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Journal of Physiology (2002), 538.3, pp. 901-910
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013280
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ABSTRACT |
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Increasing renal artery pressure (RAP) activates pressure diuresis/natriuresis and inhibits renal renin release. There is also evidence that increasing RAP stimulates release of a putative depressor hormone from the renal medulla, although this hypothesis remains controversial. We examined the relative roles of these antihypertensive mechanisms in the acute depressor responses to increased RAP in anaesthetized rabbits and rats. In rabbits, an extracorporeal circuit was established which allows RAP to be set and controlled without direct effects on systemic haemodynamics. When RAP was maintained at ~65 mmHg, cardiac output (CO) and mean arterial pressure (MAP) did not change significantly. In contrast, when RAP was increased to ~160 mmHg, CO and MAP fell 20 ± 5 % and 36 ± 5 %, respectively, over 30 min. Urine flow also increased more than 28-fold when RAP was increased. When compound sodium lactate was infused intravenously at a rate equal to urine flow, neither CO nor MAP fell significantly in response to increased RAP. In 1 kidney-1 clip hypertensive rats, MAP fell by 54 ± 10 mmHg over a 2 h period after unclipping. In rats in which isotonic NaCl was administered intravenously at a rate equal to urine flow, MAP did not change significantly after unclipping (-14 ± 9 mmHg). Our results suggest that the depressor responses to increasing RAP in these experimental models are chiefly attributable to hypovolaemia secondary to pressure diuresis/natruresis. These models therefore appear not to be bioassays for release of a putative renal medullary depressor hormone.
(Received 17 September 2001; accepted after revision 7 November 2001)
Corresponding author R. Evans: Department of Physiology, PO Box 13F, Monash University, Victoria 3800, Australia. Email: roger.evans{at}med.monash.edu.au
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INTRODUCTION |
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The kidneys play an important role in long-term blood pressure control. Thus, when renal artery pressure (RAP) is increased, renal renin release is reduced, so that the activity of the pro-hypertensive renin-angiotensin system is inhibited (Cowley, 1992). Also, urinary excretion rates of salt and water increase with elevated RAP (pressure diuresis/natriuresis), so reducing plasma volume which, if not compensated for, leads to a reduction in cardiac output (Cowley, 1992). Thirdly, evidence now exists to support the release of a putative depressor hormone from the renal medulla in response to increased RAP (Muirhead & Pitcock, 1985; Muirhead, 1990; Thomas et al. 1996; Bergström et al. 1998). Muirhead and colleagues provided the initial evidence supporting the existence of a renal medullary depressor hormone, which they dubbed medullipin. In a series of experiments spanning four decades, they showed depressor responses to transplants of medullary tissue (particularly renal medullary interstitial cells), and to administration of extracts of medullary tissue, or venous effluent of kidneys perfused at high pressure. They concluded that medullipin could be a neutral lipid hormone or pro-hormone housed in the medullary interstitial cells, and released in response to increased RAP (Muirhead & Pitcock, 1985; Muirhead, 1990). However, neither they nor others have definitively purified and chemically identified the active principle in these extracts (Brooks et al. 1994).
Experimental models have been employed to determine the physiological processes mediating release of this putative hormone, and its biological effects. The common feature of these models is that they allow RAP to be increased in vivo, and for the resulting effects on systemic haemodynamics to be observed. For example arterial pressure falls rapidly after removal of the renal artery clip in Goldblatt hypertension (Muirhead & Brooks, 1980). The depressor response to unclipping is blunted by chemical medullectomy (bromoethylamine (BEA)-pretreatment) (Bing et al. 1981), and by inhibition of intrarenal cytochrome P450-dependent arachidonate metabolism (Zou et al. 1995), consistent with the hypothesis that it is dependent, in part, on release of a lipid hormone from the renal medulla. Another approach is to establish extracorporeal circuits in anaesthetized animals, so that RAP can be set at levels greater than systemic arterial pressure. In rats, this has been achieved by cross-circulating an isolated kidney from a 'donor' rat with blood from an anaesthetized 'recipient' rat (Karlström & Göthberg, 1987). In rabbits and dogs, autoperfused kidney preparations have been used (Christy et al. 1991, 1993; Thomas et al. 1994, 1995, 1996; Evans et al. 1998b; Correia et al. 2000). In these models, depressor responses to increased RAP can be blunted or abolished by chemical medullectomy (BEA) (Christy et al. 1991), and treatments that reduce renal medullary perfusion (Bergström et al. 1995; Bergström & Evans, 1998; Correia et al. 2000). Thus, the experiments performed by Muirhead's group (Muirhead & Pitcock, 1985; Muirhead, 1990; Brooks et al. 1994), combined with the more recent physiological experimentation cited above, have provided strong circumstantial evidence for the existence of this putative renal medullary depressor hormone.
