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Am J Physiol Regul Integr Comp Physiol 278: R1-R10, 2000;
0363-6119/00 $5.00
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Vol. 278, Issue 1, R1-R10, January 2000

Determinants of the natriuresis after acute, slow sodium loading in conscious dogs

Peter Bie1 and Niels C. F. Sandgaard2

2 Department of Medical Physiology, University of Copenhagen, DK-2200 Copenhagen; and 1 Department of Physiology and Pharmacology, University of Southern Denmark, Odense, DK-5000 Odense, Denmark


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The relative importance of systemic volume, concentration, and pressure signals in sodium homeostasis was investigated by intravenous infusion of isotonic (IsoLoad) or hypertonic (HyperLoad) saline at a rate (1 µmol Na+ · kg-1 · s-1), similar to the rate of postprandial sodium absorption. IsoLoad decreased plasma vasopressin (-35%) and plasma ANG II (-77%) and increased renal sodium excretion (95-fold), arterial blood pressure (Delta BP; +6 mmHg), and heart rate (HR; +36%). HyperLoad caused similar changes in plasma ANG II and sodium excretion, but augmented vasopressin (12-fold) and doubled Delta BP (+12 mmHg) without changing HR. IsoLoad during vasopressin clamping (constant vasopressin infusion) caused comparable natriuresis at augmented Delta BP (+14 mmHg), but constant HR. Thus vasopressin abolished the Bainbridge reflex. IsoLoad during normotensive angiotensin clamping (enalaprilate plus constant angiotensin infusion) caused marginal natriuresis (9% of unclamped response) despite augmented Delta BP (+14 mmHg). Cessation of angiotensin infusion during IsoLoad immediately decreased BP (-13 mmHg) and increased glomerular filtration rate by 20% and sodium excretion by 45-fold. The results suggest that fading of ANG II is the cause of acute "volume-expansion" natriuresis, that physiological ANG II deviations override the effects of modest systemic blood pressure changes, and that endocrine rather than hemodynamic mechanisms are the pivot of normal sodium homeostasis.

volume expansion; arterial blood pressure; angiotensin II; vasopressin; atrial natriuretic peptide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE EXTRACELLULAR FLUID VOLUME is normally maintained within narrow limits despite considerable variations in daily salt and water intake. The control is based on an array of volume or pressure receptors, and the volume is determined by the actual mass of Na+, because the concentration of Na+ is adjusted rapidly and independently by changes in water turnover. Therefore, renal sodium excretion is generally considered the central regulated variable in the control of the extracellular fluid volume and, thus, of arterial blood pressure. The complex mechanisms involved in the regulation of renal sodium excretion have been studied over many decades. The effector mechanisms acting on the kidney may be divided into three categories: humoral signals, renal nerve activity, and physical factors (for review, see Ref. 19). However, a coherent understanding of the relative importance of the different control systems is not available.

It is a central concept of sodium metabolism that changes in systemic arterial blood pressure and thus in renal perfusion pressure determine the rate of renal sodium excretion, i.e., the pressure natriuresis mechanism. This concept is based on an impressive amount of experimental results obtained mainly in conscious dogs by Guyton and coworkers (for review, see Ref. 15). There is no doubt that augmentations in arterial blood pressure may be causally related to marked increases in renal sodium excretion. However, sodium excretion may also change many fold without measurable changes in arterial blood pressure (e.g., Ref. 17), possibly due to the pressure natriuresis mechanism operating according to the infinite gain principle (13). Alternatively, the primary defense against sodium loading does not involve an increase in arterial blood pressure and may, therefore, be hormonal.

The renin-angiotensin system (RAS) is the major salt-retaining hormone system (16, 17). From recent work in conscious dogs by Reinhardt and coworkers (4, 20, 24), it has been concluded that the effects of modest changes in renal perfusion pressure on sodium balance are mediated exclusively via angiotensin and aldosterone. It has also been shown that acute blockade of the RAS, e.g., by concomitant converting enzyme inhibition and aldosterone receptor blockade (3), elicits a marked natriuresis despite a decrease in arterial pressure. Thus evidence is available to support the notion that under physiological conditions the renal effects of RAS are at least as important as nonhormonal regulation via blood pressure.

