European JournalofPharmacology, 218 (1992) 319-325
319
© 1992 Elsevier Science Publishers B.V. AII rights reserved 0014-2999/92/$05.00
EJP 52572
The influence of changes in perfusion pressure and angiotensin II on the renal excretory responses to atrial natriuretic peptides A n d r z e j L. C h a m i e n i a 1 a n d E d w a r d J. J o h n s Department of Physiology, The Medical School, Birmingham B15 2T'1], UK
Received 6 January 1992; revised MS received 7 April 1992, accepted 12 May 1992
The renal actions of atriopeptin III were examined in anaesthetised rats at differing perfusion pressures before and following blockade of the renin-angiotensin system. At normal perfusion pressure 1000 ng kg i atriopeptin III caused reversible increases in glomerular filtration rate, of 20%, urine flow, absolute and fractional sodium excretions of 51-93%. Reduction of left renal perfusion pressure to 80 mm Hg decreased glomerular filtration rate by 30% and urine flow, absolute and fractional sodium excretions by 80% while atriopeptin III administration only minimally changed these variables. Concomitantly, right kidney perfusion pressure rose by 15 mm Hg which significantly increased fluid output, while the atriopeptin III induced diuresis and natriuresis were significantly larger. During infusion of captopril 900/zg kg-1 h-a when pressures at the left and right kidneys had been reduced and elevated, respectively, atriopeptin III caused larger excretory responses in both kidneys which were greater than without captopril. These result suggested that the atriopeptin III mediated natriuresis and diuresis were directly proportional to perfusion pressure and attenuated by angiotensin II. Atrial peptides; Natriuresis; Renal perfusion pressure; Angiotensin II
I. Introduction Atrial natriuretic peptides released from the heart have potent renal effects whereby they increase the excretion of both water and sodium (Needleman et al., 1989). Exactly how these compounds exert their action within the kidney is not clear, but autoradiographic studies have indicated high densities of receptors at both the glomerulus and the inner medulla (Healey and Fanistil, 1986; Koseki et al., 1986). There is now good functional evidence that high doses of these peptides can increase glomerular filtration rate which is considered to be due to dilation of glomerular mesangial cells causing increased hydraulic conductivity (Harris and Skinner, 1990) as well as to dilation of afferent and constriction of efferent arterioles (Kimura et al., 1990). However, even at low doses, which have no overt vascular effects, the atrial peptides can induce a diuresis and natriuresis. The microperfusion studies of Sonnenburg et al. (1986), Rocha and Kudo (1990) and
Correspondence to: E.J. Johns, Department of Physiology,The Medical School, Birmingham B15 2TT, UK. Tel. 44.21.414 6900, fax 44.21.414 6924. 1 Permanent address: Department of Nephrology, The Medical Academy, Debinki 7A, 80-211 Gdansk, Poland.
Schulz-Knappe et al., (1990) have shown that the peptides inhibit sodium chloride reabsorption at the inner medullary collecting tubule. However, whether the peptides have an action on sodium transport by the proximal tubules is still unclear (Bruun et al., 1991). The degree of natriuresis induced by the atrial peptides appear very variable to the extent that their physiological role has been questioned and debated (Goetz, 1990; Blaine, 1990), however, it is becoming apparent that a number of factors can determine and modulate the effectiveness of the peptides at the kidney. There is accumulating evidence from the reports of Bie et al. (1990), Salazar et al. (1987), Showalter et al. (1988) and Siragy et al. (1988) in the dog and from our own work in the rat (Chamienia and Johns, 1991) that angiotensin II can suppress the natriuretic response induced by the peptides, although the exact way in which they interact, at a glomerular or tubular site, has not been convincingly determined to date. A further factor modulating this system appears to be the perfusion pressure at the kidney. Early studies by Davis and Briggs (1987) in the rat and Seymour et al. (1987) in the dog showed that as renal perfusion pressure was reduced, the natriuretic response to the atrial peptides was severely curtailed and this was supported by a similar report in the dog by Paul et al. (1987), who reduced renal perfusion pressure both within and be-
320 yond the autoregulatory range. These results were also supported by the observations of Firth et al. (1988) in isolated perfused rat kidney. The conclusions drawn from these studies have to be qualified insofar as pressure reduction to 80 m m Hg, which is just below the autoregulatory limit for the rat kidney (Arendshorst, 1979), would promote the release of renin (Kircheim et al., 1987) and angiotensin II levels would rise in both the systemic circulation as well as the kidney itself. Therefore, it is not clear from the studies by Davis and Briggs (1987), Paul et al. (1987) and Seymour et al. (1987) whether the reduced renal excretory responses to the atrial peptides were a consequence of the reduced pressure or due to the raised production of angiotensin II or was the result of a combination of these two factors. This question was addressed in the present study by comparing the natriuretic response to atrial natriuretic peptides at prevailing pressure and when it was either reduced or raised at the kidney. An attempt was then made to define the role of the angiotensin II by undertaking studies in the presence of the converting enzyme inhibitor captopril. The results demonstrated that the renal excretory responses to atriopeptin III were pressure-dependent while the contribution of angiotensin II could still be demonstrated under these experimental conditions.