On the other hand, there has been no definitive demonstration that the acute depressor responses to increased RAP in these models are independent of the associated pressure diuresis/natriuresis. To address this issue, in the present study we determined the relative contributions of changes in cardiac output and total peripheral resistance to the depressor response to increased RAP in anaesthetized rabbits. In some rabbits, we aimed to maintain cardiac output constant when RAP was increased, by infusion of compound sodium lactate at a rate equal to urine flow ('cardiac output clamp'). This allowed us to eliminate the systemic haemodynamic effects of pressure diuresis/natriuresis. In a further group of rabbits subjected to the 'cardiac output clamp', we also tested whether inhibition of the renin-angiotensin system contributes to the depressor response to increased RAP. In analogous experiments in 1 kidney-1 clip (1K1C) hypertensive rats, we tested the effects of intravenous isotonic sodium chloride, administered at a rate equal to urine flow, on haemodynamic responses to unclipping.
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METHODS |
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Rabbit extracorporeal circuit studies
Animals. Twenty-four New-Zealand White, male rabbits were studied (2.10-2.93; mean 2.53 ± 0.03 kg). They were housed individually, in purpose built cages (500 cm high, 740 cm long and 680 cm wide) with two tiers for environmental enrichment. This housing allowed visual, but not physical, contact with rabbits in adjoining cages. The rabbits were meal fed (Evans et al. 2000) and allowed water ad libitum. The experiments were approved in advance by the Monash University Department of Physiology/Central Animal Services Animal Ethics Committee.
Preliminary surgery. Each rabbit underwent a preliminary operation for implantation of an ascending aortic flowprobe (6SB, Transonic Systems Inc., Ithaca, NY, USA) via a left thoracotomy (Shweta et al. 1999). The plug of the flowprobe was buried subcutaneously for later retrieval on the day of the acute experiment. A catheter (24 gauge, Optiva, Johnson & Johnson Medical, Brussels, Belgium) was placed in a marginal ear vein under local analgesia (1 % lignocaine, Xylocaine, Astra Pharmaceuticals, North Ryde, NSW, Australia). Anaesthesia was then induced with intravenous propofol (10 mg kg-1, Diprivan, ICI, Victoria, Australia) and after endotracheal intubation, maintained with inhaled halothane (1-4 % Fluothane, ICI). Depth of anaesthesia was monitored by testing corneal and toe-pinch reflexes. Prior to commencing the surgery itself, each rabbit was given an intramuscular injection of an antibiotic mixture containing 16 mg trimethoprim and 80 mg sulphadiazine (Tribrissen, Jurox, NSW, Australia), and a subcutaneous injection of the narcotic analgesic buprenorphine (60 µg, Temgesic, Reckitt and Coleman, NSW, Australia). Lignocaine (1 %, 2-4 ml) was instilled subcutaneously into the wound sites to enhance analgesia. Thirty millilitres of 154 mmol l-1 NaCl was given by intravenous drip during the surgery, which took 30-50 min. At the completion of the surgery, animals were closely monitored for the next 3-5 h, while they recovered in a padded box with a heat pad. Thereafter, the rabbit's wellbeing was monitored daily by visual inspection and determination of food and water intake, until the day of the acute experiment (2-3 weeks after the preliminary surgery).
Procedures on the day of the acute experiment. These were carried out under local analgesia (1 % lignocaine). The plug of the flowprobe was retrieved from its subcutaneous position, and catheters were placed in both central ear arteries (22 gauge, Optiva) and marginal ear veins (24 gauge, Optiva). The ear artery catheters were used for measurement of systemic arterial pressure, and for collection of arterial blood samples. The ear vein catheters were used for intravenous infusions of anaesthetic and physiological solutions (see below). Following a 30 min period to allow full recovery from these preparative procedures, systemic arterial pressure, cardiac output and heart rate were monitored for 30 min in the conscious state.
All subsequent experimental procedures were carried out under pentobarbitone anaesthesia (90-150 mg for induction plus 30-50 mg h-1 for maintenance, I.V.; Nembutal, Boehringer Ingelheim, Artarmon, NSW, Australia) and artificial ventilation as previously described (Bergström & Evans, 1998). The level of anaesthesia was monitored by corneal and toe pinch reflexes, and adjusted by altering the rate of infusion of pentobarbitone and, if necessary, administration of further bolus doses of 5-10 mg. Surgical procedures included implantation of a catheter via the jugular vein for measuring central venous pressure (Shweta et al. 1999), a right nephrectomy, cannulation of the left ureter for urine collection, and establishment of the extracorporeal circuit (Bergström & Evans, 1998). At the completion of the experiment, the rabbits were humanely killed with an intravenous overdose of pentobarbitone (300 mg).
