Renal Effects of High-Dose Natriuretic Peptide Receptor Blockade in Rats With Congestive Heart Failure
Abstract Previous studies suggest that elevated plasma atrial natriuretic peptide (ANP) levels participate in regulating renal excretory function in rats with congestive heart failure (CHF). To define the role of natriuretic peptides (NPs) in the regulation of renal function in CHF, the renal responses to HS-142-1 (HS), a potent NP receptor antagonist, were studied in anesthetized rats subjected to coronary ligation that developed left ventricular infarction and CHF or in sham-operated (SO) control rats. Plasma ANP levels averaged >14-fold higher in rats with CHF than in SO rats. In response to HS (20 mg/kg IV bolus), both mean arterial pressure and renal vascular resistance increased in rats with CHF but not in SO rats; glomerular filtration rate (GFR, 1.26±0.04 versus 0.76±0.11 mL/min) and renal plasma flow rate (RPF, 3.52±0.27 versus 2.70±0.32 mL/min) were significantly reduced in rats with CHF; and in SO rats, GFR (1.26±0.06 versus 1.20±0.07 mL/min) and RPF (3.98±0.21 versus 3.99±0.18 mL/min) were not significantly affected by HS. The sodium excretion rate (0.18±0.04 to 0.06±0.01 μEq/min) and fractional sodium excretion (0.01±0.02% to 0.04±0.01%) also fell markedly after HS administration in rats with CHF, but these parameters were unchanged in SO rats. These data indicate that NPs play a critical role in maintaining renal hemodynamic function and inhibiting tubule sodium reabsorption in rats with CHF, thus opposing sodium retention and preserving sodium balance in this model.
Renal dysfunction is a critical pathophysiological derangement in the syndrome of CHF, as evident from the comparative efficacy of diuretics in the clinical treatment of heart failure. Although a variety of neurohormonal factors that may adversely affect renal perfusion, filtration rate, and sodium excretion are known to be activated in heart failure, the pathophysiology of the kidney in heart failure is still incompletely understood. In particular, the role of factors having counterregulatory effects on renal function (ie, the potential to augment renal perfusion and sodium excretion) is unclear. NPs, potent regulators of body fluid homeostasis, are of particular interest in this respect. Evidence from rats with CHF suggests that in addition to impaired cardiac function, sodium retention leading to increased plasma volume and increased cardiac preload also contributes to elevated plasma ANP levels.1 2 3 Awazu et al2 have reported that acute administration of anti-ANP antibody elicited significant reductions in UNaV and FENa in rats with CHF. However, GFR and effective RPF were found to be unchanged.2 Together, these reports have suggested that ANP acts to oppose the avid renal sodium retention characteristic of CHF. A more precise definition of the role of NPs in the kidney has awaited the development of specific pharmacological antagonists of NP receptors. HS, a recently developed potent NP receptor antagonist,4 has been shown to block the effects of exogenously administered NPs and to reverse changes derived from the augmented endogenous production of NPs.5 6 7 8 Moreover, the effects of HS and anti-ANP antibody may not be equivalent.8 9 Recent reports imply that HS, a polysaccharide compound, has significant advantages over anti-ANP antibody in revealing the role of endogenous NPs in pathophysiological states, especially with respect to the regulation of renal hemodynamic function.8 9 Administration of low doses of HS (3 mg/kg) was reported to be associated with significant reductions in UNaV without significant change in GFR in experimental CHF.10 11 Interestingly, our recent observations6 suggest that doses of HS (5 mg/kg) similar in magnitude to those used in these latter studies10 11 may not be sufficient to achieve complete inhibition of the renal hemodynamic effects of NPs. The present study was therefore undertaken to determine the full extent of the participation of NPs in the regulation of renal function in CHF by assessing renal hemodynamic and excretory effects of acute NP receptor antagonism with high doses of HS in anesthetized rats with CHF induced by coronary artery ligation.