2. Materials and methods
2.1. Acute experiments The experiments were performed on male Wistar rats (mean body weight 350 g) which had been fasted overnight. The animals were anaesthetised with pentobarbitone sodium, 60 mg kg -1 body weight i.p. and maintained with a constant infusion of pentobarbitone sodium (12 mg kg -1 h - l ) . A tracheostomy was performed and polyethylene catheters were placed in the right carotid artery for the m e a s u r e m e n t of systemic arterial pressure and obtaining blood samples, and in the left femoral vein for experimental infusions. The intravenous infusion of isotonic saline (3 ml h -1) was commenced immediately and continued for the duration of the experiment. The left kidney was exposed retroperitoneally, its ureter cannulated for urine collection, and the renal artery was carefully cleared and a flowmeter probe (2.0-2.5 m m circumference, EP100 series, Carolina Medical Instruments) was placed around the artery. Especial care was exerted to avoid damage to the renal nerves. The functional integrity of the nerves was tested by applying silver wire electrodes to the coeliac/aortico-renal ganglia through which pulses of 15 V, 10 Hz and 0.2 ms were delivered for 10 s. This caused a transient fall in renal blood flow and
blanching of the kidney. If this did not occur, the kidney was considered denervated and not included in the study. Via a flank incision, the right ureter was isolated and cannulated to enable collection of urine from the right kidney. A further cannula was inserted into the abdominal aorta via the femoral artery to record left renal perfusion pressure. A thread was placed around the aorta between the two renal arteries and it was attached to a screw device such that when tightened, pressure at the left kidney was reduced which led to a rise in systemic blood pressure and therefore the perfusion pressure at which the right kidney was operating. The arterial catheters were connected to a pressure transducer (Statham P231D) and renal blood flow was measured with square wave electromagnetic flowmeter (FM 501, Carolina Medical Instruments). Both arterial pressure and renal blood flow were continuously recorded on a Grass polygraph (Model 7D, Grass Instruments, USA). After surgery was completed, a priming dose of 2 ml inulin in saline (1.5 g 100 m1-1) was given intravenously and isotonic saline infusion was replaced with one containing inulin (1.5 g 100 m l - l ) . The animals were allowed to equilibrate for two hours and then the clearance studies were undertaken.
2.2. Experimental protocols Ten clearance periods, 15 min each, were performed in two series of five clearances. Each series comprised two basal clearance periods followed by one experimental clearance, after which 15 min of stabilisation was allowed and then recovery clearances were performed. After the first series of clearances was completed, the aorta was constricted such that left renal perfusion was reduced to approximately 80 m m Hg and it was maintained at that level until the end of the experiment. Twenty five minutes later the second series of five clearance periods were undertaken. Arterial blood samples (300 izl) were taken before the first and the third and after completion of the third and the fifth clearance period in each series. The blood sample was immediately centrifuged and plasma obtained, the red ceils were resuspended in an equivalent volume of heparinised saline and reinfused into the animal. A 5 min equilibration period was allowed before the next collection period commenced. Urine was collected in pre-weighed microcentrifuge capped tubes. Atriopeptin III ( A N F 5-28, Cambridge Research Biochemicals Limited, Cambridge, England) was given as a bolus dose of 1000 ng kg-1 in 0.3 ml normal saline intravenously 2 min before experimental clearances. Two groups of animals were studied; the first group (n = 7) received the inulin in saline infusion only; in the second group of animals (n = 10), captopril was added to inulin/saline infusate at a concentration such
321
that it was delivered into t h e animal at 900 /xg kgh -1. The captopril infusion was begun after the first set of clearances were taken and was continued for 25 min before the second set of clearances were started. Bolus intravenous doses of angiotensin I (200 ng) and angiotensin II (100 ng) were given and the vasopressor and renal vasoconstrictor responses determined. These injections were repeated 20 min after the start of captopril infusion when it was found that the vasopressor and renal vasoconstrictor responses to angiotensin I, but not angiotensin II, were reduced by over 95%.
2.3. Statistical analysis All values are presented as means + S.E.M. The mean values of two basal and two recovery clearance periods were calculated for each series of five clearances and were considered as control values which were compared to the experimental value obtained during the influence of atriopeptin III. The statistical analysis was performed with analysis of variance (Sup e r A N O V A Software package for Apple Macintosh) followed by post-hoc tests (Games-Howell) for significant effects. The differences were taken to be significant when P < 0.05.