Extracorporeal circuit. Blood was withdrawn from the aorta at a rate of 110 ml min-1 by a roller pump and returned to the rabbit via two limbs; one to the renal artery and the other to the vena cava (Christy et al. 1991; Bergström & Evans, 1998). RAP was controlled by adjusting a Starling resistor incorporated into the vena caval limb, while total flow through the circuit remained constant. For example increasing the mechanical resistance in the vena caval limb using the Starling resistor diverts blood flow towards the renal limb, so increasing RAP. The circuit dead space (24 ml) was filled with 10 % w/v dextran 40 in 154 mmol l-1 NaCl (Gentran 40, Baxter Healthcare, Toonagabbie, NSW, Australia) containing 50 i.u. ml-1 heparin (Monoparin, Fisons Pharmaceuticals, Sydney, NSW, Australia). Thus, the establishment of the circuit resulted in some initial haemodilution, and consequently a relatively low haematocrit (see Results).
Once the extracorporeal circuit was established, RAP was set at ~65 mmHg for a 60 min equilibration period. A bolus dose of [3H]-inulin (4 µCi) (NEN Research Products, Sydney, NSW, Australia) was administered in 1.0 ml of 154 mmol l-1 NaCl. An infusion of polygeline solution (Haemaccel, Hoechst, Melbourne, Victoria, Australia) containing 200 i.u. ml-1 sodium heparin and 0.3 µCi ml-1 [3H]-inulin then commenced (0.18 ml kg-1min-1), which continued for the duration of the experiment. Body temperature was maintained between 36 and 38 °C. Mean arterial pressure (MAP) and central venous pressure were measured by connecting an ear artery catheter and the jugular vein catheter, respectively, to pressure transducers (Cobe, Avarda, CO, USA). Heart rate (HR) was measured by a tachometer (Model 173, Baker Medical Research Institute, Melbourne, Victoria, Australia) activated by the arterial pressure trace. Cardiac output (CO) and renal blood flow (RBF) were measured by connecting the ascending aortic flow probe, and an in-line flow probe in the renal arm of the extracorporeal circuit (Type 4N, Transonic Systems Inc.), respectively, to a compatible flowmeter (Model T208, Transonic Systems Inc.). Analogue to digital conversion of these signals, as well as measurement of plasma renin activity, plasma and urinary concentrations of [3H]-inulin and sodium, and haematocrit, were made as previously described (Bergström & Evans, 1998). [3H]-inulin clearance was used to estimate glomerular filtration rate (GFR) (Bergström & Evans, 1998). At the completion of each experiment the left kidney was removed and desiccated, and its dry weight determined. All values of RBF, GFR, urine flow (Uvol) and sodium excretion (UNaV) are therefore expressed per gram of dry kidney weight (g, mean 1.65 ± 0.05 g).
Experimental protocol. MAP, HR and CO were measured in conscious rabbits for 30 min prior to induction of anaesthesia. Haemodynamic variables were also monitored during establishment of the extracorporeal circuit, to provide detailed information about the status of the circulation under these conditions relative to conditions in the normal circulation of conscious and anaesthetized rabbits. Following establishment of the extracorporeal circuit and a 60 min equilibration period, rabbits were randomly assigned to one of the four experimental groups (n = 6 for each group). RAP was first set to ~65 mmHg for a 30 min control period in all groups. RAP was then either maintained at ~65 mmHg (group 1) or set at ~160 mmHg for 30 min (groups 2-4). This period was then followed by a 30 min recovery period (RAP ~65 mmHg). In all rabbits, urine output was determined each minute during the 90 min of the experiment. The three groups in which RAP was increased to ~160 mmHg received either no treatment (group 2), a 'cardiac output clamp', consisting of intravenous infusions of compound sodium lactate (Hartmann's solution; composition: Na+ 129 mM, K+ 5 mM, Ca2+ 2 mM, Cl- 109 mM, lactate 29 mM) to exactly match urine output each minute during the period when RAP was increased (group 3), or the combination of a 'cardiac output clamp' with a 'renin-angiotensin system clamp' (group 4), consisting of enalaprilat (2.0 mg kg-1 plus 10 µg kg-1min-1) and an intravenous infusion of angiotensin II (40-50 ng kg-1min-1; Auspep, Parkville, Victoria, Australia) titrated to restore MAP to its pre-enalaprilat level. The bolus dose of enalaprilat was administered intravenously after 30 min of stable baseline recordings following establishment of the extracorporeal circuit (that is, at the mid-point of the 60 min equilibration period), and the infusion of angiotensin II commenced 10 min later. Inhibition of angiotensin converting enzyme was confirmed by administration of bolus intravenous doses of angiotensin I (10 and 100 ng kg-1; Auspep).