Osmoreceptors are the sensory mechanism of water homeostasis. However, a number of studies indicate that hypernatremia may be associated with natriuresis (e.g., Refs. 11, 18), at least under certain conditions (2). Although the details behind this concentration-driven natriuresis are unknown, it could play a significant role in the regulation of renal sodium excretion.

The present study was designed to elucidate the relative importance of sodium concentration, extracellular fluid volume, and blood pressure in the acute natriuretic response to physiological sodium loading. Concomitant cardiovascular, hormonal, and renal responses were quantified with isotonic or hypertonic saline loading at an identical rate in terms of salt. In addition, isotonic loading was performed during acute clamping of either of the two main control systems by infusion of vasopressin or ANG II.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Six conscious female Beagle dogs weighing 12-14.5 kg were used. They were kept on a constant diet of commercial dog food (Special Diets Services, Witham, UK), receiving one meal a day at around 1400. Mean daily sodium intake was 3.1 ± 0.1 mmol/kg body wt (means ± SE). The dogs had free access to tap water, except for the night before each experiment (see below). Before the study, several surgical interventions were performed. After premedication (in mg/kg: 0.15 propionylpromazine, 0.25 methadone, and 0.02 atropine), general anesthesia was induced by propofol (4 mg/kg) and maintained by continuous infusion of propofol (550 mg/h) and a mixture of N2O and O2 (1:1). With the use of standard antiseptic procedures, both common carotid arteries were displaced into skin loops to minimize discomfort in connection with subsequent arterial puncture. In addition, a chronic episiotomy was performed to facilitate catheterization of the bladder. All dogs were awake and ambulant 1 h after surgery and had fully recovered by the following morning. After at least 4 wk, bilateral ovariectomy and hysterectomy were performed. The dogs were under constant veterinary inspection and treatment, including antibiotics for 6 days and analgesics when required. There were no complications after surgery. The dogs were trained for several months before experiments. The experimental procedures were approved by the Danish Animal Experiments Inspectorate.

Experimental Protocol

The same six dogs were used for all experiments. In each dog, experiments were performed at intervals of >1 wk. At midnight before the experiment, an electric valve controlled by a timer interrupted the water supply. The following morning, the dog was transferred to the laboratory and placed in a sling. A sterile catheter (Intracath, Becton Dickinson, Sandy, UT) was introduced into the right atrial area via the external jugular vein and used for infusions. Another catheter (Insyte-W, Becton Dickinson) was placed in the common carotid artery, allowing for continuous measurements of arterial blood pressure interrupted by periodic sampling of arterial blood. Standard electrocardiogram (ECG) electrodes were applied. A modified silicone Foley catheter (Norta, Beiersdorf, Hamburg, Germany) was used for catheterization of the bladder. An intravenous bolus of creatinine (8.2 ml approx 14 mg/kg) was given immediately followed by a continuous infusion of creatinine (7.2 ml/h approx 0.2 mg · kg-1 · min-1) for the remainder of the experiment. After a 60-min equilibration period, urine was sampled every 30 min. The bladder was flushed with 3 × 10 ml of distilled water at the end of each sampling period. Air in the bladder or drainage system was carefully avoided. After a control period (t = 0-30 min), isotonic or hypertonic salt loading was initiated and continued for 90 min (t = 30-120 min). Samples of arterial blood totaling <45 ml were obtained at 30-min intervals from t = -5 min, in EDTA tubes and at t = -5, 25, 55, 85, and 115 min for creatinine measurements. Samples for sodium, potassium, osmolality (heparinized tubes), and for hormone determinations (additives: EDTA 3 mmol/l, aprotinine 300 KIU/ml) were obtained at t = 25 and 115 min. The blood samples were centrifuged immediately at 4°C, and plasma was analyzed on the day of experiment (sodium, potassium, osmolality) or stored at -18°C until further processing. Six experimental series were performed.

Iso series. Intravenous infusion of isotonic NaCl solution (154 mmol/l) was administered at a rate of 60 µmol Na+ · kg-1 · min-1, corresponding to a flow rate of 0.39 ml · kg-1 · min-1 (n = 6).

Hyper series. An identical sodium load was administered intravenously as a hypertonic (2 mol/l NaCl) solution at a flow rate of 0.03 ml · kg-1 · min-1(n = 6).