3. Results
The effect of atriopeptin III on blood pressure and renal function before and then during perfusion pressure manipulation in the first group of animals is shown in table 1. Atriopeptin III had no effect on blood pressure, heart rate or left renal blood flow in either the first or second part of the study. At the left kidney the first test of atriopeptin III caused a re-
versible increase in glomerular filtration rate, of 20% (P < 0.05) which was associated with significant increases in urine flow (P < 0.001), absolute sodium excretion (P < 0.01), and fractional sodium excretion (P < 0.001). Pressure reduction at the left kidney to 80 mm Hg caused a significant (P < 0.001) fall in glomerular filtration rate of some 30%, while basal levels of urine flow, absolute and fractional sodium excretions were markedly reduced (P < 0.001) by some 80%. Administration of the second dose of atriopeptin III caused small increases in left urine flow and absolute sodium excretion and small reduction in fractional sodium excretion which did not reach statistical significance and were much smaller than the responses obtained at the normal renal pressure and a comparison of the responses are shown in fig. 1. The basal level of glomerular filtration rate and excretory variables of the right kidney were very comparable to those of the left kidney (table 1). The right kidney responded to the first test of atriopeptin III with non-significant rise in glomerular filtration rate, of approximately 19%, and reversible increases (all P < 0.001) in urine flow, absolute and fractional sodium excretions which were not statistically different in magnitude to those recorded in the left kidney. The constriction of the aorta to reduce perfusion pressure at the left kidney increased pressure at the right kidney by approximately 20 mm Hg (P < 0.001) which caused an approximate doubling of basal levels of urine flow, absolute and fractional sodium excretions (all P < 0.001). Under these conditions, the second dose of atriopeptin III had no effect on right kidney glomerular filtration rate, but caused significant (all P < 0.001) reversible increases in urine flow, absolute and fractional sodium excretions which were significantly larger (all P < 0.001) than during the first atriopeptin III
TABLE 1 Effect of two doses of atriopeptin III, 1000 ng k g - 1, on blood pressure and renal function before and during reduction in perfusion pressure to 80 mm Hg at the left kidney. Basal 1
Exptl 1
Rec 1
Basal 2
Exptl 2
Rec 2
117 + 2 5.1 + 0.4 318 + 10 1.2 _+ 0.2 8.5 ± 1.4 1.79+ 0.2 1.04+ 0.12
112 +2 5.2 +0.5 307 ±9 1.4 ±0.1 a 15.9 _+2 c 3.41 ±0.31 b 1.72+0.18 c
114 +0 5.0 +0.5 310 ±9 1.1 ±0.1 8.7 +1.3 1.98_+0.22 1.31±0.17
80 + 0 4.4 ± 0.3 313 ± 14 0.8 _+ 0.1 2.0 ± 0.3 0.17_+ 0.04 0.15± 0.04
79 ± 0 4.3 ± 0.3 311 + 15 0.9 ± 0.1 2.4 + 0.5 0.19± 0.06 0.14_+ 0.04
80 ± 0 4.4 + 0.3 313 +_ 14 0.8 + 0.1 2.2 ± 0.6 0.18+ 0.08 0.15 ± 0.04
121 + 1.3 + 6.4 + 1.45+ 0.77-+
117 _+2 1.4 _+0.1 13.5 ±2.3 c 3.13_+0.38 c 1.56±0.19 c
117 _+1 1.1 _+0.1 7.1 +1.3 1.69-+0.31 1.15+0.24
136 _+ 3 1.2 _+ 0.1 15.7 _+ 3.5 3.94± 0.65 2.44_+ 0.38
132 _+ 3 125 + 1.3 ± 0.1 1.2 + 32.3 _+ 6.6 c 17.1 + 7.50+ 1.30 c 4.40+ 3.99_+ 0.61 c 2.66+
Left kidney Perfusionpressure, m m H g Renalbloodflow, m l m i n - X g -1 Heart rate, b m i n - t Glomerular filtration rate, m l m i n - l g i Urineflowrate,/~lmin-lg I Absolute sodium excretion,/.Lmol min -1 g-~ Fractional sodium excretion, %
Right kidney Perfusion pressure, mm Hg Glomerular filtration rate, m l m i n - l g i U r i n e f l o w r a t e , / x l m i n - l g -1 Absolute sodium excretion,/zmol min 1 g-1 Fractional sodium excretion, %
2 0.1 0.9 0.19 0.08
Signifcantly different from respective control values, a p < 0.05, b p < 0.01, c p < 0.001.
2 0.1 3.2 0.64 0.36
322
A UV p.I min "lg -1
8-
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**
I I
10"
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64 2
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gruel r a i n - l g - 1
1.5.
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0.0
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AFE Na %
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NORMAL PRESSURE
REDUCED PRESSURE
Fig. 1. This figure presents the changes in urine flow (AUV), absolute sodium excretion (AUNaV) and fractional sodium excretion (AFENa) in the left kidney after intravenous bolus of atriopeptin III, 1000 ng kg-1 at normal and reduced pressures in the group infused with vehicle throughout (open histograms, n = 7) or given captopril, 900 ~g kg - I h - I , in the second half of the experiment (slashed histograms, n = 10). * P < 0.05; * * P < 0.01.