Unclipping of 1K1C hypertensive rats
Animals. Male Wistar rats (200-220 g) were purchased from the Möllegård Breeding Centre (Stensved, Denmark), and housed 2-4 per cage, in a room maintained between 23 and 25 °C with a 12 h light/dark cycle. Standard rat chow (R-34, Lactamin, Vadstena, Sweden) and water were provided ad libitum. The study was performed after prior approval from the Ethics Committee for Animal Experimentation at Göteborg University.
Surgical and experimental methodology. Under ketamine (58 mg kg-1 I.P.; Parke Davis, Warner Lambert Nordic AB, Solna, Sweden) and xylazine (7 mg kg-1 I.P., Bayer Sweden AB, Göteborg, Sweden) anaesthesia, a silver clip (inner diameter 0.2 mm, width 1.5 mm) was positioned around the left renal artery and the right kidney was removed (Bergström et al. 2001). Buprenorphine (0.03 mg kg-1, Temgesic, Scherring-Plough AB, Stockholm, Sweden) was administered post-operatively for analgesia.
Four to six weeks later, the terminal acute experiment was performed under sodium thiobutabarbitone (120 mg kg-1, I.P., Inactin, Research Biochemicals International, Natick, MA, USA) anaesthesia. Anaesthesia was monitored throughout the surgery and experiment by periodically testing corneal and toe-pinch reflexes, and supplemented if necessary by additional intravenous bolus doses (5-10 mg kg-1) of thiobutabarbitone. The trachea was cannulated (PE-240), the tail artery was cannulated (PE 50) for measurement of MAP, the right jugular vein was cannulated (PE 50), with the tip of the cannula positioned near the right atrium, for measurement of CVP and infusion of bovine serum albumin (2 % w/v in 154 mmol l-1 NaCl, 4 ml h-1) throughout the surgery and experiment, and the left ureter was cannulated (PE-10) for collection of urine. Heparinized 154 mmol l-1 NaCl (5 i.u. ml-1, 1.2 ml h-1) was infused via the tail artery catheter to maintain its patency. At the completion of the experiment, each rat was humanely killed with an intravenous overdose of thiobutabarbitone (50 mg).
Experimental protocol. Ninety minutes after completion of the surgery, the anaesthetized rats were randomized to three different experimental groups. In group 1, the renal artery clip was manipulated but not removed, while in groups 2 and 3 the renal artery clip was removed. Group 3 was given 154 mmol l-1 NaCl intravenously every 5 min, at a volume equal to urine flow over the preceding 5 min, across the 2 h experimental period following removal of the renal artery clip. Thus, these experimental groups were analogous to groups 1, 2 and 3 in the rabbit extracorporeal circuit experiment. Urinary sodium concentration was measured by flame photometry as previously described (Bergström et al. 2001), in pooled samples from the 30 min control period, and each of the two 60 min periods after unclipping or sham unclipping. Haematocrit was measured in 100 µl blood samples taken 30 min before the unclipping/sham unclipping procedure and at the completion of the experiment . All values of Uvol and UNaV are expressed per gram of wet kidney weight (g, mean 1.57 ± 0.05 g).
Statistical analyses
Data collected during the preparative phase of the rabbit experiment were subjected to analysis of variance, partitioned to make specific comparisons between each state (conscious, anaesthetized and 'circuit established'), and between animals receiving the 'angiotensin II clamp' (group 4) and control animals (groups 1, 2 and 3). P values were conservatively adjusted using the Ryan-Holm-Sidák procedure to account for the fact that six comparisons were made within this analysis (Ludbrook, 1998).
In the rabbit experiment, we compared the levels of variables during the final 15 min of the period of increased RAP, with the final 15 min of the control period. In the rat experiment, we compared the levels of variables during the final 15 min of the experiment (105-120 min after unclipping) with those during the 30 min control period. Our specific hypotheses were that the changes in these variables between these two time periods would differ between the experimental groups. We therefore used unpaired t tests to specifically compare the changes in group 1 with group 2, group 2 with group 3, and (in the rabbit experiment) group 3 with group 4. P values were conservatively adjusted using the Ryan-Holm-Sidák (Ludbrook, 1998) procedure to account for the fact that multiple comparisons were made (three for the rabbit experiment and two for the rat experiment). P ≤0.05 was considered to be statistically significant.
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RESULTS |
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Baseline haemodynamic variables in rabbits
Levels of haemodynamic variables in conscious and anaesthetized states were similar to those we have observed previously (Evans & Bergström, 1998; Shweta et al. 1999). MAP was 23 ± 3 mmHg less, stroke volume (SV) was 0.19 ± 0.06 ml kg-1 less, CO was 27 ± 12 ml min-1 kg-1 less, and HR was 35 ± 11 beats min-1 greater in the anaesthetized compared with the conscious state. Once the extracorporeal circuit was established, MAP and CO returned to levels similar to the conscious state, although HR remained elevated (by 39 ± 7 beats min-1) compared with the conscious state (Table 1). Resting levels of all haemodynamic variables, including RBF and renal vascular resistance (RVR), were closely similar in rabbits treated with enalaprilat/angiotensin II (group 4; 'angiotensin clamp') compared with those in rabbits not given this treatment (Table 1). The enalaprilat treatment completely abolished increases in MAP in response to 10 and 100 ng kg-1 angiotensin I, which averaged 3 ± 1 % and 8 ± 2 %, respectively, in rabbits from groups 1-3, and -7 ± 3 and 1 ± 3 %, respectively, in rabbits from group 4.
