Pressure Natriuresis After Adrenomedullin in Anesthetized Spontaneously Hypertensive Rats
Abstract Adrenomedullin (ADM), a peptide with potent vasodilatory and natriuretic actions, is elevated in patients with essential hypertension. Because pharmacological doses of ADM result in renal vasodilation and natriuresis, it has been suggested that ADM may play a modulatory role in hypertension through potential actions on renal pressure natriuresis. However, it is unclear whether elevation of plasma ADM within the pathophysiological range has similar actions. To determine the effects of pathophysiological doses of ADM on blood pressure and on the relationship between renal perfusion pressure (RPP) and renal hemodynamics and sodium excretion, renal function was determined at RPPs of 80, 105, 130, and 155 mm Hg in spontaneously hypertensive rats (SHR) infused with ADM at 50 ng · kg−1 · min−1 (ADM-50, n=5) and at 100 ng · kg−1 · min−1 (ADM-100, n=5) and in control SHR (n=5). Decreasing RPP from 155 to 80 mm Hg in control SHR decreased (P<.05) absolute sodium excretion from 0.81±0.25 to 0.04±0.02 μEq/min, fractional sodium excretion from 0.32±0.11% to 0.06±0.04%, and urine flow rate from 11.5±2.8 to 1.03±0.31 μL/min. ADM infusion elevated (P<.05) plasma ADM levels in ADM-infused SHR (679±47 pg/mL in ADM-50, 858±79 in ADM-100) compared with control (79.5±27.8). However, although reduction of RPP from 155 to 80 mm Hg in ADM rats decreased absolute sodium excretion (ADM-50, 0.98±0.10 to 0.09±0.04 μEq/min; ADM-100, 0.95±0.09 to 0.07±0.02 μEq/min), fractional sodium excretion (ADM-50, 0.31±0.03% to 0.17±0.04%; ADM-100, 0.33±0.02% to 0.09±0.01%), and urine flow (ADM-50, 13.6±1.4 to 1.73±0.75 μL/min; ADM-100, 13.5±1.5 to 1.07±0.16 μL/min), these decreases were not different from values found in controls. Renal plasma flow and glomerular filtration rate were also similar in control and ADM-treated SHR at each level of RPP. Thus, acute increases in ADM to levels found in pathophysiological conditions have no effect on blood pressure, pressure natriuresis, or renal autoregulation in the SHR. These findings do not support the hypothesis that ADM serves as a modulating factor in hypertension, at least in the SHR.
Adrenomedullin is a novel 52–amino acid peptide originally isolated from human pheochromocytoma.1 Studies have shown that levels of ADM are elevated in the plasma of patients with hypertension, renal failure, and heart failure compared with normal subjects,2 3 and infusion of ADM has hypotensive and diuretic effects in animals.4 5 6 7 Thus, ADM may participate in fluid volume homeostasis and may play a protective role in hypertension. However, animal studies demonstrating an effect of ADM have infused large doses that possibly increase plasma ADM levels greatly beyond levels observed in pathophysiological states in humans. Furthermore, it has been well established that the kidney plays a central role in long-term control of blood pressure through the relationship between RPP and sodium excretion and that this pressure-natriuresis relationship is altered in both human and experimental hypertension.8 Thus, the acute hypotensive actions of ADM may not result in long-term reductions in blood pressure unless ADM normalizes the shifted pressure-natriuresis relationship in hypertension. The goal of this study was to determine the acute effects of pathophysiological blood levels of ADM on blood pressure and the relationship between RPP, renal hemodynamics, and sodium excretion in SHR.
Twelve-week-old male SHR (Harlan Sprague Dawley, Inc, Indianapolis, Ind) were fed regular rat chow and allowed tap water ad libitum. All animals were housed according to institutional guidelines, and the studies were approved by the University of Mississippi Medical Center Animal Care and Use Committee. On the day of study, rats were anesthetized with thiobutabarbital sodium 100 mg/kg IP (Inactin, Promonto GmBH) and placed on a warming pad to maintain body temperature at 37°C. PE-240 tubing was inserted through a tracheostomy to maintain an open airway. The left jugular vein was cannulated with PE-50 tubing for intravenous infusion. The left carotid artery and the left femoral artery were cannulated with PE-50 polyethylene catheters for continuous measurement of arterial pressure and blood sampling. The carotid and femoral arterial catheters were connected to strain gauges (model P23DC, COBE), and arterial pressures above and below the renal artery were recorded on a polygraph (MFE Instruments). A midline abdominal incision was made and the bladder cannulated with a flare-tipped PE-50 tube for urine collection. An electronic servo-controlled Silastic balloon occluder manufactured in our laboratory was placed around the aorta above the superior mesenteric artery.