challenge: for urine flow, 15.86 + 4.13 versus 6.81 + 1.51 /xl min -1 g - l ; absolute sodium excretion, 3.33 + 0.99 versus 1.56 + 0.23/xmol min-1 g-1; and fractional sodium excretion 1.44 + 0.60 versus 0.59 + 0.18%. A comparison of the responses are shown in fig. 2. Table 2 contains the data obtained from the second group of rats in which the converting enzyme inhibitor was infused during the second part of the study when perfusion pressure to the left kidney was reduced to 80 m m Hg. Blood pressure, heart rate and left renal blood flow were not affected to any great extent by the administration of the first dose of atriopeptin III. In the first part of the study, the atriopeptin III caused a small 10% rise in left kidney glomerular filtration rate which was associated with significant reversible increases in urine flow (P < 0.001), absolute sodium excretion (P < 0.01) and fractional sodium excretion (P < 0.001) which were similar in magnitude to those observed in the first group of rats. During the second part of the study, administration of the captopril and concomitant reduction in left renal perfusion pressure to 80 m m Hg, was associated with a reduction in basal glomerular filtration rate of some 30% and much larger decreases in urine volume, absolute and fractional sodium excretions (all P < 0.001) of some 70-80%. Administration of the second dose of atriopeptin III under these conditions increased left glomerular filtration rate by 40% (P < 0.01) which was associated with a significant (P < 0.05) increase in urine volume and increases in absolute and fractional sodium excretions which just failed to reach statistical significance. Although these tubular responses were smaller than those obtained in the first part of the study, they were larger than those obtained at 80 m m Hg in the absence of converting enzyme inhibitor (table 1), i.e. for urine flow
TABLE 2 Effect of two doses of atriopeptin III, 1000 ng kg 1, on blood pressure and renal function before and during reduction in perfusion pressure to 80 mm Hg at the left kidney and converting enzyme inhibition with captopril, 900 p.g k g - ] h 1. Basal 1
Exptl 1
Rec 1
Captopril 900/zg k g - 1 h - 1 Basal 2
Exptl 2
Rec 2
80 _+ 0 4.7 _+ 0.5 298 + 10 0.7 + 0.08 1.7 4- 0.3 0.17+ 0.09 0.19_+ 0.10
80 + 0 80 _+ 0 4.7 _+ 0.4 4.7 _+ 0.5 298 _+13 306 _+12 1.0 -+ 0.08 b 0.6 + 0.05 3.7 -+ 0.9 a 1.2 -+ 0.2 0.53+ 0.256 0.09_+ 0.02 0.34_+ 0.13 0.11_+ 0.03
126 + 2 1.2 + 0.1 26.1 + 2.7 7.90+ 0.80 4.93_+ 0.48
121 + 2 121 + 3 1.3 + 0.1 1.2 + 0.1 52.6 + 4.3 c 23.7 + 1.6 15.05_+ 1.1 c 7.67_+ 0.48 8.92_+ 0.86 c 4.88_+ 0.26
Left kidney Perfusion pressure, mm Hg Renal blood flow, ml m i n - 1 g - 1 Heart rate, b m i n - 1 Glomerular filtration rate, ml m i n - t g-1 Urine flow rate,/zl min -1 g-1 Absolute sodium excretion, g mol m i n - t g-1 Fractional sodium excretion, %
112 _+ 1 4.4 _+ 0.4 293 _+10 1.1 _+ 0.2 6.4 _+ 0.6 1.50 + 0.22 0.94 + 0.12
111 _+ 2 111 _+ 1 4.5 _+ 0.4 4.6 + 0.4 281 _+17 278 + 12 1.2 _+ 0.04 1.1 _+ 0.05 14.4 -+ 1.1 c 5.9 -+ 0.6 3.25+_ 0.29 c 1.41-+ 0.15 1.88+ 0.16 c 0.95+ 0.09
Right kidney Perfusion pressure, mm Hg 115 _+ Glomerular filtration rate, ml m i n - i g-1 1.1 _+ Urine flow rate, ~1 min - t g-1 6.9 + Absolute sodium excretion,/z mol m i n - 1 g - 1 1.62 -+ Fractional sodium excretion, % 1.06+
1 0.0 0.5 0.15 0.11
1 114 _+ 0.1 1.1 + 2.0 c 6.5 + 0.47 c 1.60+ 2.33+ 0.36 c 1.10+
114 + 1.2 + 16.6 + 3.79+
Significantly different from respective control values, a p < 0.05, b p < 0.01, c p < 0.001.
2 0.1 0.5 0.15 0.12
323
40-
I
**
I
AUV p.I min" I g . 1
["**"1
30. 20 100 I
AU
V Na I~mol rain" 1 g- 1
8-
glomerular filtration rate to increase transiently by 8%, while at the same time the magnitude of reversible rises in urine flow, absolute and fractional sodium excretions were significantly larger than those obtained in the first part of the study (P < 0.001), and from those obtained at the higher pressure when captopril was not given (P < 0.01). A comparison of these responses is shown in fig. 2.
* - - 1 V**'I •T .
4. Discussion
//// ///.,
64
T
i
2.
i/// ,-//., ,-i/~ ////
//// ////
0
AFE Na
5.0-
I- ** -I
-
4.0-
i///
2.
3.02.01.00.~ NORMAL PRESSURE
RAISED PRESSURE
Fig. 2. This figure presents the changes in urine flow (AUV), absolute sodium excretion (AUrqaV) and fractional sodium excretion (AFENa) in the right kidney after intravenous bolus of atriopeptin lII, 1000 ng kg-1 at normal and increased pressures in the group infused with vehicle throughout (open histograms, n = 7) or given captopril, 900 p.g kg -1 h -1, in the second half of the experimen[ (slashed histograms, n = 10). * P < 0.05; ** P < 0.01.