Responses to increased RAP in rabbits
Group 1. In these animals, in which RAP was maintained at ~65 mmHg for the entire 90 min of the experiment, levels of renal haemodynamic (Fig. 1), renal excretory (Fig. 2) and systemic haemodynamic (Fig. 3 and Fig. 4) variables, and levels of plasma renin activity (Fig. 5) remained relatively stable.
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Figure 1. Renal haemodynamic responses to increased renal arterial pressure (RAP) Symbols and error bars represent the mean ± S.E.M. of average levels during each of the six 15 min experimental periods (n = 6). In group 1 ( | ||
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Figure 2. Renal excretory responses to increased renal artery pressure Symbols, error bars and treatments are as for Fig. 1. GFR, glomerular filtration rate; FF, filtration fraction; FEvol, fractional excretion of urine; Uvol, urine flow; FENa, fractional sodium excretion; UNaV, sodium excretion. | ||
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Figure 3. Systemic haemodynamic responses to increased renal artery pressure Symbols, error bars and treatments are as for Fig. 1. MAP, mean arterial pressure; SVR, systemic vascular resistance; CVP, central venous pressure. Percentage changes in MAP were calculated for the last 5 min of periods 2-6 relative to the last 5 min of the preceding period. | ||
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Figure 4. Systemic haemodynamic responses to increased renal artery pressure Symbols, error bars and treatments are as for Fig. 1. SV, stroke volume; CO, cardiac output; HR, heart rate; HCT, haematocrit. | ||
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Figure 5. Responses of plasma renin activity to increased renal artery pressure Columns and error bars represent the mean ± S.E.M of plasma renin activity at the ends of periods 1, 3 ( | ||
Group 2. When RAP was increased to ~160 mmHg, RBF increased from 12 ± 2 to 44 ± 5 ml min-1 g-1 and RVR was reduced from 5.8 ± 0.7 to 3.8 ± 0.4 mmHg ml-1 min g (Fig. 1). At the same time, GFR increased from 1.5 ± 0.5 to 5.7 ± 1.0 ml min-1 g-1, Uvol increased from 0.07 ± 0.04 to 1.88 ± 0.22 ml min-1 g-1, UNaV increased from 9 ± 5 to 186 ± 14 µmol min-1 g-1, and the fractional excretions of sodium and urine increased from 7 ± 1 to 41 ± 2 % and from 9 ± 1 to 45 ± 3 %, respectively (Fig. 2). Filtration fraction did not change significantly (Fig. 2).
There was also a dramatic depressor response to increasing RAP. MAP fell from 83 ± 4 to 54 ± 5 mmHg, and systemic vascular resistance (SVR) decreased from 0.63 ± 0.04 to 0.52 ± 0.05 mmHg ml-1 min g. CVP was also reduced from 2.4 ± 1.7 to -0.2 ± 1.3 mmHg (Fig. 3). CO fell from 132 ± 6 to 105 ± 4 ml min-1 kg-1 and SV fell from 0.49 ± 0.02 to 0.39 ± 0.01 ml kg-1. Haematocrit increased by 1.5 ± 0.6 %. HR remained relatively constant across the 90 min experimental period (Fig. 4).
Group 3 ('cardiac output clamp'). When RAP was increased to ~160 mmHg and compound sodium lactate was infused at a rate equivalent to urine flow, RBF increased, and RVR fell, similarly to that seen in group 2 (Pgroup ≥ 0.18; Fig. 1). Increases in GFR, Uvol, UNaV, and the fractional excretions of sodium and urine were also similar to those observed in group 2 (Pgroup ≥ 0.15; Fig. 2). In contrast to group 2, neither SV, CO, MAP, SVR nor haematocrit changed significantly, although CVP fell from 2.0 ± 1.2 to 1.3 ± 1.0 mmHg. Between-group comparisons indicated clear differences in responses to increasing RAP to ~160 mmHg in groups 2 and 3. Thus, the reductions in MAP (group 2, 30 ± 5 mmHg versus group 3, 7 ± 3 mmHg; Pgroup = 0.005), CO (group 2, 28 ± 7 ml kg-1 min-1 versus group 3, 3 ± 4 ml kg-1 min-1; Pgroup = 0.04) and CVP (group 2, 2.6 ± 0.5 mmHg versus group 3, 0.7 ± 0.2 mmHg; Pgroup = 0.01) were less in rabbits receiving the compound sodium lactate infusion.