Iso+AVP series. Vasopressin (AVP, Ferring, Malmö, Sweden) was added to the isotonic saline to a concentration of 1.54 ng/ml. Consequently, the rate of infusion of AVP was 600 pg · kg-1 · min-1. The experiments were otherwise identical to the Iso series (n = 6).

Iso+ANG II series. Endogenous ANG II production was inhibited by enalapril maleate (Sigma, St. Louis, MO) 2 mg/kg iv at t = -60 min. The injection was immediately followed by a continuous infusion of ANG II (Sigma) at a rate of 3 ng · kg-1 · min-1. If necessary, the rate was adjusted so that mean arterial blood pressure (MABP) was returned as rapidly as possible to the individual control level of the dog in the experiment. This infusion rate (in different dogs varying between 2.5 and 3.5 ng · kg-1 · min-1) was then maintained throughout the experiment. After 1 h of stabilization at normal blood pressure the experiment was performed as in the Iso series (n = 6).

Iso+stopANG II. The protocol was a copy of the Iso+ANG II series, except that the infusion of ANG II was stopped at t = 90 min, i.e., 30 min before the termination of the saline loading. Consequently, plasma ANG II must have decreased rapidly in this period, because endogenous ANG II production was still inhibited by enalaprilate (n = 4).

Time control. All procedures were identical to those of the Iso series, except that the loading was not performed (n = 6).

Hemodynamics

Arterial blood pressure and ECG were measured continuously by a pressure transducer (Statham P50) connected to a clinical monitor (Dialogue 2000, Danica Elektronik, Rødovre, Denmark). The pressure signal was digitized at 300-Hz sampling frequency, and MABP was calculated over a 7-s time window. The monitor calculated heart rate on the basis of the ECG. Data were sampled every 10 s by computer and subsequently averaged over 30-min periods.

Analyses

The concentration of sodium and potassium ions in plasma and urine was measured by flame photometry (IL243 flame photometer, Instrumentation Laboratory, Lexington, MA). Plasma and urine osmolality were determined by freezing-point depression (Advanced Osmometer, model 3D3, Advanced Instruments, Needham Heights, MA). Plasma protein concentration was measured by a refractometer (Clinical refractometer T2-NE, Atago, Tokyo, Japan), and colloid osmotic pressure was determined by a colloid osmometer (Colloid Osmometer 4400, Wescor, Logan, UT). Concentrations of creatinine in urine and plasma were measured by Jaffé's reaction modified from Bonsnes and Taussky (5).

Extraction. Hormone concentrations in plasma and urine were measured by radioimmunoassays after extraction (8). Hormone data have not been corrected for incomplete recovery.

AVP. This was measured using an antibody (AB3096) produced in this laboratory at a final dilution of 1:800,000. Otherwise, the assay was performed according to Emmeluth et al. (9). Cross reactivity was determined for a number of analogous peptides: [Lys8]-vasopressin, oxytocin, and pressinoic acid all <0.001%, [deamino-Cys1,D-Arg8]-vasopressin <0.07%, and [Arg8]-vasotocin <0.25%. The detection limit was <0.2 pg/ml, and the mean recovery of unlabeled AVP added to plasma was 66%. Intra- and interassay coefficients of variation were <8%.

Endothelin-1. The concentrations in plasma and urine were determined using a specific antibody (RAS 6901) purchased from Peninsula Laboratories. The procedure was described previously (8). The detection limit was ~0.4 pg/ml, and the mean extraction recovery of unlabeled endothelin-1 added to plasma was 90%. Intra- and interassay coefficients of variation were 5 and 7%, respectively.

Atrial natriuretic peptide (ANP). A specific antibody (AB95069/3) produced in this laboratory was used in a final dilution of 1:27,000 according to the procedure of Schütten et al. (23). The detection limit was 1.5 pg/ml, and the mean extraction recovery of unlabeled ANP added to plasma was 74%. The intra-assay coefficient of variation was 6%.