2.2+0.7 versus 0.31_+0.13 /~1 min -1 g - t (p
319
© 1992 Elsevier Science Publishers B.V. AII rights reserved 0014-2999/92/$05.00
EJP 52572
The influence of changes in perfusion pressure and angiotensin II on the renal excretory responses to atrial natriuretic peptides A n d r z e j L. C h a m i e n i a 1 a n d E d w a r d J. J o h n s Department of Physiology, The Medical School, Birmingham B15 2T'1], UK
Received 6 January 1992; revised MS received 7 April 1992, accepted 12 May 1992
The renal actions of atriopeptin III were examined in anaesthetised rats at differing perfusion pressures before and following blockade of the renin-angiotensin system. At normal perfusion pressure 1000 ng kg i atriopeptin III caused reversible increases in glomerular filtration rate, of 20%, urine flow, absolute and fractional sodium excretions of 51-93%. Reduction of left renal perfusion pressure to 80 mm Hg decreased glomerular filtration rate by 30% and urine flow, absolute and fractional sodium excretions by 80% while atriopeptin III administration only minimally changed these variables. Concomitantly, right kidney perfusion pressure rose by 15 mm Hg which significantly increased fluid output, while the atriopeptin III induced diuresis and natriuresis were significantly larger. During infusion of captopril 900/zg kg-1 h-a when pressures at the left and right kidneys had been reduced and elevated, respectively, atriopeptin III caused larger excretory responses in both kidneys which were greater than without captopril. These result suggested that the atriopeptin III mediated natriuresis and diuresis were directly proportional to perfusion pressure and attenuated by angiotensin II. Atrial peptides; Natriuresis; Renal perfusion pressure; Angiotensin II
I. Introduction Atrial natriuretic peptides released from the heart have potent renal effects whereby they increase the excretion of both water and sodium (Needleman et al., 1989). Exactly how these compounds exert their action within the kidney is not clear, but autoradiographic studies have indicated high densities of receptors at both the glomerulus and the inner medulla (Healey and Fanistil, 1986; Koseki et al., 1986). There is now good functional evidence that high doses of these peptides can increase glomerular filtration rate which is considered to be due to dilation of glomerular mesangial cells causing increased hydraulic conductivity (Harris and Skinner, 1990) as well as to dilation of afferent and constriction of efferent arterioles (Kimura et al., 1990). However, even at low doses, which have no overt vascular effects, the atrial peptides can induce a diuresis and natriuresis. The microperfusion studies of Sonnenburg et al. (1986), Rocha and Kudo (1990) and
Correspondence to: E.J. Johns, Department of Physiology,The Medical School, Birmingham B15 2TT, UK. Tel. 44.21.414 6900, fax 44.21.414 6924. 1 Permanent address: Department of Nephrology, The Medical Academy, Debinki 7A, 80-211 Gdansk, Poland.
Schulz-Knappe et al., (1990) have shown that the peptides inhibit sodium chloride reabsorption at the inner medullary collecting tubule. However, whether the peptides have an action on sodium transport by the proximal tubules is still unclear (Bruun et al., 1991). The degree of natriuresis induced by the atrial peptides appear very variable to the extent that their physiological role has been questioned and debated (Goetz, 1990; Blaine, 1990), however, it is becoming apparent that a number of factors can determine and modulate the effectiveness of the peptides at the kidney. There is accumulating evidence from the reports of Bie et al. (1990), Salazar et al. (1987), Showalter et al. (1988) and Siragy et al. (1988) in the dog and from our own work in the rat (Chamienia and Johns, 1991) that angiotensin II can suppress the natriuretic response induced by the peptides, although the exact way in which they interact, at a glomerular or tubular site, has not been convincingly determined to date. A further factor modulating this system appears to be the perfusion pressure at the kidney. Early studies by Davis and Briggs (1987) in the rat and Seymour et al. (1987) in the dog showed that as renal perfusion pressure was reduced, the natriuretic response to the atrial peptides was severely curtailed and this was supported by a similar report in the dog by Paul et al. (1987), who reduced renal perfusion pressure both within and be-
320 yond the autoregulatory range. These results were also supported by the observations of Firth et al. (1988) in isolated perfused rat kidney. The conclusions drawn from these studies have to be qualified insofar as pressure reduction to 80 m m Hg, which is just below the autoregulatory limit for the rat kidney (Arendshorst, 1979), would promote the release of renin (Kircheim et al., 1987) and angiotensin II levels would rise in both the systemic circulation as well as the kidney itself. Therefore, it is not clear from the studies by Davis and Briggs (1987), Paul et al. (1987) and Seymour et al. (1987) whether the reduced renal excretory responses to the atrial peptides were a consequence of the reduced pressure or due to the raised production of angiotensin II or was the result of a combination of these two factors. This question was addressed in the present study by comparing the natriuretic response to atrial natriuretic peptides at prevailing pressure and when it was either reduced or raised at the kidney. An attempt was then made to define the role of the angiotensin II by undertaking studies in the presence of the converting enzyme inhibitor captopril. The results demonstrated that the renal excretory responses to atriopeptin III were pressure-dependent while the contribution of angiotensin II could still be demonstrated under these experimental conditions.
