial pressure increased 9±4 mm Hg in association with a twofold to threefold rise in plasma renin activity. Additionally, glomerular filtration rate, renal plasma flow, sodium excretion, and fractional sodium excretion decreased to 70±5%, 64±5%, 31±4%, and 38±6% of control, respectively, while filtration fraction increased 15±2%. In contrast to the pronounced short-term effects of norepinephrine on renal function, during chronic norepinephrine infusion, all indexes of renal function returned to control levels. However, elevations in both plasma renin activity and mean arterial pressure were sustained and on day 7 were 2.3±0.6 ng angiotensin I/mL per hour (control, 0.5±0.1) and 110±7 mm Hg (control, 90±3). In dogs with fixed plasma levels of Ang II, acute and chronic changes in renal function induced by norepinephrine were similar to those in control dogs except that acute reductions in glomerular filtration rate tended to be more severe, and changes in filtration fraction and fractional sodium excretion were either attenuated or abolished. Moreover, in the absence of a rise in plasma Ang II concentration, mean arterial pressure did not change either acutely or chronically during norepinephrine infusion. These findings suggest a critical role for Ang II in mediating the hypertension associated with elevated levels of renal adrenergic stimulation that have little or no long-term effect on renal blood flow.
There has been a long-standing interest in the neural mechanisms that may be involved in the pathogenesis of hypertension. Since the kidneys are thought to play a dominant role in long-term blood pressure control, one pathway that could link the sympathetic nervous system to renal function is the renal nerves.1 Although the subject is controversial,2 evidence exists for enhanced adrenergic tone to the kidneys in both experimental hypertension3 4 and human essential hypertension.5 Furthermore, renal denervation delays or attenuates the rise in arterial pressure in several experimental models of hypertension.2 Although these studies support a potential role of the renal nerves in the pathogenesis of hypertension, they provide little insight into the renal mechanisms that may be important in mediating neurally induced hypertension.
The acute renal responses to renal adrenergic stimulation have been well characterized and include vasoconstriction, increased tubular reabsorption of sodium, and enhanced renin release. The direct effects of renal adrenergic stimulation on renal hemodynamics and sodium excretion are achieved by activation of α-adrenergic receptors; additionally, indirect effects may result from the generation of angiotensin II (Ang II). However, the quantitative importance of Ang II in indirectly mediating the acute renal hemodynamic and sodium excretory responses to renal adrenergic stimulation is controversial.6 7 8 9 10 11 Moreover, studies have not been designed to determine the role of Ang II in indirectly mediating the long-term effects of renal adrenergic stimulation on renal function and arterial pressure.
We designed the present study to examine the role of the renin-angiotensin system in mediating the renal and hypertensive effects of chronic renal adrenergic stimulation. To mimic the physiological effects of chronic renal adrenergic stimulation, norepinephrine was chronically infused into the renal artery of uninephrectomized dogs at a rate that produced no significant long-term reductions in glomerular filtration rate (GFR) or renal plasma flow. Dogs were studied either under control conditions when the renin-angiotensin system was functional or after chronic blockade of the renin-angiotensin system with captopril. To prevent baseline changes in arterial pressure, fluid volume, and renal function from influencing the subsequent responses to norepinephrine during captopril administration, plasma Ang II concentration was fixed at normal levels by continuous infusion of Ang II. Thus by comparing the responses in both groups of dogs, we were able to assess the importance of long-term increases in plasma Ang II concentration in indirectly mediating the long-term effects of renal adrenergic stimulation on renal hemodynamics, excretory function, and arterial pressure.
Ten female dogs weighing 21.5±0.9 kg were used in this study. All procedures were carried out in accordance with institutional and National Institutes of Health guidelines regarding the use of laboratory animals. Dogs were sedated with acepromazine (3 mg/kg) and anesthetized with pentobarbital sodium (30 mg/kg IV). With the use of aseptic techniques, a right nephrectomy was performed, and Tygon catheters (0.05-inch ID, 0.09-inch OD; Norton Plastics) were implanted in the femoral arteries and veins. The tips of the femoral arterial and venous catheters were located distal to the origin of the renal artery and in the vena cava, respectively. Additionally, a small-bore Tygon catheter (0.04-inch ID, 0.07-inch OD) was placed in the renal artery of the remaining kidney, and an episiotomy was performed to facilitate catheterization of the urinary bladder as described previously.12 13
All catheters were tunneled subcutaneously and exteriorized between the scapulae. Except during chronic saline infusion, patency of the renal arterial catheters was maintained by flushing daily with sterile isotonic saline and by filling the catheters with heparin (1000 U/mL). The femoral catheters were flushed two to three times weekly and filled with heparin.