Group 4 ('cardiac output clamp' plus 'angiotensin clamp'). Responses in this group were indistinguishable from those in group 3 (Figs 1-5; Pgroup ≥ 0.08).
Plasma renin activity in rabbits
Plasma renin activity did not change significantly over the course of the experiment in any of the four groups (Fig. 5).
Cumulative Na+ balance in rabbits
When RAP was maintained at ~65 mmHg (group 1), a slightly positive cumulative sodium balance (0.7 ± 0.3 mmol Na+) was observed during the period 30-60 min after commencing the experiment. Group 2, in which RAP was increased to ~160 mmHg during this period, developed a markedly negative sodium balance (-14.4 ± 2.2 mmol Na+, Pgroup = 0.004 compared with group 1). In group 3, in which RAP was set to ~160 mmHg during this period, and compound sodium lactate was infused at a rate equal to urine flow, cumulative sodium balance was not significantly different from that in group 1 (-3.7 ± 2.6 mmol Na+; Pgroup ≥ 0.14). However, despite volume replacement and a stable MAP in group 4, a small but significant negative sodium balance developed (-6.7 ± 1.9 mmol Na+; Pgroup = 0.03 compared with group 1).
Responses to unclipping of 1K1C hypertensive rats
Compared to control levels, MAP had fallen by 54 ± 10 mmHg during the period 105-120 min after unclipping in group 2 rats. This was associated with increased Uvol (from 4 ± 2 to 156 ± 26 µl min-1 g-1) and UNaV (from 0.2 ± 0.1 to 17.1 ± 3.3 µmol min-1 g-1) during the first 60 min after unclipping. In the second hour after unclipping, Uvol (58 ± 11 µl min-1 g-1) and UNaV (7.6 ± 2.1 µmol min-1 g-1) reduced towards baseline levels. In contrast, Uvol and UNaV remained relatively stable in group 1 rats, in which the clip was manipulated but not removed, and MAP did not change (+3 ± 6 mmHg change). In group 3 rats, which received 154 mmol l-1 NaCl intravenously after unclipping, at a rate exactly matched to Uvol, Uvol remained elevated for the 2 h of the study (249 ± 46 and 265 ± 57 µl min-1 g-1, respectively), as did UNaV (30.4 ± 5.9 and 32.8 ± 6.6 µmol min-1 g-1, respectively). MAP fell by 14 ± 9 mmHg in group 3, significantly less than that observed in group 2 (Pgroup = 0.049), and not significantly different from the response observed after sham unclipping in group 1 (Pgroup = 0.14). Neither HR nor CVP changed significantly in any of the groups across the course of the experiment (Fig. 6). Haematocrit fell similarly in all groups, averaging 43 ± 1 % during the 30 min control period, and 37 ± 2 % at the completion of the experiment.
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Figure 6. Systemic haemodynamic responses and urine output after removing the clip from the renal artery (unclipping) of 1 kidney-1 clip hypertensive rats Groups are: sham-unclipping (group 1; | ||
In group 1, cumulative sodium balance was slightly positive (+1.03 ± 0.20 mmol Na+). Group 2, in which the clip was removed from the renal artery, developed a negative sodium balance compared with group 1 (-0.76 ± 0.41 mmol Na+, Pgroup = 0.04). In group 3, in which the clip was removed and isotonic NaCl was infused at a rate equal to Uvol, cumulative sodium balance was positive and significantly greater than that in group 1 (+3.10 ± 0.31 mmol Na+, Pgroup = 0.006).
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DISCUSSION |
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Our important new finding was that in two models that have been used for studying the putative antihypertensive hormonal function of the kidney, the acute depressor responses to increased RAP were abolished or greatly blunted when urinary fluid excretion was matched with intravenous infusions of isotonic salt solutions. We conclude that the depressor responses to increased RAP in these models can be chiefly accounted for by reduced CO, most probably secondary to increased salt and water excretion, rather than release of a putative renal medullary depressor hormone.
Because our conclusions draw heavily on results obtained using the rabbit extracorporeal circuit model, our first aim was to assess the status of systemic haemodynamics under these experimental conditions. MAP and CO were substantially reduced and HR increased in anaesthetized rabbits, when compared to the conscious state. In contrast, CO and MAP in rabbits with an established extracorporeal circuit were closely similar to values observed in the conscious state, and remained stable when RAP was maintained at ~65 mmHg over the 90 min course of the experiment. Thus, our observations of the responses to increased RAP are unlikely to be confounded by the haemodynamic conditions of our experiment. Furthermore, the 'cardiac output clamp' (rabbits in groups 3 and 4) effectively maintained haematocrit, SV and CO in the face of increased Uvol, when RAP was increased to ~160 mmHg. The aim of the 'angiotensin clamp' (group 4) was to provide inhibition of endogenous angiotensin II generation, while avoiding the potentially confounding effects of hypotension and renal vasodilatation that normally attend inhibition of angiotensin converting enzyme. This aim was met, as evidenced by the abolition of responses to intravenous angiotensin I, while resting systemic and renal haemodynamics were similar to those in anaesthetized rabbits from the other three groups.