2. Materials and methods
2.1. Acute experiments The experiments were performed on male Wistar rats (mean body weight 350 g) which had been fasted overnight. The animals were anaesthetised with pentobarbitone sodium, 60 mg kg -1 body weight i.p. and maintained with a constant infusion of pentobarbitone sodium (12 mg kg -1 h - l ) . A tracheostomy was performed and polyethylene catheters were placed in the right carotid artery for the m e a s u r e m e n t of systemic arterial pressure and obtaining blood samples, and in the left femoral vein for experimental infusions. The intravenous infusion of isotonic saline (3 ml h -1) was commenced immediately and continued for the duration of the experiment. The left kidney was exposed retroperitoneally, its ureter cannulated for urine collection, and the renal artery was carefully cleared and a flowmeter probe (2.0-2.5 m m circumference, EP100 series, Carolina Medical Instruments) was placed around the artery. Especial care was exerted to avoid damage to the renal nerves. The functional integrity of the nerves was tested by applying silver wire electrodes to the coeliac/aortico-renal ganglia through which pulses of 15 V, 10 Hz and 0.2 ms were delivered for 10 s. This caused a transient fall in renal blood flow and
blanching of the kidney. If this did not occur, the kidney was considered denervated and not included in the study. Via a flank incision, the right ureter was isolated and cannulated to enable collection of urine from the right kidney. A further cannula was inserted into the abdominal aorta via the femoral artery to record left renal perfusion pressure. A thread was placed around the aorta between the two renal arteries and it was attached to a screw device such that when tightened, pressure at the left kidney was reduced which led to a rise in systemic blood pressure and therefore the perfusion pressure at which the right kidney was operating. The arterial catheters were connected to a pressure transducer (Statham P231D) and renal blood flow was measured with square wave electromagnetic flowmeter (FM 501, Carolina Medical Instruments). Both arterial pressure and renal blood flow were continuously recorded on a Grass polygraph (Model 7D, Grass Instruments, USA). After surgery was completed, a priming dose of 2 ml inulin in saline (1.5 g 100 m1-1) was given intravenously and isotonic saline infusion was replaced with one containing inulin (1.5 g 100 m l - l ) . The animals were allowed to equilibrate for two hours and then the clearance studies were undertaken.
2.2. Experimental protocols Ten clearance periods, 15 min each, were performed in two series of five clearances. Each series comprised two basal clearance periods followed by one experimental clearance, after which 15 min of stabilisation was allowed and then recovery clearances were performed. After the first series of clearances was completed, the aorta was constricted such that left renal perfusion was reduced to approximately 80 m m Hg and it was maintained at that level until the end of the experiment. Twenty five minutes later the second series of five clearance periods were undertaken. Arterial blood samples (300 izl) were taken before the first and the third and after completion of the third and the fifth clearance period in each series. The blood sample was immediately centrifuged and plasma obtained, the red ceils were resuspended in an equivalent volume of heparinised saline and reinfused into the animal. A 5 min equilibration period was allowed before the next collection period commenced. Urine was collected in pre-weighed microcentrifuge capped tubes. Atriopeptin III ( A N F 5-28, Cambridge Research Biochemicals Limited, Cambridge, England) was given as a bolus dose of 1000 ng kg-1 in 0.3 ml normal saline intravenously 2 min before experimental clearances. Two groups of animals were studied; the first group (n = 7) received the inulin in saline infusion only; in the second group of animals (n = 10), captopril was added to inulin/saline infusate at a concentration such
321
that it was delivered into t h e animal at 900 /xg kgh -1. The captopril infusion was begun after the first set of clearances were taken and was continued for 25 min before the second set of clearances were started. Bolus intravenous doses of angiotensin I (200 ng) and angiotensin II (100 ng) were given and the vasopressor and renal vasoconstrictor responses determined. These injections were repeated 20 min after the start of captopril infusion when it was found that the vasopressor and renal vasoconstrictor responses to angiotensin I, but not angiotensin II, were reduced by over 95%.
2.3. Statistical analysis All values are presented as means + S.E.M. The mean values of two basal and two recovery clearance periods were calculated for each series of five clearances and were considered as control values which were compared to the experimental value obtained during the influence of atriopeptin III. The statistical analysis was performed with analysis of variance (Sup e r A N O V A Software package for Apple Macintosh) followed by post-hoc tests (Games-Howell) for significant effects. The differences were taken to be significant when P < 0.05.