After a 1-week convalescence period, the dogs were placed in metabolic pens in a room maintained at 22±3°C with a 12-hour light/dark cycle. They were fitted with an aluminum and canvas backpack containing a pressure transducer (model P23 ID, Statham Laboratories, Inc) positioned at heart level.12 The electrical connections to the pressure transducers and the infusion lines were brought to the top of the cage through a flexible tube attached to the top of the backpack. Isotonic saline (240 mL/d) was infused continuously into a venous catheter with a tubing pump (model 375A, Sage Instruments). Also, an infusion pump (model 944, Harvard Apparatus) was used to continually infuse isotonic saline (60 mL/d plus 20 U/mL heparin) into the renal arterial catheter. For the infusion into the renal artery, 100 feet of PE-50 tubing was connected in series with the renal arterial catheter. The high resistance provided by this tubing prevented intermittent fluctuations in the renal arterial infusion that would occur in response to transient changes in arterial pressure or vertical postural changes of the dogs. Both the renal arterial and venous infusion lines were connected to disposable filters (Cathivex, Millipore) to prevent passage of bacteria and other contaminants. The dogs were given free access to water and maintained on a fixed daily diet of two 15.5-oz cans of prescription heart diet (H/D, Hill’s Pet Products, Inc) supplemented with 5 mL vitamin syrup (V.A.L. Syrup, Fort Dodge Laboratories). Two cans of H/D provide less than 5 mmol sodium and approximately 60 mmol potassium. Thus, with the saline infusion, sodium intake was approximately 50 mmol/d. Water consumption was monitored, and 24-hour urine samples were collected at 1:30 pm, 30 minutes after feeding. Rectal temperature was monitored daily, and amoxicillin (250 mg BID, Warner Chilcott Laboratories), dicloxicillin (250 mg BID, Bristol Laboratories), and a trimethoprim (400 mg) and sulfamethoxazole (80 mg) combination (Sulfatrim, BID; Schein Pharmaceutical, Inc) were given prophylactically.
Before the control period, the dogs were conditioned to handling during a training and equilibration period that lasted approximately 10 days. They were trained to lie quietly on the cage floor for up to 1 hour and also to stand in a supporting sling for 2 to 3 hours for measurement of renal function. Experiments were begun only after adequate training and only after balance of sodium, potassium, and water was achieved. During the training, control, experimental, and recovery periods, arterial pressure was continuously recorded from a femoral arterial catheter with a polygraph (model 7D, Grass Instruments) and a personal computer (model 6300, AT&T) equipped with an analog-to-digital convertor. The analog output from the Grass polygraph was sampled at 200 Hz for a duration of 12 seconds, once a minute, 24 h/d. The digitized data for each 12-second burst were immediately processed to compute mean arterial pressure (MAP) and heart rate. The daily values for MAP and heart rate were determined from the average of the 1140 sample points collected during the 19 hours from 1 pm to 8 am.
Norepinephrine (Levophed, Winthrop Pharmaceuticals) was infused continuously for 7 days at 0.1 μg/kg per minute into the renal artery of two groups of dogs. In the control dogs, the renin-angiotensin system was functional; in a second group of dogs, the plasma Ang II concentration was fixed at control levels by continuous intravenous infusion of the converting enzyme inhibitor captopril (Bristol-Myers Squibb Pharmaceutical Research Institute) and Ang II ([Asp1,Val5]Ang II, Ciba-Geigy Corp) for 7 days before norepinephrine infusion. Captopril and Ang II were infused at 14 μg/kg per minute and 0.58±0.04 ng/kg per minute, respectively. This rate of captopril infusion chronically inhibits endogenous Ang II formation in dogs.12 13 The above infusion rate of Ang II was necessary to maintain MAP, GFR, renal plasma flow, and sodium excretion at control levels during captopril administration.
In control dogs with a functional renin-angiotensin system, norepinephrine was infused after a 3-day control period during which renal function was measured twice. Renal function was subsequently measured on days 1, 4, and 7 of norepinephrine infusion. After 7 days, the norepinephrine infusion was terminated, and renal function was measured on days 1, 4, and 7 of the recovery period.
In the second group of dogs in which the renin-angiotensin system was fixed at control levels, renal function was measured twice during a 3-day control period before an 18-day infusion of captopril plus Ang II was begun. After 7 days of captopril plus Ang II infusion, renal function was determined before the 7-day norepinephrine infusion into the renal artery was initiated. Then, as in the control dogs, measurements of renal function were made on days 1, 4, and 7 of norepinephrine infusion and on days 1 and 4 after the norepinephrine infusion was stopped. A final determination of renal function was made 5 to 7 days after the captopril plus Ang II infusion was terminated.