ANG II. To determine ANG II immunoreactivity in plasma, a specific antibody (Ab-5-030682) produced by P. Christensen was used in a final dilution of 1:1,100,000. Cross reactivity with ANG I was <0.1%. Plasma samples were incubated with antibody for 24 h and with tracer 125I-labeled ANG II (kindly provided by the Department of Clinical Physiology, Glostrup Hospital, Denmark) for another 24 h. A sediment of free antigen was made by adding a charcoal-plasma suspension, and, after centrifugation, radioactivity of the supernatant was measured. The detection limit was 1.4 pg/ml, and the mean extraction recovery of unlabeled ANG II added to plasma was 88%. Intra- and interassay coefficients of variation were 5 and 11%, respectively.

Statistics

Data are presented as means ± SE. The results were evaluated by one-way ANOVA for repeated measurements within groups and by two-way ANOVA for repeated measurements between groups. Possible inhomogeneity of variance was assessed by use of Levene's test. When present, the data were logarithmically transformed before ANOVA. If the results of the ANOVA showed significance, all differences between means were investigated systematically by Newman-Keuls test. P values smaller than 0.05 were considered to indicate significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Systemic Hemodynamics

In the different series of experiments, the initial values of MABP were very similar. MABP increased in response to the sodium load but to rather different extents (Table 1). The increases observed in the Hyper, Iso+AVP, and Iso+ANG II series were markedly larger than those observed in the Iso series. Notably, cessation of ANG II infusion during saline infusion was followed by a rapid decrease in MABP to control level. Thus the blood pressure increase was exaggerated by the increase in plasma sodium concentration and by addition of AVP or ANG II.

                              
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  		Table 1.   Hemodynamic variables

Control heart rates were low. During the Iso series, heart rate increased markedly (+33%) despite the concomitantly elevated MABP (Table 1), a phenomenon known as the Bainbridge reflex. This response was unaffected by ANG II. However, heart rate did not change at all in the Hyper and Iso+AVP series in which identical increases in blood pressure occurred.

Plasma Composition

In the Iso series, plasma protein concentration and plasma oncotic pressure decreased by ~20% without measurable changes in plasma sodium or osmolality (Fig. 1, Table 2). In the Hyper series, plasma sodium concentration increased by ~9 mmol/l and plasma osmolality increased by ~17 mosmol/kgH2O. However, oncotic pressure was only reduced by 14%, i.e., significantly less than the change measured during the Iso series. During Iso+AVP, the reductions in oncotic pressure and plasma protein concentration were further augmented, possibly because of the reduction in fluid excretion caused by the infused AVP.


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  		Fig. 1.   A and B: plasma osmolality and plasma vasopressin (AVP). , Control value, t = 25 min; , end infusion value, t = 115 min. C and D: urine flow and free water. , Control period, t = 0-30 min; , infusion periods, t = 30-120 min. Values are means ± SE. Significant differences (P < 0.05): * different from preinfusion level of same series, dagger  different from control series, § different from isotonic series.


                              
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  		Table 2.   Plasma electrolytes, protein, oncotic pressure, and hormones

Hormones

ANG II. In the different series, plasma concentrations of ANG II averaged ~17 pg/ml during control conditions and decreased to very similar values during Iso, Hyper, and Iso+AVP (3.7 ± 0.5, 3.8 ± 0.7, and 3.5 ± 0.5 pg/ml, respectively; Fig. 2). The control data of the Iso+ANG II series (continuous infusion of ANG II after angiotensin-converting enzyme inhibition) demonstrate that supranormal plasma ANG II concentrations (44 ± 2 pg/ml) were required to maintain normal blood pressure.


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  		Fig. 2.   A: plasma ANG II. , Control value, t = 25 min; , end infusion value, t = 115 min. B-D: glomerular filtration rate (GFR), rate of sodium excretion (Excre), and fractional (Fract) sodium excretion. , Control period, t = 0-30 min; , infusion periods, t = 30-120 min. Values are means ± SE. Significant differences (P < 0.05): * different from preinfusion level of same series, dagger  different from control series, § different from isotonic series.

AVP. In the Iso series, plasma AVP decreased significantly from 1.14 ± 0.29 to 0.74 ± 0.17 pg/ml or by 35% (Fig. 1), whereas a 12-fold elevation was generated by the hypertonic saline in the Hyper series. The infusion of isotonic saline concomitant with AVP (Iso+AVP series) produced plasma AVP concentrations of 30 ± 2 pg/ml, i.e., significantly higher than those seen during the Hyper series. The metabolic clearance rate was found to be 13.3 ± 1.1 ml · kg body wt-1 · min-1, including appropriate correction for incomplete recovery during extraction.