3. Results
The effect of atriopeptin III on blood pressure and renal function before and then during perfusion pressure manipulation in the first group of animals is shown in table 1. Atriopeptin III had no effect on blood pressure, heart rate or left renal blood flow in either the first or second part of the study. At the left kidney the first test of atriopeptin III caused a re-
versible increase in glomerular filtration rate, of 20% (P < 0.05) which was associated with significant increases in urine flow (P < 0.001), absolute sodium excretion (P < 0.01), and fractional sodium excretion (P < 0.001). Pressure reduction at the left kidney to 80 mm Hg caused a significant (P < 0.001) fall in glomerular filtration rate of some 30%, while basal levels of urine flow, absolute and fractional sodium excretions were markedly reduced (P < 0.001) by some 80%. Administration of the second dose of atriopeptin III caused small increases in left urine flow and absolute sodium excretion and small reduction in fractional sodium excretion which did not reach statistical significance and were much smaller than the responses obtained at the normal renal pressure and a comparison of the responses are shown in fig. 1. The basal level of glomerular filtration rate and excretory variables of the right kidney were very comparable to those of the left kidney (table 1). The right kidney responded to the first test of atriopeptin III with non-significant rise in glomerular filtration rate, of approximately 19%, and reversible increases (all P < 0.001) in urine flow, absolute and fractional sodium excretions which were not statistically different in magnitude to those recorded in the left kidney. The constriction of the aorta to reduce perfusion pressure at the left kidney increased pressure at the right kidney by approximately 20 mm Hg (P < 0.001) which caused an approximate doubling of basal levels of urine flow, absolute and fractional sodium excretions (all P < 0.001). Under these conditions, the second dose of atriopeptin III had no effect on right kidney glomerular filtration rate, but caused significant (all P < 0.001) reversible increases in urine flow, absolute and fractional sodium excretions which were significantly larger (all P < 0.001) than during the first atriopeptin III
TABLE 1 Effect of two doses of atriopeptin III, 1000 ng k g - 1, on blood pressure and renal function before and during reduction in perfusion pressure to 80 mm Hg at the left kidney. Basal 1
Exptl 1
Rec 1
Basal 2
Exptl 2
Rec 2
117 + 2 5.1 + 0.4 318 + 10 1.2 _+ 0.2 8.5 ± 1.4 1.79+ 0.2 1.04+ 0.12
112 +2 5.2 +0.5 307 ±9 1.4 ±0.1 a 15.9 _+2 c 3.41 ±0.31 b 1.72+0.18 c
114 +0 5.0 +0.5 310 ±9 1.1 ±0.1 8.7 +1.3 1.98_+0.22 1.31±0.17
80 + 0 4.4 ± 0.3 313 ± 14 0.8 _+ 0.1 2.0 ± 0.3 0.17_+ 0.04 0.15± 0.04
79 ± 0 4.3 ± 0.3 311 + 15 0.9 ± 0.1 2.4 + 0.5 0.19± 0.06 0.14_+ 0.04
80 ± 0 4.4 + 0.3 313 +_ 14 0.8 + 0.1 2.2 ± 0.6 0.18+ 0.08 0.15 ± 0.04
121 + 1.3 + 6.4 + 1.45+ 0.77-+
117 _+2 1.4 _+0.1 13.5 ±2.3 c 3.13_+0.38 c 1.56±0.19 c
117 _+1 1.1 _+0.1 7.1 +1.3 1.69-+0.31 1.15+0.24
136 _+ 3 1.2 _+ 0.1 15.7 _+ 3.5 3.94± 0.65 2.44_+ 0.38
132 _+ 3 125 + 1.3 ± 0.1 1.2 + 32.3 _+ 6.6 c 17.1 + 7.50+ 1.30 c 4.40+ 3.99_+ 0.61 c 2.66+
Left kidney Perfusionpressure, m m H g Renalbloodflow, m l m i n - X g -1 Heart rate, b m i n - t Glomerular filtration rate, m l m i n - l g i Urineflowrate,/~lmin-lg I Absolute sodium excretion,/.Lmol min -1 g-~ Fractional sodium excretion, %
Right kidney Perfusion pressure, mm Hg Glomerular filtration rate, m l m i n - l g i U r i n e f l o w r a t e , / x l m i n - l g -1 Absolute sodium excretion,/zmol min 1 g-1 Fractional sodium excretion, %
2 0.1 0.9 0.19 0.08
Signifcantly different from respective control values, a p < 0.05, b p < 0.01, c p < 0.001.
2 0.1 3.2 0.64 0.36
322
A UV p.I min "lg -1
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NORMAL PRESSURE
REDUCED PRESSURE
Fig. 1. This figure presents the changes in urine flow (AUV), absolute sodium excretion (AUNaV) and fractional sodium excretion (AFENa) in the left kidney after intravenous bolus of atriopeptin III, 1000 ng kg-1 at normal and reduced pressures in the group infused with vehicle throughout (open histograms, n = 7) or given captopril, 900 ~g kg - I h - I , in the second half of the experiment (slashed histograms, n = 10). * P < 0.05; * * P < 0.01.