When RAP was increased from 65 to 160 mmHg in the extracorporeal circuit model, RBF and GFR increased, and RVR decreased. This apparent absence of autoregulatory behaviour probably reflects the relatively narrow range of autoregulation in this preparation, which extends only from about 80 to about 110 mmHg (G. A. Eppel & R. G. Evans, unpublished observations). The great merit of this preparation is that it allows RAP to be increased in vivo, to levels greater than systemic arterial pressure. In the present study, this allowed us to investigate the factors contributing to the acute depressor response to increased RAP.
When RAP was increased in anaesthetized rabbits that had not received the 'cardiac output clamp' (group 2), MAP fell. This depressor response was associated with increased Uvol and UNaV, negative sodium balance, increased haematocrit, reduced CVP, SV and CO, and a small but significant reduction in SVR. Thus, it appears to result predominately from reduced CO mediated by pressure diuresis/natriuresis. This hypothesis was confirmed in rabbits treated with the 'cardiac output clamp' (group 3). In this group cumulative sodium balance was maintained, so that reductions in haematocrit, SV and CO were prevented, and no significant depressor response was observed. The response to increased RAP in rabbits from group 2 resembles that to haemorrhage in conscious rabbits after sino-aortic baroreceptor denervation, in which SVR falls as CO is reduced (Schadt & Ludbrook, 1991; Evans et al. 2001). In contrast, the usual response to haemorrhage or acute central hypovolaemia in unanaesthetized rabbits (and indeed all mammals in which it has been studied), consists of two distinct phases. In the first (compensatory) phase MAP is maintained in the face of a falling CO, chiefly by reflex increases in sympathetic vasomotor drive and so SVR. This is followed by a decompensatory phase in which this reflex sympathetic activation fails, and SVR and MAP fall precipitously (Schadt & Ludbrook, 1991). However, some general anaesthetic agents blunt the compensatory phase, presumably by inhibiting baroreceptor-mediated increases in sympathetic vasomotor drive (Evans et al. 2001). Pentobarbitone anaesthesia also greatly blunts cardiovascular reflexes (Morita et al. 1987), so it is hardly surprising that SVR did not increase in response to increased RAP in rabbits from group 2. SVR actually fell in response to increased RAP in rabbits from group 2, which, like the reduction in SVR seen during haemorrhage in sino-aortic baroreceptor denervated rabbits, might be secondary to the depressor response itself, perhaps through local autoregulatory mechanisms (Schadt & Ludbrook, 1991). Consistent with this, the 'cardiac output clamp' also abolished the progressive reduction in SVR during increased RAP.
Our experiments using 1K1C hypertensive rats complement our rabbit experiments, in that they show that the depressor response to unclipping is associated with transient diuresis and natriuresis, and is greatly blunted when fluid and sodium depletion is prevented by administration of isotonic saline. However, in contrast to the rabbit experiments, our volume replacement regimen in rats resulted in a slightly positive cumulative sodium balance. This is unlikely to have confounded our observations, since administration of even large volumes of isotonic saline has little effect on MAP in normovolaemic, Inactin-anaesthetized, rats (Keeler & Wilson, 1989). Our observations therefore contrast with those of Neubig and Hoobler (1975), who found similar depressor responses to unclipping in 1K1C hypertensive rats, regardless of whether sodium balance was maintained by intravenous infusion of isotonic saline. However, they are consistent with studies showing that the normalization of arterial pressure after unclipping is delayed or blunted by a surgical uretero-caval anastamosis or saline loading (Liard & Peters, 1970; Muirhead & Brooks, 1980). All of these earlier studies were confounded by the fact that unclipping was performed under relatively long-acting anaesthesia (pentobarbitone or ether), from which the animals recovered during the experimental period. This was obviated in the present study by performing the entire study under tightly controlled and stable (Inactin) anaesthesia. We therefore conclude that the acute depressor response to increasing RAP by unclipping 1K1C hypertensive rats is most probably chiefly due to reduced extracellular fluid volume, resulting from pressure diuresis/natriuresis.