ANP. Plasma ANP concentrations increased marginally during the Iso series (59 ± 5 to 67 ± 6 pg/ml), but not to a level significantly higher than that of the control series (Table 2). During the Hyper, Iso+AVP, and time control series, no significant changes in plasma ANP were observed. In the Iso+ANG II series, the preinfusion plasma ANP levels unexpectedly were elevated by 47% compared with control series. In addition, the increase in plasma ANP from this elevated level in response to the isotonic saline infusion also was exaggerated and averaged some 39 pg/ml, i.e., approximately five times higher than observed in IsoLoad series.

Renal Variables

Glomerular filtration rate. The clearance of creatinine was used as a measure of glomerular filtration rate (GFR). In the Iso, Hyper, and Iso+AVP series, in which the plasma concentrations of ANG II were allowed to decrease, consistent elevations in GFR were observed (Fig. 2). In contrast, GFR remained statistically unchanged during Iso+ANG II, despite the exaggeration of the concomitant saline-mediated increase in MABP. However, a trend toward an increase was evident during the first periods of saline infusion. In the Iso+stopANG II series when the angiotensin infusion was terminated, a significant increase in GFR occurred from 36 ± 4 to 44 ± 3 ml/min (n = 4, P < 0.005, paired t-test), whereas MABP concomitantly fell from 118 ± 3 to 110 ± 4 mmHg (Table 1).

Urine flow. The dogs were mildly dehydrated because of the withdrawal of water at midnight before the experiment. Consequently, control urine flows were low, on the order of 0.12-0.22 ml/min (Fig. 1). During the Iso series, plasma AVP decreased (see above) and urine flow increased to 1.4 ± 0.2 ml/min. Despite the 12-fold elevation of plasma AVP in the Hyper series, urine flow also increased in this series; the increase was about four-fold to 0.62 ± 0.10 ml/min. However, this result reflects an increase in osmolar clearance larger than the concomitant decrease in free water clearance, the changes being +1.12 and -0.66 ml/min, respectively. When isotonic loading was combined with hormone infusion, so that the decrease in plasma AVP or in plasma ANG II was prevented, a time course of urine flow very similar to that of the Hyper series was observed, irrespective of the hormone infused. Remarkably, when ANG II infusion was stopped at t = 90 min, urine flow in the subsequent period increased to levels (1.43 ± 0.17 ml/min) very similar to those observed in the Iso series (1.36 ± 0.19 ml/min).

Sodium excretion. Renal sodium excretion was low and stable in the time control experiments, and increased 95-fold (from 1.6 ± 0.4 to 153 ± 29 µmol/min) in the Iso series (Fig. 2). Estimated on the basis of the changes in plasma oncotic pressure, the hypertonic saline (Hyper series) did not produce the same degree of volume expansion as observed during the Iso series. In addition, the increase in urine flow in the Hyper series was about one-third of that observed in the Iso series. However, the increase in renal sodium excretion was very similar to that of the Iso series. AVP clamping did not alter the natriuretic response to isotonic saline. Remarkably, the angiotensin clamping (Iso+ANG II series) eliminated some 90% of the natriuretic response, as sodium excretion increased from 2.0 ± 0.5 to only 16 ± 9 µmol/min although the concomitant increase in MABP in this series (+13 mmHg) was significantly larger than that of the Iso series (+6 mmHg). In the Iso+stopANG II series, cessation of ANG II infusion was followed by an immediate increase in sodium excretion from 1.5 ± 0.3 to 70 ± 15 µmol/min (n = 4, Fig. 3), concomitant with a clear-cut decrease in MABP (-8 mmHg). The acute effects of ANG II on sodium excretion appear completely dissociated from MABP.