Experiments for measurement of renal function were begun at approximately 8 am by placing the dogs in the recumbent position on the cage floor. After the dogs had rested 45 minutes on the cage floor, two arterial blood samples were collected. A 5-mL blood sample was collected into a chilled tube containing Na2EDTA for the measurement of resting plasma renin activity (PRA) and plasma concentrations of norepinephrine and epinephrine. A second 1.5-mL blood sample was collected for determination of hematocrit and plasma concentrations of sodium, potassium, and protein. The blood sample for measurement of PRA was not taken during the captopril plus Ang II infusion. Subsequently, the dogs were placed in a supporting sling and the urinary bladder was catheterized. The sling was adjusted so that the dogs could stand; however, the dogs usually permitted their weight to be supported by the sling while their legs hung passively.
After the dogs had been 45 minutes in the sling and MAP and heart rate had stabilized, another set of arterial blood samples was collected in control dogs for determination of the effects of posture on PRA and plasma catecholamine concentration. Then, GFR and effective renal plasma flow were estimated from the clearances of 125I-iothalamate (Glofil, Isotex Diagnostics) and 131I-iodohippurate (Hippuran, Syncor International Corp), respectively, as previously described.13 During each experiment, three consecutive 20-minute clearance periods were conducted for determination of GFR and renal plasma flow. At the end of each clearance period, the bladder was flushed twice with a total of 20 mL sterile distilled water, and the wash was added to the urine collected. A 1.5-mL arterial blood sample was taken at the midpoint of each clearance period for determination of the plasma concentrations of 125I-iothalamate and 131I-iodohippurate. On the days when the norepinephrine infusion was either begun or ended (day 7 of norepinephrine infusion), three additional clearance periods were performed for determination of the transient changes in renal function. An additional blood sample was taken during the last clearance period for determination of the attendant changes in PRA and plasma catecholamine concentration.
The norepinephrine infusate was prepared fresh twice daily and contained ascorbic acid (1 mg/mL) as an antioxidant and heparin (20 U/mL). In addition, the syringes and infusion lines were shielded from light with aluminum foil to minimize photooxidation. The stability of norepinephrine in the infusion system was assessed with the use of high-performance liquid chromatographic (HPLC) techniques for measurement of the norepinephrine content of the fresh infusate and the infusate after it passed through the high-resistance infusion lines 12 hours later. Under these conditions, the norepinephrine concentration of the infusate taken from the infusion lines was 91±3% of the level measured in the fresh solution from the syringe.
PRA was measured by radioimmunoassay14 and is expressed as nanograms Ang I generated per milliliter plasma per hour of incubation. Plasma and urine concentrations of sodium and potassium were determined by flame photometry (IL 943, Instrumentation Laboratories), plasma protein concentration by refractometry (American Optical), and hematocrit by a micromethod (Autocrit II, Clay Adams).
Plasma norepinephrine and epinephrine concentrations were determined by HPLC as previously described.15 In brief, dihydroxybenzylamine (Sigma Chemical Co) was added to each sample as an internal standard. Plasma samples (2.0 mL) were deproteinized (0.2 mL of 1.0N HClO4), centrifuged for 60 minutes at 50 000g, decanted, and stored at −80°C. For the assay, the catecholamines were extracted onto alumina oxide at pH 8.6. The alumina was washed with deionized water and eluted with 0.1N HClO4. The eluate was injected into the HPLC system (BAS 200, Bioanalytical Systems), and norepinephrine and epinephrine were measured by electrochemical detection at an electrode potential of −700 mV with the use of computerized digital data collection and peak integration techniques (Axxiom Chromatography, Inc). Values are expressed as picograms per milliliter plasma.
Results are expressed as mean±SEM. Experimental and recovery data were compared with control using ANOVA with Dunnett’s t test for repeated measures.16 Control values were averaged to calculate a single control value.
Baseline Values for Control Dogs and Dogs With Fixed Plasma Ang II Concentration
Table 1⇓ shows the baseline values for MAP and heart rate (19-hour averages); 24-hour urinary sodium, potassium and volume excretion; water consumption; and renal hemodynamics in five control dogs and five dogs chronically infused with captopril plus Ang II. There were no significant differences in the baseline values for control dogs and for dogs with fixed plasma Ang II concentration before captopril plus Ang II infusion. Moreover, in dogs with fixed plasma Ang II concentration, there were no significant changes in any of the above baseline values or in any daily measurements during the initial 7 days of captopril plus Ang II infusion. Thus, during captopril plus Ang II infusion, MAP, heart rate, water and electrolyte balances, and renal function before intrarenal infusion of norepinephrine did not change significantly.