Materials and Methods
Adult male Munich-Wistar rats weighing 250 to 270 g were used in the studies. The method for producing myocardial infarction is similar to that originally described by Maclean et al.12 In brief, under brevital anesthesia (50 mg/kg), a left thoracotomy was performed, the heart was exteriorized, and the left coronary artery was ligated between the pulmonary trunk and the left auricle. The heart was then returned to the thoracic cavity, and the chest was closed (n=23). SO rats (n=25) underwent thoracotomy and closure of the thorax only. In addition, 26 normal Munich-Wistar rats, without any surgical intervention before the clearance experiments, were studied to control for the thoracotomy procedure. Rats received standard rat chow (Rodent Lab Chow 5001, Ralston Purina Co) and water ad libitum for the 3 to 4 weeks between the surgery and the acute clearance studies.
On the day of the clearance experiment, rats were anesthetized with Inactin (0.1 g/kg IP) and placed on a thermostatically controlled heating table to maintain body temperature between 36°C and 37°C. After tracheostomy, the left femoral artery was cannulated for measurement of MAP and HR and to allow intermittent blood sampling at the midpoint of each clearance period. The right femoral vein was cannulated for infusion of 8% inulin and 1% PAH in isotonic saline, initially at a rate of 3.3 mL/h for the first 30 minutes and then at a rate of 20 μL/min continuously throughout each experiment. The left ureter was cannulated for collection of urine; the bladder was cannulated to allow free drainage of urine from the right side. After a 1-hour stabilization period, two 20-minute baseline urine collections were made in all groups before the experimental interventions. Each rat then received either an intravenous bolus dose of vehicle (0.15 mL isotonic saline) or HS (20 mg/kg) in 0.15 mL isotonic saline. This high dose of HS has been shown to produce practically complete inhibition of the renal responses to exogenous ANP and saline volume expansion.8 13 Ten minutes later, two 20-minute urine samples were collected. At the end of the experiments, the right carotid artery was cannulated with a PE-50 catheter connected to a pressure transducer. By carefully advancing the catheter tip until the characteristic pressure tracing of left ventricular pressure was obtained, LVDP was measured in each rat. The heart was then removed from the thoracic cavity, weighed, and dissected into three equal transverse sections, each ≈0.5 cm thick. The midsection of each heart was then examined. When compared with noninfarcted myocardium, a pale scarred portion of the anterior left ventricular wall was evident in rats subjected to coronary ligation. The infarcted sector was measured and expressed as a percentage of the entire ventricular area (ie, scarred plus viable myocardium), thus providing an approximate estimate of the extent of left ventricular infarction. This model of CHF is well established,12 14 15 and the left ventricular scars in the rats with CHF were so grossly evident that this method for rough estimation of ventricular infarct size was deemed satisfactory for our purposes.
Plasma ANP concentrations were determined by using radioimmunoassay as described previously.16 In brief, ≈5 mL of blood was collected from the abdominal aortic trunk in five rats from each vehicle-treated group at the end of the experiments and mixed with 15 mg EDTA and aprotinin (1500 kallikrein-inactivating units). After centrifugation, the supernatant was collected for subsequent extraction and estimation of ANP levels.
The concentrations of inulin and PAH were determined by using the anthrone method17 and a colorimetric technique,18 respectively. Urine volume was determined gravimetrically, and GFR and RPF were assessed as clearances of inulin and PAH, respectively, calculated from standard formulas. The concentrations of sodium in plasma and urine were measured by flame photometry. RVR was calculated as MAP divided by renal blood flow.
All data are expressed as mean±SEM. Differences between the baseline and experimental observations within groups were compared by Student’s paired t test. Comparisons between vehicle- or HS-treated groups were made by ANOVA, followed by Sheffé’s test where appropriate.19 Significance was accepted at P<.05.
Body weights were similar in rats with CHF and normal rats (Table 1⇓). No significant differences in kidney weights were found among the three groups of rats. Heart weight, however, was considerably higher in rats with CHF compared with both normal and SO rats. MAP was significantly lower in rats with CHF compared with normal and SO rats. LVDP was much higher in rats with CHF than in normal and SO rats. Pleural effusions, left ventricular dilatation, and scarring were observed only in rats with CHF and not in normal or SO rats. In rats with CHF, the extent of left ventricular infarction ranged between 35% and 50%. Basal plasma levels of ANP were significantly higher in rats with CHF than in normal or SO rats, as shown in Table 1⇓.