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  		Fig. 3.   Renal sodium excretion as function of mean arterial blood pressure in individual sampling periods before and during saline infusion. Arrows indicate time course within experiments. A: relationships occurring during isotonic infusion (circles), hypertonic infusion (squares), and isotonic infusion with vasopressin (triangles). B: relationships occurring during isotonic infusion during ongoing ANG II infusion (circles) and identical experiments in which the infusion of angiotensin was stopped before last sampling period (squares); time control data are marked by triangles. Symbols represent mean values. * Significant increment in sodium excretion over preceding period. Filled symbols indicate significant increment in blood pressure over preceding period.

Hormone excretion rates. The urinary excretion rates of AVP increased in all series (Table 3). Notably, urinary excretion of AVP also increased in the Iso series where plasma levels of AVP decreased by 35%. No changes were observed in the rates of endothelin-1 excretion in any of the series.

                              
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  		Table 3.   Renal variables


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present results elucidate the importance of endocrine factors and blood pressure for the initial response to an intravenous salt load, which, with respect to amount and rate, is similar to the intestinal absorption of sodium occurring postprandially. The results provide substantial evidence supporting the notions 1) that the renin-angiotensin-aldosterone system plays a pivotal role in the regulation of renal sodium excretion and 2) that this regulation is independent of AVP and, most remarkably, of concomitant changes in MABP.

The design of the experiments included highly standardized conditions with respect to food and water intake as well as animal quarters. Conscious animals were used, because any kind of anesthesia perturbs the osmo- and volume control systems. The dogs were trained for months to ensure sedate, stable control conditions. The potentially disturbing influence of sex hormones was eliminated through bilateral ovariectomy. Chronic catheters were avoided to eliminate the risk of infections and other complications associated with chronic instrumentation. Mild dehydration produced by withdrawal of drinking water 9 h before the experiment was included to reduce spontaneous variations in renal function. In the control periods and throughout the time control experiments, urine osmolality measured ~900-1,200 mosmol/kgH2O, i.e., roughly half-maximal concentration in dogs. The control rates of sodium excretion were low, with mean values ranging from 1.2 to 3.2 µmol/min, corresponding to 2-5 mmol/day. This is less than the salt intake of the dogs (~40 mmol/day), probably because the dogs fasted for 18-20 h before the experiments. In any case, this stable, antinatriuretic state provided a suitable background for the study of natriuretic stimuli.

The NaCl load generated a severe suppression of plasma angiotensin concomitant with a modest increase in arterial blood pressure. The increase in sodium excretion appeared to an overwhelming degree to be controlled by the suppression of ANG II and not by the simultaneous blood pressure elevation. When ANG II was not allowed to decrease in response to saline infusion, sodium excretion increased only marginally, although the concomitant elevation in MABP was exaggerated (see Fig. 3). Furthermore, when the ANG II infusion was turned off during isotonic expansion, sodium excretion increased profusely, although MABP concomitantly decreased to control level. Although indicating dominance of ANG II over blood pressure, the quantitative limitations to these findings must be realized. Plasma ANG II concentrations varied during the different experimental procedures from 0.25 to 2.5 times control, whereas the maximal increase in MABP was 13%. Also, the results only describe the acute interaction between the RAS and blood pressure. It has been shown convincingly in conscious dogs that large, acute increases in renal perfusion pressure (60 mmHg) produce marked natriuresis under conditions where plasma ANG II is maintained by constant infusion (16).

The concept that over days and weeks sodium excretion is determined by changes in arterial blood pressure is based on a large body of evidence (e.g., Ref. 17). However, the present results seem to indicate that this concept should be adjusted to leave room for an acute and dominating role of ANG II as long as blood pressure is close to normal. Figure 3 shows the relationship between MABP and sodium excretion in the present experiments. From the figure, it is apparent that the pressure-natriuresis curve generated by saline infusion is quite flat when physiological levels of ANG II are maintained by infusion of the peptide. During the Hyper, Iso, and Iso+AVP series, remarkably large and remarkably similar increases in sodium excretion were observed concomitant with substantial decreases in plasma ANG II to almost identical levels. However, the blood pressure responses were significantly different. In two series of experiments (Hyper and Iso+AVP), the increase in MABP was approximately double that seen in the Iso series. This observation is difficult to explain by shifting of pressure-natriuresis curves. Rather, it supports the notion that acute, physiological changes in plasma ANG II are capable of controlling sodium excretion even when blood pressure is changed in directions counteracting the antinatriuretic effect of this hormone.