Effects of Posture on PRA and Plasma Catecholamine Concentration
Table 2⇓ illustrates the effects of posture on PRA and on the plasma concentration of norepinephrine before, during, and after chronic intrarenal infusion of norepinephrine. Under all conditions, plasma norepinephrine and epinephrine (control, 110±34 pg/mL) concentrations during standing did not change significantly. However, under control conditions and during the recovery period, standing induced a twofold to fourfold increase in PRA. In addition, although MAP tended to increase slightly during standing, changes in MAP and heart rate were not statistically significant. In contrast to the control and recovery periods, during chronic intrarenal norepinephrine infusion, standing did not increase PRA above the stimulated recumbent values (as discussed below). Additionally, there were no significant postural effects on MAP or heart rate during norepinephrine infusion.
Acute Responses to Intrarenal Arterial Infusion of Norepinephrine
Figs 1⇓ and 2⇓ summarize the short-term effects of intrarenal norepinephrine infusion on MAP, urinary sodium excretion, and renal hemodynamics in control dogs and in dogs in which the renin-angiotensin system was fixed at control levels. In control dogs, MAP, heart rate, GFR, renal plasma flow, and filtration fraction were relatively constant throughout the control period, whereas absolute and fractional excretion of sodium tended to decrease with time. Subsequently, during the first 60 minutes of intrarenal norepinephrine infusion, MAP increased 9±4 mm Hg above control (control, 96±4 mm Hg) in association with a twofold to threefold increase in PRA (from 1.5±0.4 to 3.8±0.6 ng Ang I/mL per hour), a fall in heart rate from 78±4 to 70±3 beats per minute, and a threefold to fourfold increase in plasma norepinephrine concentration (from 175±15 to 667±85 pg/mL). During the first 20 minutes of norepinephrine infusion, GFR (control, 35±2 mL/min) and renal plasma flow (control, 102±3 mL/min) fell to 70±5% and 64±5% of control, respectively, and filtration fraction increased to 15±2% above control. Throughout the remainder of the 1-hour clearance experiment, GFR and renal plasma flow recovered modestly to control values, and filtration fraction remained elevated. During the initial 20 minutes of norepinephrine infusion, there were also pronounced decrements in both urinary sodium (control, 23±5 μmol/min) and fractional sodium excretion (control, 0.45±0.23%) to 48±10% and 65±13% of control, respectively. However, in contrast to the time-dependent increments in both GFR and renal plasma flow, reductions in both urinary sodium and fractional sodium excretion tended to become more pronounced over time. Although not shown in Figs 1⇓ and 2⇓, urinary potassium excretion also decreased during intrarenal norepinephrine infusion, falling to 45±10% of control (control, 19±5 μmol/min); fractional potassium excretion (control, 9.76±1.70%) did not change significantly. Finally, hematocrit (control, 38±2%) and plasma concentrations of sodium (control, 147±1 mmol/L), potassium (control, 4.2±0.1 mmol/L), and protein (control, 6.2±0.1 mg/dL) did not change significantly during the first 60 minutes of intrarenal norepinephrine infusion in control dogs.
In dogs chronically infused with captopril plus Ang II, as in control dogs, MAP, heart rate, and renal hemodynamics were relatively constant throughout the control period before norepinephrine infusion; however, in contrast to control dogs, time-dependent reductions in urinary sodium and fractional sodium excretion were either attenuated or abolished (Figs 1⇑ and 2⇑). Moreover, in dogs in which plasma Ang II concentration was unable to increase above control levels, the arterial pressure, renal hemodynamic, and renal excretory responses to norepinephrine were altered. First, the acute rise in arterial pressure (control, 95±5 mm Hg) and the fall in heart rate (control, 69±5 beats per minute) associated with norepinephrine infusion in the control dogs were abolished during captopril plus Ang II infusion. Second, although reductions in renal plasma flow were comparable in both groups of dogs during norepinephrine infusion, there was a tendency for a greater fall in GFR when plasma Ang II concentration was fixed at control levels; consequently, during chronic administration of captopril plus Ang II, filtration fraction failed to increase acutely during norepinephrine infusion as it did in control dogs. In addition, although comparable antinatriuretic responses to norepinephrine occurred in control dogs and in dogs infused with captopril plus Ang II (control, 33±4 μmol/min), reductions in fractional excretion of sodium (control, 0.64±0.13%) were attenuated approximately 50% in the absence of a rise in plasma Ang II concentration. The effects of norepinephrine on urinary potassium excretion (control, 25±7 μmol/min) and fractional potassium excretion (control, 17.78±5.20%) were similar in both groups of dogs. Finally, as in control dogs, norepinephrine infusion had no significant effect on hematocrit (control, 33±2%) or on the plasma concentrations of sodium (control, 145±1 mmol/L), potassium (control, 4.2±0.1 mmol/L), and protein (control, 6.6±0.2 mg/dL).