After completion of surgery, 2 mL of 0.9% NaCl containing 5% bovine serum albumin was infused to compensate for the loss of fluid during surgery. All rats received a jugular venous infusion of 0.9% NaCl containing [125I]iothalamate (Isotex Diagnostics) (0.5 μCi · kg−1 · min−1) and [131I]iodohippurate (Syncor International Corp) (0.1 Ci · kg−1 · min−1) at a rate of 2.0 mL/h to allow measurement of GFR and RPF, respectively. After a 60-minute stabilization period, rats were randomly divided into three groups. Two experimental groups (n=5 rats/group) received ADM (rat adrenomedullin, Phoenix Pharmaceuticals) added to the maintenance saline solution to provide a rate of 50 or 100 ng · kg−1 · min−1 (ADM-50 and ADM-100, respectively). Control rats (n=5) received the maintenance infusion as ADM vehicle throughout the experiment. The ADM was prepared fresh daily. Preliminary studies demonstrated that bolus infusion of ADM at a dose of 1 μg/kg reduced blood pressure in this preparation. Twenty minutes after initiation of ADM or vehicle, RPP in each rat was adjusted to 155, 130, 105, or 80 mm Hg in random order by means of the servo-controlled occluder.9 After a 10-minute stabilization period, urine samples were collected during a 15-minute experimental period at each RPP. Blood samples were obtained from the carotid artery before and after the urine collection for determination of plasma concentration of sodium and 125I and 131I radioactivities. At the end of each experiment, blood was drawn from the carotid artery into ice-chilled tubes containing disodium EDTA for the assay of plasma ADM concentration. This blood was centrifuged at 3000g for 20 minutes at 4°C and stored at −20°C until analyzed. Rats were then killed using a venous injection of potassium chloride while still under anesthesia.
ADM concentration in plasma was measured by radioimmunoassay.3 In brief, 1 mL of plasma was extracted on a C-18 cartridge and eluted with 75% methanol containing 1% trifluoroacetic acid. Samples and standards were incubated with 100:1 antibody raised against ADM(1-52) at 4°C for 24 hours. 125I-labeled ADM was added and incubated another 24 hours at 4°C. Free and bound fractions were then separated by addition of a second antibody and centrifuged. Radioactivity of the pellet was measured with a gamma counter (Packard Instrument). Minimal detectable concentration for this assay was 0.5 pg per tube, and the half-maximal inhibition dose of radio-iodinated ligand binding by ADM was 10 pg per tube. Recovery was 72±2%, and intra-assay and interassay variations were 10% and 12%, respectively.
Urine volume was determined gravimetrically. Sodium con- centrations in plasma and urine were measured by flame pho- tometry (Instrumentation Laboratories). Radioactivities of [125I]iothalamate and [131I]iodohippurate in plasma and urine were determined with a gamma counter (Packard Instrument). Determination of sodium concentration and radioactivities of [125I]iothalamate and [131I]iodohippurate in plasma and urine as well as urine flow rate permitted calculation of GFR, RPF, and urinary sodium excretion rate by standard expressions.
Values are expressed as mean±SEM. Statistical comparisons between values at different RPPs within a single group were determined with the use of ANOVA for repeated measures followed by Tukey test. Statistical significance between groups was determined using ANOVA followed by Bonferroni test. A value of P<.05 was considered significant.
Continuous infusion of ADM into SHR at either 50 or 100 ng · kg−1 · min−1 elevated (P<.05) plasma ADM concentrations compared with control SHR (Table⇓). Although SHR infused at the 100 ng · kg−1 · min−1 rate had higher plasma levels of ADM than SHR infused at 50 ng · kg−1 · min−1, this difference did not reach statistical significance. Blood pressure before activation of the servo occluder was not different between SHR infused with ADM at either rate and control SHR (Table⇓).