The notion of dominance of the RAS over moderate changes in pressure is supported by results of experiments over 4 days performed in chronically instrumented dogs by Reinhardt and coworkers (20). The experiments included servo-controlled reduction in renal arterial pressure concomitant with ANG II and aldosterone infusion or converting enzyme inhibition. A constant reduction of renal perfusion pressure to 80% of normal induced transient sodium retention, transient elevation in plasma aldosterone, and stabilization of systemic arterial blood pressure 30 mmHg above control after 4 days, i.e., total body sodium and blood pressure were reset to higher, but stable, values, so-called pressure escape (20). The increases in total body sodium and in arterial blood pressure could be prevented by chronic infusion of captopril (4). Maintenance of chronically elevated levels of plasma aldosterone by infusion suppressed the pressure escape (24). Infusion of angiotensin together with aldosterone further suppressed the pressure escape despite continued activation of natriuretic mechanisms such as ANP (24). These results favor the notion that the RAS, also under chronic conditions, controls renal sodium excretion irrespective of the effect of modest changes in renal perfusion pressure. In humans, Singer et al. (25) showed that the increase in sodium excretion after an isotonic saline infusion is markedly inhibited when plasma ANG II is prevented from decreasing by a continuous infusion. Thus, with respect to sodium excretion, the effects of modest changes in blood pressure (e.g., <30%) are easily overridden by changes in the activity of the RAS well within the physiological working range.

The mechanisms involved in the direct, antinatriuretic effect of ANG II may include 1) contraction of glomerular arterioles by modulation of the tubuloglomerular feedback mechanism (afferent) and directly (efferent), 2) reduction of the glomerular ultrafiltration coefficient, 3) change in proximal sodium reabsorption, and 4) inhibition of distal sodium reabsorption directly or indirectly via changes in medullary hemodynamics. Although very clear with regard to the overall antinatriuretic effect, the present results are not suited to elucidate the relative importance of the various intrarenal actions of ANG II.

The role of ANP in normal sodium homeostasis is still uncertain. Results of Zimmerman et al. (26) suggested that during acute volume expansion, ANP plays a role in modulating sodium excretion but that other factors are important during stable volume expansion. Cowley et al. (6) reported data to indicate that ANP may play a minor role during isotonic saline infusion in normal and atrial-resected dogs. In the present study, plasma ANP increased (by a very modest 14%) only during the Iso series. Remarkably, enalapril plus replacement of ANG II in Iso+ANG II series increased the control values of plasma ANP by 47% compared with the Iso series without measurable changes in the control values of sodium excretion. Furthermore, the isotonic saline infusion in Iso+ANG II series increased plasma ANP by an additional 44% from the already elevated control level, whereas sodium excretion barely increased. These results appear incompatible with a significant role of ANP in the initial adjustments of renal sodium excretion triggered by physiological sodium loading.

Recent studies indicated that endothelin-1 may contribute to the regulation of sodium excretion (10, 22). However, the present results do not suggest a relationship between endothelin-1 and sodium excretion. Whether this is a matter of differences in routes of administration of saline (intravenous vs. intracarotid) or in the amounts of sodium infused needs further investigation.

In the present study, the reductions of plasma oncotic pressure might affect the dynamics of glomerular filtration and tubular reabsorption and thereby increase renal sodium excretion. Cowley and Skelton (7) showed that saline infusion to conscious, renal-denervated dogs decreased colloid osmotic pressure and elicited natriuresis in contrast to the same degree of volume expansion with whole blood, during which sodium excretion hardly increased. They suggested that a decrease in colloid osmotic pressure is essential for the natriuresis during volume expansion. Our results indicate that a decrease in plasma oncotic pressure is not sufficient to increase sodium excretion during isotonic expansion. In the Iso+ANG II series, where plasma ANG II was clamped at normotensive levels, only a very small natriuresis was observed concomitant with the usual decrease in plasma protein concentration. The absence of substantial natriuresis after infusion of blood (7) may possibly be related to the amount of ANG II infused with the blood. It is not known how much ANG II is required to inhibit renal sodium excretion. If, in the present study, the natriuresis is due almost exclusively to the severe reductions in plasma and kidney ANG II levels, the quantitative relationship is truly impressive. Control ANG II concentrations were ~17 pg/ml plasma. Decreases in plasma ANG II by ~13 pg/ml were associated with increases in sodium excretion of some 150 µmol/min. The daily sodium intake (approx 40 mmol) corresponds to an average rate of excretion of ~28 µmol/min. If linearity is assumed, it then appears that a constant decrease in ANG II of 2.5 pg/ml plasma would suffice to increase renal sodium excretion by an amount equivalent to that normally ingested. Although the control systems are not linear and the deviations in plasma ANG II concentrations are not constant, the order of magnitude of this result serves to emphasize the power of the RAS under physiological conditions.