Chronic Responses to Intrarenal Arterial Infusion of Norepinephrine
Figs 3⇓ and 4⇓ summarize the effects of chronic intrarenal arterial norepinephrine infusion on MAP, PRA, 24-hour urinary sodium excretion, and renal hemodynamics. In control dogs, MAP increased 12±4 mm Hg during the first day of infusion; subsequently, MAP increased progressively throughout the 7-day infusion period and on day 7 was elevated 20±7 mm Hg above control (to 110±7 mm Hg). Heart rate tended to decrease approximately 12% during chronic norepinephrine infusion, but the changes were not statistically significant. In association with the chronic increase in MAP, there was a sustained increase in PRA throughout the 7-day norepinephrine infusion period: recumbent PRA values were approximately fivefold greater than control during norepinephrine infusion (Table 2⇑). In addition, plasma norepinephrine concentration increased fourfold to fivefold during chronic intrarenal arterial infusion of norepinephrine (Table 2⇑), whereas plasma epinephrine concentration (control, 110±34 pg/mL) was unchanged. When the norepinephrine infusion was terminated on day 7, MAP, PRA, and plasma norepinephrine concentration returned to control levels during the first day of the recovery period.
In contrast to the pronounced short-term effects of norepinephrine on renal hemodynamics (Fig 2⇑), in control dogs there were no significant long-term changes in GFR, renal plasma flow, or filtration fraction during either chronic norepinephrine infusion or the postinfusion recovery period (Fig 4⇑). In addition, although norepinephrine induced sodium retention during the first hour of infusion in control dogs (Fig 1⇑), there was no net retention of sodium during the initial 24 hours of norepinephrine infusion (Fig 3⇑). Furthermore, 24-hour urinary sodium excretion did not change significantly throughout the 7 days of norepinephrine infusion and throughout the postinfusion recovery period. Similarly, 24-hour urinary potassium excretion did not change during either the norepinephrine infusion or the recovery period. Plasma protein concentration (control, 6.2±0.1 mg/dL) tended to increase during norepinephrine infusion and after 7 days was 6.6±0.1 mg/dL. Hematocrit (control, 38±2%) decreased to 35±2% after the first day of norepinephrine infusion before increasing to above control levels (day 7, 41±2%). Both hematocrit and plasma protein concentration returned to control values during the postinfusion recovery period. Finally, plasma concentrations of sodium (control, 147±1 mmol/L) and potassium (control, 4.2±0.1 mmol/L) did not change significantly during norepinephrine infusion or the subsequent recovery period.
In marked contrast to the hypertension induced by norepinephrine infusion in control dogs, when plasma Ang II concentration was fixed at control levels, MAP (and heart rate) did not change significantly during chronic intrarenal arterial infusion of norepinephrine (Fig 3⇑). In addition, whereas sodium and water balance was maintained during chronic norepinephrine infusion when the renin-angiotensin system was functional, in the absence of a rise in plasma Ang II concentration above control levels, loss of sodium (30±4 mmol) and water (267±32 mL) occurred on day 1, before sodium and water balance was subsequently achieved. Conversely, during the 24-hour period after the norepinephrine infusion was terminated, sodium (26±2 mmol) and water (130±9 mL) retention occurred. A natriuresis also occurred on recovery day 5 when the captopril plus Ang II infusion was terminated. After the first recovery day, one dog in the captopril plus Ang II group was excluded from analysis because of a leak in the renal artery infusion line. Finally, as in control dogs, GFR, renal plasma flow, and filtration fraction did not change significantly (Fig 4⇑), nor did urinary potassium excretion during chronic intrarenal infusion of norepinephrine.
The effects of chronic norepinephrine infusion on hematocrit and on the plasma concentrations of sodium, potassium, and protein during chronic administration of captopril plus Ang II were similar to those that occurred in control dogs. During chronic infusion, plasma protein concentration (control, 6.6±0.2 mg/dL) increased to 6.8±0.2 mg/dL by day 7; however, this rise was not statistically significant. Hematocrit (control, 33±2%) did not decrease on day 1 as it did in control dogs, but it did increase thereafter, being 37±2% on day 7. Additionally, plasma concentrations of sodium (control, 145±1 mmol/L) and potassium (control, 4.2±0.1 mmol/L) did not change significantly during either the experimental or recovery periods.
Acute Responses to Cessation of Chronic Intrarenal Arterial Infusion of Norepinephrine
In general, the acute renal excretory and renal hemodynamic responses to cessation of norepinephrine infusion on day 7 were qualitatively similar in control dogs and in dogs chronically administered captopril plus Ang II (Figs 5⇓ and 6⇓). In both groups of dogs, MAP, heart rate, renal hemodynamics, and urinary electrolyte excretion were stable during the last hour of norepinephrine infusion. During the hour after norepinephrine infusion was stopped, MAP decreased progressively in control dogs and in dogs treated with captopril plus Ang II, falling from 116±6 to 104±4 and from 98±5 to 91±6 mm Hg, respectively, by the last clearance period. Heart rate tended to increase during this time, but the changes were not statistically significant. In control dogs, the hypotensive response was associated with a fall in PRA from 3.1±0.8 to 1.4±0.2 ng Ang I/mL per hour.