Changes from basal values in MAP and RVR after HS administration in normal and SO rats and in rats with CHF are shown in Fig 1⇓. In response to HS, MAP and RVR rose significantly in rats with CHF but not in the other groups (Fig 1⇓ and Table 2⇓). In several rats from each group, HR was measured. From similar basal values, HR was unchanged after either vehicle (298±8 to 292±5 bpm) or HS (285±18 to 285±15 bpm) administration in rats with CHF. Similarly, HR was unchanged by vehicle or HS administration in normal and SO rats. Changes from average basal values of GFR, RPF, FENa, and UNaV after the administration of HS to all rats are shown in Fig 2⇓. Whereas basal levels of GFR were similar among all groups, HS administration was associated with a significant reduction in GFR, averaging 41% in rats with CHF (Table 2⇓ and Fig 2⇓). In contrast, GFR remained unchanged in both normal and SO rats. Baseline RPF was found to be slightly lower in rats with CHF compared with normal and SO rats. After HS administration, RPF fell by 23% in rats with CHF but was unaltered in normal and SO rats. FF was higher in rats with CHF at basal values compared with the other two rat groups. After HS administration, FF was significantly reduced in rats with CHF, whereas in both normal and SO rats, FF was not significantly changed. RVR was significantly elevated after HS administration only in the CHF group. HS administration was also associated with significant reductions in urinary flow rate (≈27%) in rats with CHF (Table 2⇓). Striking decreases in UNaV (54%) were observed in rats with CHF. Neither urinary flow rate nor UNaV was changed in the normal or SO rats after HS administration. FENa was similar among all groups of rats and was reduced significantly (57%) after HS in rats with CHF but was unchanged after vehicle administration. FENa was unchanged in normal and SO rats after HS administration.
Congestive heart failure is characterized by increased levels of circulating vasoconstrictive and sodium-retaining factors, such as vasopressin, catecholamines, angiotensin II, and aldosterone. The activity of renal sympathetic nerves20 21 and the production of locally generated vasoactive and antinatriuretic factors such as endothelin are also increased.22 23 Despite the adverse nature of this milieu to the kidney, especially when combined with the lower arterial perfusion pressures typical of heart failure, GFR is comparatively well maintained, albeit at lower levels of renal blood flow and increased levels of sodium reabsorption.14 24 25 Preservation of renal clearance function under such conditions is dependent on the increased activity of counterregulatory hormones such as ANP and locally produced mediators such as prostaglandin E2 and nitric oxide. It has been suggested that ANP, elevated because of higher cardiac filling pressures, may be one of the principal factors activated to oppose vasoconstriction and renal sodium retention in CHF.1 2 3 ANP is a potent regulator of body fluid homeostasis,26 whose pharmacological administration promotes natriuresis by inhibiting tubule sodium reabsorption and elevating GFR.27 In addition, ANP may promote renal excretion of fluid and sodium indirectly by inhibition of the renin-angiotensin system.28 Our present data are in general agreement with those reported previously2 10 11 showing that NPs are important for the promotion of natriuresis in rats with CHF. The data obtained with anti-ANP antibody, however, suggested that the renal hemodynamic effects of ANP were comparatively weak in CHF2 and that the principal target site for ANP in heart failure was the renal tubule (where it acts to decrease sodium reabsorption). Using a specific pharmacological antagonist of NP receptors, the present study reveals the actions of endogenous NPs in CHF from a more direct perspective.
First, we observed that when basal ANP levels in plasma were elevated, both MAP and RVR were significantly increased in rats with CHF after NP receptor blockade, an effect not seen in normal or SO rats. These data strongly suggest that contrary to the previous reports,2 10 11 NPs function as important renal and systemic vasodilators opposing the various vasoconstrictive neurohormonal factors activated in CHF.