The experimental procedures did generate increases in renal perfusion pressure. Therefore, the results may appear less conclusive with regard to the balance between ANG II and blood pressure. Other protocols may provide more convincing evidence. First, the sodium load can be decreased to a magnitude not associated with changes in blood pressure. Second, the loading protocol may be repeated under conditions where renal blood pressure is controlled. This can be obtained by administration of a vasodilator maintaining systemic blood pressure constant, thus avoiding chronic instrumentation, or by constriction of the arteries supplying the kidneys, thus avoiding the systemic use of exogenous substances other than saline. Some of these possibilities have been exploited (1, 21).

The Bainbridge reflex, i.e., an increase in heart rate in response to saline infusion, was described more than 80 years ago (for review, see Ref. 12). In the present study, the reflex appeared very active during volume expansion. However, during infusion of hypertonic saline, which elevated plasma AVP by induction of endogenous secretion or of isotonic saline plus exogenous AVP, the response was completely abolished. These data strongly suggest that AVP inhibits the Bainbridge reflex. It is difficult, however, to analyze further the mechanism involved in this inhibition on the basis of the present data.

In summary, this study shows that during concomitant, physiological deviations in 1) extracellular fluid volume, 2) arterial blood pressure, and 3) concentrations in plasma of ANG II and AVP, acute changes in sodium excretion are predominantly determined by ANG II. In addition, the results leave little room for a substantial role for neither other hormones, such as AVP, ANP, endothelin-1, and urodilatin, nor for physical factors, e.g., colloid osmotic pressure.

Perspectives

The multiple mechanisms involved in the physiological regulation of sodium excretion may be thought of as a set of boxes within boxes, the innermost box representing the primary control system of highest precision. So far this primary regulation, and, as such, the first line of defense toward sodium loading, has not been unquestionably identified. The present data point toward the intrarenal actions of ANG II as the effector part of the primary control system. Changes in activity of the RAS within 15% as estimated from the changes in plasma ANG II may severely influence renal sodium excretion. It is tempting to suggest that for small pressure changes, the so-called pressure natriuresis mechanism is an expression of changes in the intrarenal actions of ANG II and that the apparently "infinite gain" (13, 14) is due to the extreme sensitivity and lack of adaptation of this response. It is not known, however, whether the controlled variable is intra- or extrarenal. Therefore, it is difficult to hypothesize whether the sensory mechanism is sensitive to extracellular fluid volume (extrarenal) or to an intratubular solute concentration (intrarenal). Studies identifying the controlled variable, the involved sensory mechanism, and the hierarchy of the intrarenal actions of ANG II seem warranted.


    ACKNOWLEDGEMENTS

The expert technical assistance of Sigurd K. Hansen in the dog laboratory and of Birthe Lynderup Christensen, Trine Eidsvold, Inge H. Pedersen, and Barbara Sørensen with the analyses is gratefully appreciated. AVP was received as a generous gift from Dr. Hans Vilhardt, Department of Medical Physiology, University of Copenhagen. Aprotinine was kindly provided by Novo Nordisk. Dr. H. W. Reinhardt and Dr. Ole Skøtt provided valuable constructive comments to the manuscript.


    FOOTNOTES

The work was supported by grants from The Danish Medical Research Council, the Novo Nordisk Foundation, and the Velux Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. Bie, Dept. of Physiology and Pharmacology, Odense University, 21 Winsløwparken, DK-5000 Odense C, Denmark (E-mail: bie{at}imbmed.sdu.dk).

Received 15 March 1999; accepted in final form 26 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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