Our results also confirm and extend previous evidence indicating that the renin-angiotensin system plays little or no role in mediating the depressor response to increased RAP in the rabbit extracorporeal circuit model (Christy et al. 1993), since systemic haemodynamic responses to increased RAP in rabbits receiving both the 'cardiac output clamp' and the 'angiotensin clamp' were indistinguishable from those in the group receiving only the 'cardiac output clamp'. This hypothesis is further supported by our observation of unchanged plasma renin activity throughout the course of the experiment. Plasma renin activity does fall when RAP is increased in relatively long experimental protocols using this model (Bergström & Evans, 1998; Correia et al. 2000). However, the relatively long circulating half-life of renin probably prevented substantial changes in its circulating activity across the relatively short time-course of the present experiment.
Our present observations prompt reinterpretation of our previous studies employing the rabbit extracorporeal circuit model, because observations regarding the depressor response to increased RAP had previously been interpreted in the context of release of a putative renomedullary depressor hormone. For example the fact that the depressor response to increased RAP was abolished in chemically medullectomized (BEA-treated) rabbits (Christy et al. 1991), and in rabbits in which medullary blood flow was reduced by medullary interstitial infusion of noradrenaline (Correia et al. 2000), was taken as evidence supporting the notion that increased medullary blood flow mediates release of the putative renal medullary depressor hormone in response to increased RAP. However, these treatments also blunted the pressure diuresis/natriuresis response (Christy et al. 1991; Correia et al. 2000), which probably made important contributions to their effects on the systemic haemodynamic responses to increased RAP .
In contrast, our present results are difficult to reconcile with our previous observation that medullary interstitial infusion of the V1-agonist [Phe2,Ile3,Orn8]-vasopressin blunted the depressor response to increased RAP, since the pressure diuresis/natriuresis response during V1-receptor stimulation was, if anything, slightly greater than that of control rabbits (Bergström & Evans, 1998; Evans et al. 1998a). Interpreted in the light of our present findings, this previous observation could possibly reflect an effect of V1-receptor activation on the systemic haemodynamic response to hypovolaemia. This notion is consistent with the proposed roles of both central nervous system (Johnson et al. 1988) and peripheral (Schadt & Ludbrook, 1991) V1-receptors in recovery from severe hypovolaemia. Furthermore, because significant systemic spill-over occurs when agents are infused into the renal medullary interstitum of rabbits (Evans et al. 1998a; Correia et al. 1999), [Phe2,Ile3,Orn8]-vasopressin could have gained access to these sites when administered via this route.
In conclusion, the results of this study indicate that the depressor responses to increased RAP, in both the extracorporeal circuit model in rabbits and after unclipping in 1K1C hypertensive rats, is chiefly due to hypovolaemia secondary to pressure diuresis/natriuresis, and not to release of a putative renal medullary depressor hormone. This conclusion is based on our finding that the depressor responses to increasing RAP in these models are abolished or greatly blunted when urinary fluid losses are replaced by intravenous infusions of isotonic salt solutions. These models therefore are not suitable bioassays for the putative renal medullary depressor hormone, even though they provide a presumed stimulus for its release - increased RAP. This calls for reinterpretation of previous studies by ourselves (Christy et al. 1991, 1993; Bergström et al. 1995, 1998, 2001; Thomas et al. 1994, 1995, 1996; Bergström & Evans, 1998; Evans et al. 1998b; Correia et al. 2000) and others (Muirhead & Brooks, 1980; Bing et al. 1981; Muirhead & Pitcock, 1985; Karlström & Göthberg, 1987; Muirhead, 1990; Zou et al. 1995) using these models. Furthermore, although our findings do not provide direct evidence against the existence of a putative renal medullary depressor hormone, they do reinforce the dominance of pressure diuresis/natriuresis in mediating the acute antihypertensive function of the kidney.
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Acknowledgements
This work was supported by grants from the National Health and Medical Research Council of Australia (977713, 143785), the National Heart Foundation of Australia (G 00M 0633), the Ramaciotti Foundations (A6370), the Swedish Medical Research Council (12580), the Inga Britt and Arne Lundberg Foundation and the Swedish National Heart and Lung Foundation.
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), RAP was maintained at ~65 mmHg for the entire 90 min of the experiment. In groups 2-4, RAP was increased to ~160 mmHg during periods 3 and 4 (30 min in total). Group 2 (
) received no further treatment, but groups 3 (
) and 4 (
) received an intravenous infusion of compound sodium lactate equal to urine flow, during periods 3 and 4 ('cardiac output clamp'). In addition, group 4 had been pre-treated with the angiotensin converting enzyme inhibitor enalaprilat (2 mg kg-1 plus 10 µg kg-1 min-1 I.V.) and also received an intravenous infusion of angiotensin II (40-50 ng kg-1 min-1) to restore mean arterial pressure and renal blood flow to baseline levels ('angiotensin clamp'; see Table 1). RBF, renal blood flow; RVR, renal vascular resistance. In this and subsequent figures, some symbols are obscured because the data points are coincident.
) and 5 (n = 6). Groups are as for Fig. 1. Ang I, angiotensin I.