Although GFR and renal plasma flow were not lower than control values on day 7 of norepinephrine infusion, during the 20-minute period immediately after termination of norepinephrine infusion, GFR and renal plasma flow increased 15% to 22% in both control dogs and dogs infused with captopril plus Ang II before gradually falling to control levels; filtration fraction tended to decrease during this time. Despite the hypotension, urinary sodium and fractional sodium excretion increased approximately twofold in control dogs immediately after termination of the norepinephrine infusion, whereas in dogs with fixed plasma Ang II concentration, these variables did not change significantly. Also, in control dogs, urinary and fractional potassium excretion increased progressively to 44±14% and 38±5% above control, respectively, 40 to 60 minutes after the norepinephrine infusion was stopped. Similar changes in urinary and fractional potassium excretion occurred in dogs infused with captopril plus Ang II once the norepinephrine infusion was terminated, but in general, these changes were not statistically significant. Finally, there were no significant acute changes in hematocrit or plasma concentrations of sodium, potassium, and protein in either group of dogs during the hour after norepinephrine infusion.
Postmortem Renal Morphology
Postmortem examination showed the norepinephrine-infused kidneys from control dogs and dogs infused with captopril plus Ang II to appear normal, with no thrombi and only occasional minute areas of focal ischemia. The infused kidneys from the control dogs and the dogs infused with captopril plus Ang II weighed 47.0±2.3 and 48.7±2.1 g, respectively.
It is well established that acute renal adrenergic stimulation decreases urinary sodium excretion. The antinatriuretic effects of renal adrenergic stimulation are mediated directly by (1) an influence on renal tubular cells to promote sodium reabsorption and (2) vasoconstriction, which decreases GFR and renal blood flow. Additionally, activation of the renal nerves increases renin release at a level of stimulation below that required to increase sodium reabsorption and cause vasoconstriction. Therefore, since Ang II has potent vasoconstrictor and antinatriuretic effects, it would be expected that some of the renal actions associated with renal adrenergic stimulation are mediated indirectly via Ang II generation. Unfortunately, the design of many experimental studies does not allow a quantitative assessment of the multiple pathways by which adrenergic stimulation alters sodium excretion and renal hemodynamics, especially under chronic conditions. In the present study, the renal actions of acute and chronic renal adrenergic stimulation were compared in dogs with a functional renin-angiotensin system and in dogs in which plasma levels of Ang II were fixed at normal levels. Since plasma Ang II concentration was maintained at normal levels during captopril administration by continuous intravenous infusion of Ang II, we were able to assess the role of endogenous Ang II in mediating the effects of renal adrenergic stimulation on renal function and arterial pressure in the absence of the confounding influence of hypovolemia, hypotension, and renal vasodilation, responses associated with blockade of the renin-angiotensin system by administration of either captopril alone or Ang II receptor antagonists. Under the conditions of the present study, the findings indicate that the hypertension induced by chronic renal adrenergic stimulation is not due to the direct sodium-retaining effects of norepinephrine but rather is indirectly mediated via Ang II generation.
Studies in both the dog and rat have shown that chronic intrarenal infusion of norepinephrine, at doses that have little or no effect on MAP when given intravenously, produce chronic hypertension.17 18 19 Although these studies do not prove that enhanced renal nerve activity contributes to the pathogenesis of hypertension, they do provide evidence that chronic renal adrenergic stimulation decreases renal excretory capability and increases the level of arterial pressure required to achieve long-term sodium and water balance. However, in the previous studies in dogs, much higher infusion rates of norepinephrine (approximately threefold) were used than used in the present study. These higher norepinephrine infusion rates produced or would be expected to produce rather substantial acute and chronic reductions in renal blood flow.11 17 18 Furthermore, when renal function was measured during the recovery period after chronic intrarenal norepinephrine infusion, renal function was permanently impaired.18 The present findings indicate that chronic hypertension can be achieved by more physiological levels of renal adrenergic stimulation that have little or no long-term effects on either GFR or renal plasma flow.
Although numerous studies have shown that renal adrenergic stimulation increases renin release in the short term, there is relatively little information on the long-term effects of renal sympathetic stimulation on renin release. Previous studies in dogs chronically infused with norepinephrine into the renal artery fail to clarify this issue. In the study by Cowley and Lohmeier,18 4-day norepinephrine infusion (0.27 μg/kg per minute) did produce chronic increments in PRA and arterial pressure as long as the norepinephrine was administered directly into the renal artery and not intravenously. Furthermore, adrenergic stimulation of renin release was sustained during intrarenal norepinephrine infusion irrespective of whether the dogs were maintained on a high or low sodium intake. In contrast, in a study of longer duration (14 days), Katholi et al17 reported that a similar intrarenal infusion rate of norepinephrine (0.285 μg/kg per minute) produced chronic hypertension but failed to stimulate PRA beyond 6 days. It is conceivable that the differential long-term PRA responses to norepinephrine in these two studies may reflect differences in the duration of renal adrenergic stimulation, but it is also important to note that in the study by Katholi et al, blood samples for PRA were taken from dogs that were standing in a supporting sling.