challenge: for urine flow, 15.86 + 4.13 versus 6.81 + 1.51 /xl min -1 g - l ; absolute sodium excretion, 3.33 + 0.99 versus 1.56 + 0.23/xmol min-1 g-1; and fractional sodium excretion 1.44 + 0.60 versus 0.59 + 0.18%. A comparison of the responses are shown in fig. 2. Table 2 contains the data obtained from the second group of rats in which the converting enzyme inhibitor was infused during the second part of the study when perfusion pressure to the left kidney was reduced to 80 m m Hg. Blood pressure, heart rate and left renal blood flow were not affected to any great extent by the administration of the first dose of atriopeptin III. In the first part of the study, the atriopeptin III caused a small 10% rise in left kidney glomerular filtration rate which was associated with significant reversible increases in urine flow (P < 0.001), absolute sodium excretion (P < 0.01) and fractional sodium excretion (P < 0.001) which were similar in magnitude to those observed in the first group of rats. During the second part of the study, administration of the captopril and concomitant reduction in left renal perfusion pressure to 80 m m Hg, was associated with a reduction in basal glomerular filtration rate of some 30% and much larger decreases in urine volume, absolute and fractional sodium excretions (all P < 0.001) of some 70-80%. Administration of the second dose of atriopeptin III under these conditions increased left glomerular filtration rate by 40% (P < 0.01) which was associated with a significant (P < 0.05) increase in urine volume and increases in absolute and fractional sodium excretions which just failed to reach statistical significance. Although these tubular responses were smaller than those obtained in the first part of the study, they were larger than those obtained at 80 m m Hg in the absence of converting enzyme inhibitor (table 1), i.e. for urine flow
TABLE 2 Effect of two doses of atriopeptin III, 1000 ng kg 1, on blood pressure and renal function before and during reduction in perfusion pressure to 80 mm Hg at the left kidney and converting enzyme inhibition with captopril, 900 p.g k g - ] h 1. Basal 1
Exptl 1
Rec 1
Captopril 900/zg k g - 1 h - 1 Basal 2
Exptl 2
Rec 2
80 _+ 0 4.7 _+ 0.5 298 + 10 0.7 + 0.08 1.7 4- 0.3 0.17+ 0.09 0.19_+ 0.10
80 + 0 80 _+ 0 4.7 _+ 0.4 4.7 _+ 0.5 298 _+13 306 _+12 1.0 -+ 0.08 b 0.6 + 0.05 3.7 -+ 0.9 a 1.2 -+ 0.2 0.53+ 0.256 0.09_+ 0.02 0.34_+ 0.13 0.11_+ 0.03
126 + 2 1.2 + 0.1 26.1 + 2.7 7.90+ 0.80 4.93_+ 0.48
121 + 2 121 + 3 1.3 + 0.1 1.2 + 0.1 52.6 + 4.3 c 23.7 + 1.6 15.05_+ 1.1 c 7.67_+ 0.48 8.92_+ 0.86 c 4.88_+ 0.26
Left kidney Perfusion pressure, mm Hg Renal blood flow, ml m i n - 1 g - 1 Heart rate, b m i n - 1 Glomerular filtration rate, ml m i n - t g-1 Urine flow rate,/zl min -1 g-1 Absolute sodium excretion, g mol m i n - t g-1 Fractional sodium excretion, %
112 _+ 1 4.4 _+ 0.4 293 _+10 1.1 _+ 0.2 6.4 _+ 0.6 1.50 + 0.22 0.94 + 0.12
111 _+ 2 111 _+ 1 4.5 _+ 0.4 4.6 + 0.4 281 _+17 278 + 12 1.2 _+ 0.04 1.1 _+ 0.05 14.4 -+ 1.1 c 5.9 -+ 0.6 3.25+_ 0.29 c 1.41-+ 0.15 1.88+ 0.16 c 0.95+ 0.09
Right kidney Perfusion pressure, mm Hg 115 _+ Glomerular filtration rate, ml m i n - i g-1 1.1 _+ Urine flow rate, ~1 min - t g-1 6.9 + Absolute sodium excretion,/z mol m i n - 1 g - 1 1.62 -+ Fractional sodium excretion, % 1.06+
1 0.0 0.5 0.15 0.11
1 114 _+ 0.1 1.1 + 2.0 c 6.5 + 0.47 c 1.60+ 2.33+ 0.36 c 1.10+
114 + 1.2 + 16.6 + 3.79+
Significantly different from respective control values, a p < 0.05, b p < 0.01, c p < 0.001.
2 0.1 0.5 0.15 0.12
323
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glomerular filtration rate to increase transiently by 8%, while at the same time the magnitude of reversible rises in urine flow, absolute and fractional sodium excretions were significantly larger than those obtained in the first part of the study (P < 0.001), and from those obtained at the higher pressure when captopril was not given (P < 0.01). A comparison of these responses is shown in fig. 2.
* - - 1 V**'I •T .
4. Discussion
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5.0-
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Fig. 2. This figure presents the changes in urine flow (AUV), absolute sodium excretion (AUrqaV) and fractional sodium excretion (AFENa) in the right kidney after intravenous bolus of atriopeptin lII, 1000 ng kg-1 at normal and increased pressures in the group infused with vehicle throughout (open histograms, n = 7) or given captopril, 900 p.g kg -1 h -1, in the second half of the experimen[ (slashed histograms, n = 10). * P < 0.05; ** P < 0.01.
2.2+0.7 versus 0.31_+0.13 /~1 min -1 g - t (p