Second, the most important novel finding of the present study was that NP receptor blockade, in addition to reducing RPF, resulted in striking reductions in GFR (>40%) in rats with CHF. Consequently, the observed reductions in UNaV in response to NP receptor blockade in rats with CHF were ascribable to decreases in the filtered sodium load in addition to decreases in fractional (and absolute) tubule reabsorption of sodium. Therefore, data from the present study strongly implicate maintenance of GFR as a major role for NPs in rats with CHF. The apparent discrepancies in these findings and previous reports using anti-ANP antibody2 and low-dose HS10 11 may have arisen because (1) there were different target sites for HS and anti-ANP antibody (ie, tissue receptors versus circulating peptide), (2) there was more effective inhibition by HS of locally synthesized NPs, which may be relatively shielded from the circulation and hence from binding to antibody,6 7 and (3) the doses of HS may have been relatively greater than the doses of anti-ANP antibody used in the previous study and were clearly more than sixfold greater than those used by Nishikimi et al.10 and Wada et al.10 11 In other regards, the experimental protocols used in our study and in the other studies2 10 11 were similar. Our previous experience with HS, in a variety of models in which ANP levels are known to be elevated, has revealed that at lower doses, HS administration elicits reductions in UNaV without effecting changes in renal hemodynamics.6 8 13 At higher doses, however, reductions in GFR have been observed consistently, the magnitude of the reduction being dependent on the model studied. Therefore, the absence of a significant effect of HS on GFR in CHF reported by Nishikimi et al and Wada et al may be explained by the lower dose of HS (3 mg/kg) used in their studies. Indeed, their data do appear to show a reduction in GFR of ≈20%, which did not achieve statistical significance. Therefore, in light of our previous experience establishing the effects of low (5 mg/kg) and high (20 mg/kg) doses of HS, we interpret our present data as being consistent with the published reports but adding new insight into the pathophysiology of NPs in heart failure by virtue of the higher doses of HS used achieving more complete blockade of renal NP receptors. The lack of effect of HS in normal anesthetized rats is consistent with our previous observations6 13 and imply that NPs do not play a major role in regulating systemic or renal hemodynamics or sodium excretion in the “hydropenic” rat preparation. However, a small but significant effect of NPs is evident when the preparation maintains “euvolemic” conditions.13 Studies in conscious rats to address the longer-term physiological role of NPs have yet to be undertaken.
Several studies have reported attenuated renal excretory responses to exogenous ANP in CHF,29 30 a finding presumed to be due to neurohormonal activation of renal vasoconstrictors or downregulation of ANP receptors.22 31 32 As CHF progresses, these factors may combine to further limit the renal responses to ANP. The contribution of endogenous NPs to this equation is unclear. Our data suggest that the major roles of NPs in heart failure are to maintain GFR and reduce tubule sodium reabsorption, thus effectively counterbalancing the renal vasoconstrictive and direct antinatriuretic tubule effects of, for example, catecholamines and angiotensin II.
In summary, we showed that MAP and RVR were significantly increased in rats with CHF after NP receptor blockade but not in SO and normal control rats. In response to HS, GFR and RPF were significantly reduced in rats with CHF, whereas in normal and SO rats, GFR and RPF were unchanged. UNaV and FENa fell significantly after HS administration in rats with CHF but not in normal and SO rats. In contrast to earlier reports,2 10 11 our data indicate that NPs play a critical role both in maintaining renal hemodynamics and filtered sodium load and in inhibiting tubule sodium reabsorption in anesthetized rats with CHF. These potent dual effects on sodium excretion imply that NPs are of major importance in maintaining sodium homeostasis in experimental CHF.
Selected Abbreviations and Acronyms
|ANP||=||atrial natriuretic peptide|
|CHF||=||congestive heart failure|
|FENa||=||fractional excretion of sodium|
|GFR||=||glomerular filtration rate|
|LVDP||=||left ventricular diastolic pressure|
|MAP||=||mean arterial pressure|
|RPF||=||renal plasma flow rate|
|RVR||=||renal vascular resistance|
|UNaV||=||urinary sodium excretion rate|
This study was supported by grants from the National Institutes of Health (DK-35930 and DK-30410). We are indebted to the Pharmaceutical Research Laboratories of Kyowa Hakko Kogyo Co of Japan for their gift of HS-142-1 used in these experiments. The expert technical assistance of Miguel A. Zayas is also appreciated.
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
- Received January 12, 1995.
- Accepted August 7, 1995.
- © 1995 American Heart Association, Inc.
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