Fig 1⇓ shows the relationship between RPP and urinary flow rate in all rat groups. Decreasing RPP from 155 mm Hg to lower perfusion pressures decreased (P<.05) urinary flow rate in each rat group. In control SHR, urine flow rate was 11.47±2.80 μL/min at an RPP of 155 mm Hg and 1.03±0.31 μL/min at an RPP of 80 mm Hg. In ADM-50 SHR, urine flow rate was 13.6±1.39 μL/min at an RPP of 155 mm Hg and 1.73±0.75 μL/min at an RPP of 80 mm Hg. In ADM-100 SHR, urine flow rate was 13.5±1.45 μL/min at an RPP of 155 mm Hg and 1.07±0.16 μL/min at an RPP of 80 mm Hg. Urine flow rate was not different between groups at equivalent RPPs.
As depicted in Fig 2⇓, decreasing RPP from 155 mm Hg to lower perfusion pressures decreased (P<.05) absolute sodium excretion in all groups. In control SHR, absolute urinary sodium excretion was 0.81±0.25 mmol/min at an RPP of 155 mm Hg and 0.04±0.02 mmol/min at an RPP of 80 mm Hg. Absolute urinary sodium excretion in ADM-50 SHR was 0.98±0.10 mmol/min at an RPP of 155 mm Hg and 0.09±0.04 mmol/min at an RPP of 80 mm Hg. Absolute urinary sodium excretion in ADM-100 SHR was 0.95±0.09 mmol/min at an RPP of 155 mm Hg and 0.07±0.02 mmol/min at an RPP of 80 mm Hg. Absolute urinary sodium excretion was not different between groups at equivalent RPPs. Fractional urinary sodium excretion demonstrated a similar pattern (Fig 3⇓).
GFR at an RPP of 155 mm Hg was 1.87±0.20 mL/min in control SHR, 2.21±0.08 mL/min in ADM-50 SHR, and 2.06±0.14 mL/min in ADM-100 SHR. RPF at an RPP of 155 mm Hg was 4.24±0.29 mL/min in control SHR, 4.37±0.20 mL/min in ADM-50 SHR, and 4.08±0.27 mL/min in ADM-100 SHR. Thus, ADM had no effect on either GFR or RPF. Decreasing RPP from 155 to lower perfusion pressures decreased (P<.05) GFR and RPF in control SHR and in both ADM-infused SHR groups. Furthermore, each sequential reduction in RPP resulted in a significant (P<.05) reduction in GFR or RPF in the ADM-infused SHR. There were no significant differences in GFR or RPF between the three groups at equivalent RPPs. Thus, the blunted autoregulation of GFR and RPF in SHR was not improved by ADM (Figs 4⇓ and 5⇓).
The present study shows that in SHR, acute increases in plasma ADM levels approximately 10-fold higher than levels reported in pathophysiological conditions in humans do not lower blood pressure, increase sodium excretion, alter renal hemodynamics, or improve abnormal pressure natriuresis in SHR. Thus, ADM at plasma concentrations comparable to those reported in pathological conditions is not a modulator of blood pressure or renal function in this model. The observations from this study are not in accordance with animal studies of other investigators, which have reported both hypotensive and natriuretic actions of ADM.4 5 6 7
One potential explanation for the discrepancy between the findings in our study and those of previous investigators is the difference in doses of ADM administered. Previous studies have examined the effects of ADM infused systemically at rates between 1.7 and 17 μg · kg−1 · min−1 in rats5 6 7 or infused intrarenally at rates between 0.25 and 25 ng · kg−1 · min−1 in dogs.4 In rats, ADM at these doses has been observed to decrease blood pressure, increase GFR, and increase urinary sodium excretion. Studies of regional hemodynamics performed at these infusion rates have found increases in blood flow to most internal organs, including the kidney. Blood flow to the brain was unchanged, whereas blood flow to skeletal muscle was increased in one study and decreased in another after ADM administration.5 6 Although plasma levels of ADM achieved in these studies were not determined, measurement of plasma ADM levels in normal humans and in humans with hypertension, renal failure, or heart failure suggests that plasma levels in these conditions are in the picograms per milliliter range.2 3 The current study infused doses of ADM at rates intended to achieve plasma ADM concentrations 6- to 10-fold higher than that observed in control SHR and 10-fold higher than pathophysiological levels reported in human cardiovascular and renal diseases. At these rates, ADM had no effect on blood pressure, renal hemodynamics, or sodium excretion. Thus, effects reported from the previous studies in rats are likely the result of pharmacological doses of ADM.5 6 7 This conclusion would support early dose-response studies by Ishiyama et al,10 who found that huge increases in plasma ADM level were required to elicit any physiological