The current results demonstrate that under control conditions, but not during chronic renal adrenergic stimulation by norepinephrine, standing in a supporting sling increases renin release above recumbent levels (Table 2⇑). Since standing did not increase the plasma concentrations of either norepinephrine or epinephrine, the present findings and those from earlier studies in conscious dogs20 21 22 indicate that posturally induced increments in PRA are mediated via activation of the renal nerves and, in addition, are abolished when the level of renal adrenergic stimulation is elevated before the orthostatic stress. Therefore, if one compares the standing PRA values from dogs before and during chronic norepinephrine infusion (Table 2⇑), as did Katholi et al,17 no apparent differences exist. On the other hand, if resting, recumbent PRA values are compared (Table 2⇑ and Fig 3⇑), it is clear that PRA was higher during chronic norepinephrine infusion than when there was little neurogenic stimulation of the kidneys. Thus, the present findings indicate that chronic renal adrenergic stimulation does in fact produce sustained activation of the renin-angiotensin system, raising the possibility that elevations in plasma Ang II concentration may contribute at least in part to the long-term effects of renal sympathetic stimulation on renal function and arterial pressure.
To directly assess the contribution of elevated plasma Ang II levels to the hypertension induced by chronic renal adrenergic stimulation, we compared responses in dogs with a functional renin-angiotensin system with those in dogs with fixed plasma levels of Ang II. The results were unequivocal. In control dogs, PRA increased fourfold to fivefold during chronic norepinephrine infusion, and MAP increased approximately 20 mm Hg. In contrast, in dogs that were unable to increase plasma Ang II concentration above control levels when norepinephrine was infused intrarenally, MAP remained at normotensive levels. These results suggest a critical role for Ang II in mediating the long-term hypertensive effects of renal adrenergic stimulation.
The long-term changes in urinary sodium excretion associated with intrarenal norepinephrine infusion are consistent with the long-term arterial pressure responses and indicate that the Ang II generated during renal adrenergic stimulation shifts renal pressure natriuresis to higher levels, eventuating in hypertension.1 During intrarenal norepinephrine infusion, peripheral plasma levels of norepinephrine increased approximately fourfold (Table 2⇑). When plasma Ang II concentration was fixed at control levels, sodium and water were lost on day 1 of norepinephrine infusion in the absence of a change in MAP; conversely, on day 1 of the recovery period, sodium and water were retained. These excretory responses would be expected from a vasoconstrictor agent that increases peripheral resistance (and decreases vascular capacitance) without altering renal pressure natriuresis.1 In contrast, when the renin-angiotensin system was functional, sodium balance was maintained during intrarenal norepinephrine infusion, despite the concomitant hypertension. This indicates that the Ang II generated during chronic renal adrenergic stimulation decreased renal excretory capability and increased the level of arterial pressure required to achieve sodium balance. The chronic antinatriuretic effects of Ang II were further manifested immediately after the intrarenal norepinephrine infusion was stopped (Fig 5⇑); during this time, natriuresis occurred in association with reductions in PRA and MAP. In comparison, sodium excretion did not increase when the norepinephrine infusion was terminated in dogs with fixed plasma levels of Ang II (despite smaller reductions in MAP than in control dogs). Taken together, the long-term changes in arterial pressure and sodium balance in response to norepinephrine infusion suggest that the direct effects of renal adrenergic stimulation on pressure natriuresis are relatively weak compared with the indirect effects mediated by endogenously generated Ang II.
Although the hypertensive effects of norepinephrine occurred in the absence of significant changes in renal hemodynamics, it is important to note that moderate reductions in renal plasma flow may have been obscured during chronic norepinephrine infusion because renal function was measured while the dogs were in a standing position. Because standing normally induces a modest reduction in renal plasma flow (approximately 15%) in dogs that is mediated in part by the generation of Ang II,23 it is likely that passive standing decreased renal plasma flow to a greater extent during the control and recovery periods than during chronic norepinephrine infusion when there were no postural effects on renin release (Table 2⇑). Therefore, it is possible that modest reductions in renal plasma flow occurred during chronic norepinephrine infusion that were masked by disproportionately greater acute falls in renal plasma flow in response to standing during the control and recovery periods than during sustained renal adrenergic stimulation by intrarenal norepinephrine infusion.