response. In dogs, on the other hand, ADM infused into the renal artery at doses as low as 1 ng · kg−1 · min−1 have been reported to increase blood pressure, renal blood flow, and urinary sodium excretion.4 At higher doses, GFR is also increased. These effects occurred during infusion of ADM into the renal artery at rates that should have resulted in intrarenal concentrations similar to those achieved in our study. The reason for the difference between observations in the dog and our findings in the rat is unclear. However, because intrarenal ADM increased blood pressure in the dog, clearly there are effects of ADM that are unique to that species or result from the intrarenal route of administration. Neither study, however, supports a role for pathophysiological plasma levels of ADM as a major factor in reducing blood pressure. It should be acknowledged, however, that rat ADM mRNA has been found to be expressed in heart, adrenal gland, lungs, kidney, spleen, small intestine, and vascular smooth muscle whereas specific binding sites for ADM have been found in many organs.11 12 13 14 15 Thus, ADM may function as a local regulatory factor, and circulating ADM may have no functional significance. Development of specific ADM antagonists or studies in animals with the deletion of the gene controlling ADM production will be necessary to clarify the role of ADM in the regulation of cardiovascular function.
A second possible explanation for the lack of an effect of ADM in the current study is that the SHR is unresponsive or refractory to the effects of ADM. This seems unlikely because ADM was present in plasma of SHR receiving ADM vehicle, and this level was higher than that previously reported in normotensive Sprague-Dawley rats.11 Because the antibody used to determine ADM levels in the present study is highly specific for ADM, it is likely that hypertension in rats is associated with elevations of plasma ADM levels, just as it is in humans.2 He and associates5 have shown that acute intravenous infusion of 5 μg · kg−1 · min−1 of ADM produced twice the percentage reduction in mean arterial pressure in SHR as it did in Sprague-Dawley rats. ADM-induced increases in solid organ blood flows, including the kidney, were also similar in SHR and Sprague-Dawley rats in that study. Similar hemodynamic responses to equivalent pharmacological doses of ADM have also been reported between anesthetized normotensive rats and SHR by others.10 Thus, SHR can respond to supraphysiological doses of ADM in a manner similar to normotensive rats, supporting the hypothesis that postreceptor effects of ADM are similar in normotensive rats and SHR. It is still possible, however, that the effects of pathophysiological doses of ADM are blunted in SHR as a result of chronic exposure to higher circulating levels of ADM.
The pressure-natriuresis relationship is altered in known human and experimental hypertension models, including SHR,8 14 and it has been proposed that this resetting of pressure natriuresis is a primary cause of hypertension.15 It has been shown in this laboratory that the normalization of abnormal pressure natriuresis was closely related to the prevention of hypertension in Dahl rats.16 17 Theoretically, the hypotensive and natriuretic properties of ADM should ameliorate the abnormality in the pressure-natriuresis relationship if ADM significantly contributed to sustained reductions in blood pressure in hypertensive animals. However, ADM did not affect pressure natriuresis in the present study. Furthermore, preliminary studies in our lab have found continuous infusion of ADM at the rate of 20 ng · kg−1 · min−1 for 5 days did not reduce 24-hour blood pressure in SHR (unpublished observations, 1996).
In conclusion, acute infusion of ADM at rates that achieve pathophysiological blood levels has no effect on blood pressure, renal pressure natriuresis, or renal hemodynamics in the SHR. These findings do not support the hypothesis that ADM plays an antihypertensive role in hypertension.
Selected Abbreviations and Acronyms
|ADM-50||=||rats given adrenomedullin 50 ng · kg−1 · min−1|
|ADM-100||=||rats given adrenomedullin 100 ng · kg−1 · min−1|
|GFR||=||glomerular filtration rate|
|RPF||=||renal plasma flow|
|RPP||=||renal perfusion pressure|
|SHR||=||spontaneously hypertensive rats|
- Received March 17, 1997.
- Revision received April 17, 1997.
- Accepted April 30, 1997.
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