It is likely that the chronic renal hemodynamic response to norepinephrine was influenced not only by Ang II generation but by local regulatory mechanisms and by changes in arterial pressure as well. In the present study, rather substantial reductions in GFR and renal plasma flow occurred acutely in response to adrenergic stimulation, but these changes were not sustained chronically despite the continuous infusion of norepinephrine. In fact, considerable escape from adrenergically mediated vasoconstriction occurred during the first hour of norepinephrine infusion, and within 24 hours both GFR and renal plasma flow had returned to control levels. Moreover, the acute increases in both GFR and renal plasma flow to above control levels when the chronic infusion of norepinephrine was terminated (Fig 6⇑) indicate that the vasoconstrictor influence of norepinephrine was sustained chronically but was offset by vasodilator counterregulatory mechanisms. This escape from adrenergically mediated vasoconstriction may be mediated by increased release of vasodilators such as prostaglandins24 25 or endothelium-derived nitric oxide.26 Increased arterial pressure may also have contributed to the restoration of renal plasma flow (and filtration fraction) during chronic norepinephrine infusion in dogs with a functional renin-angiotensin system because these renal hemodynamic changes persist during long-term intravenous infusion of Ang II only when renal perfusion pressure is not allowed to increase.27 28 Thus, in the present study, the hypertension induced by Ang II may have prevented chronic differences in renal plasma flow and filtration fraction in dogs with and without high plasma levels of Ang II.
The present studies also indicate that Ang II plays an important role in mediating the acute renal actions of adrenergic stimulation. Because filtration fraction increased acutely during norepinephrine infusion (and GFR tended to be better preserved) when the renin-angiotensin system was intact but not when plasma Ang II concentration was maintained at control levels, it appears that the Ang II generated during acute renal adrenergic stimulation is vital to the maintenance of GFR, presumably via its ability to constrict efferent arterioles.7 8 9 29 30 Furthermore, reductions in fractional sodium excretion were clearly attenuated (by approximately 50%) during acute norepinephrine infusion when increments in plasma Ang II concentration were prevented by captopril infusion, suggesting an important role of the renin-angiotensin system in mediating increased tubular reabsorption of sodium during acute adrenergic stimulation. However, despite smaller reductions in fractional sodium excretion in captopril-treated dogs, the acute antinatriuretic response to norepinephrine was comparable in controls and in dogs administered captopril because there was a greater fall in GFR when the renin-angiotensin system was nonfunctional.
In previous studies, the role of Ang II in decreasing renal blood flow during acute adrenergic stimulation has been variable.7 8 9 11 Because reductions in renal plasma flow during norepinephrine infusion were comparable in captopril-treated and control dogs, the present findings do not indicate a contribution of Ang II to the renal blood flow response to acute renal adrenergic stimulation. However, this finding differs from that in a recent study conducted in our laboratory in recumbent, conscious dogs.11 This previous study indicated that Ang II plays a significant role in mediating reductions in renal blood flow during intrarenal norepinephrine infusion when basal levels of renal nerve activity and PRA are low before acute renal adrenergic stimulation. The seemingly disparate results from these two studies may reflect differential activation of the renin-angiotensin system before norepinephrine infusion. That is, under conditions in which the renin-angiotensin system is activated (whether by standing or by anesthesia and surgical stress), the additional Ang II generated during subsequent renal adrenergic stimulation may have little influence on the magnitude of the adrenergically induced fall in renal blood flow.
In conclusion, the present findings indicate that baseline levels of neural tone and circulating Ang II may modify the renal responses to Ang II generated during acute adrenergic stimulation. Under conditions of elevated renal nerve activity and renin release, including passive standing, further increases in the plasma levels of Ang II during subsequent renal adrenergic stimulation appear to have a diminished influence on renal blood flow and sodium excretion compared with responses when basal neural tone and renin secretion are low. Moreover, the present results suggest that Ang II plays a critical role in mediating the chronic hypertension associated with levels of renal adrenergic stimulation that have little chronic influence on renal blood flow. This essential role of Ang II in mediating neurogenic hypertension may account for the efficacy of angiotensin-converting enzyme inhibitors in reducing blood pressure in experimental models of hypertension in which increased renal nerve activity is believed to contribute importantly to the pathogenesis of the disease.31 32 33
This research was supported by National Heart, Lung, and Blood Institute grants HL-11678 and HL-51971. Dr Reinhart is a recipient of a National Research Service Award from the National Institutes of Health, Bethesda, Md. We thank the Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ, for their generous donation of captopril.
Reprint requests to Glenn A. Reinhart, PhD, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216-4505. E-mail reinhart@ fiona.umsmed.edu.
- Received August 15, 1994.
- Revision received October 3, 1994.
- Accepted January 11, 1995.
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