1 Vascular Biology and Hypertension Program, Division of Cardiovascular Disease, Department of Medicine, and 2 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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Pathophysiological features of both primary aldosteronism and pseudohyperaldosteronism are hyperactive amiloride-sensitive epithelial Na+ channels (ENaC) and refractory hypertension. Peripheral blood lymphocytes express ENaC, which functions and is regulated similarly to ENaC expressed by renal principal cells. Thus it was hypothesized that individuals with either of these hypertensive etiologies could be identified by assessment of the function and regulation of peripheral blood lymphocyte ENaC, by whole cell patch clamp. We also tested the hypothesis that specific inhibition of hyperactive ENaC with amiloride could ameliorate the hypertension. To test these hypotheses, we solicited blood samples from normotensive, controlled hypertensive, and refractory hypertensive individuals. Lymphocytes were examined electrophysiologically to determine whether ENaC was hyperactive. All positive findings were from refractory hypertensive individuals. Nine refractory hypertensive patients had amiloride added to their hypertensive therapy. Amiloride normalized the blood pressure of four subjects. These individuals all had hyperactive ENaC. Amiloride had no effect on individuals with normal ENaC. These findings suggest that whole-cell patch clamp of peripheral blood lymphocytes can be used to identify accurately and rapidly hypertensive individuals who will respond to amiloride therapy.
sodium channel; lymphocytes; aldosterone
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INTRODUCTION |
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THE RENAL CORTICAL COLLECTING DUCT reabsorbs salt and water by processes that are regulated by vasopressin and aldosterone. The rate-limiting step in hormone-regulated Na+ reabsorption is the flow of Na+ down its electrochemical gradient through amiloride-sensitive epithelial Na+ channels (ENaC) in the collecting duct of the nephron. During periods of salt or water deprivation, circulating vasopressin and aldosterone levels increase, stimulating ENaC and thereby increasing Na+ reabsorption. ENaC also is expressed by peripheral blood lymphocytes (5). Lymphocyte ENaC appears to be regulated by the same cellular signal transduction pathways used by renal principal cells (1, 3-5, 18). Thus human renal ENaC function can be assessed indirectly by electrophysiological examination of peripheral blood lymphocytes.
Three subunits, ,
, and
, of ENaC have been cloned (6,
7, 13, 14). Subsequently, it was found that polymorphisms in
-ENaC caused pseudohyperaldosteronism (Liddle's disease)
(17). The pathophysiology induced by ENaC polymorphisms is
primarily severe hypertension, despite low aldosterone levels
(17). A premature stop mutation in the
-ENaC subunit
gene underlies the disorder in the proband (17).
Additional polymorphisms in the
- and
-ENaC subunits that produce
a similar hypertensive phenotype also have been described
(10). The physiological mechanism and its
pathophysiological consequences in pseudohyperaldosteronism have been
reviewed recently (16).
Primary aldosteronism also produces hypertension (8). We recently demonstrated an acute activation of ENaC by aldosterone that was not inhibited by spiranolactone (18). However, amiloride completely inhibited all of the ENaC current activated by aldosterone (18). Recent studies have indicated that primary aldosteronism may be a more common cause of refractory hypertension than was previously thought, accounting for up to 10.5% of all hypertensives and up to 25% of patients with refractory hypertension (12). The use of plasma aldosterone-to-renin ratios for the determination of primary aldosteronism has improved the accurate diagnosis of this disorder (15). At the cellular level, primary aldosteronism and pseudohyperaldosteronism are both characterized by inappropriate activity of ENaC (5, 18). In most cases, the inappropriately activated Na+ channels are inhibited by amiloride, a K+-sparing diuretic (5, 11, 18). Thus it was hypothesized that amiloride may be of specific benefit to a subset of hypertensive individuals with constitutively activated ENaC caused by either aldosteronism or gain of function polymorphisms in ENaC itself (Liddle's disease).
Prior studies by this laboratory have demonstrated that peripheral
blood lymphocytes express Na+ channels that are
indistinguishable from those expressed by Na+-reabsorbing
renal epithelial cells (1, 3-5). Recent RT-PCR analysis of lymphocyte mRNA confirmed the presence of messenger RNA for
- and
-ENaC subunits (5). Also, immunofluorescence analysis with anti-
-ENaC antibodies indicated the presence of ENaC
in the plasma membranes of normal lymphocytes and lymphocytes from
individuals with Liddle's disease (5). Abnormal
Na+ channel activity has been observed in transformed
lymphocytes obtained from patients with pseudohyperaldosteronism and
also in purified ENaC from individuals with genetically confirmed
pseudohyperaldosteronism (2, 11). The abnormal basal
activation found in these studies was consistent with the
pathophysiological mechanism of excessive salt reabsorption. Also, in
vitro studies now have demonstrated directly the acute, nongenomic
activation of ENaC in both lymphocytes and renal principal cells by
aldosterone (18). Therefore, the electrophysiological
characteristics of lymphocytes isolated from the peripheral blood of
hypertensive individuals have been hypothesized to be a useful
predictor for this specific hypertensive etiology.
Peripheral blood lymphocytes are readily available from a small blood sample (10 ml). Human renal principal cells (the source of the excessive salt retention) are not available for electrophysiological examination. One cellular signal transduction pathway shared by lymphocytes and renal principal cells utilizes adenosine 3',5'-cyclic monophosphate (cAMP). Elevation of cellular cAMP increases the Na+ flux through ENaC in both lymphocytes and renal principal cells (1, 3-5). In the absence of stimuli, these channels are closed (nonconducting). Therefore, it is possible to assess the functional status of the channels (closed or activated) by testing the response to cAMP directly by using whole cell patch clamp. The magnitude of the basal whole cell Na+ conductance and the response of the Na+ conductance to stimulation with a membrane-permeable analog of cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, are both necessary for an accurate determination. One complication is that the physical pressure of the patch pipette can induce channel activation. Thus one routinely observes activated ENaC in normal cells. This source of variability means that there is a possibility of false positive determinations. However, this limitation can be overcome by assessing the Na+ conductance of multiple cells from a single sample.
On the basis of these criteria, we determined the functional ENaC status (normal or abnormal) of lymphocytes obtained from three distinct phenotypic groups. One group was normotensive. A second group was made up of individuals with controlled hypertension, and the third group consisted of individuals with refractory hypertension. In a subset of volunteers with refractory hypertension, we determined whether lymphocyte Na+ conductance accurately predicted an antihypertensive response, or lack of response, to amiloride. All electrophysiological screens and a pilot efficacy trial were performed with the electrophysiologists blinded as to volunteer identity, blood pressure status, medication status, age, sex, race, and clinical phenotypic group. The clinicians were blinded to the electrophysiological findings. We also performed the same electrophysiological tests on lymphocytes from an individual with primary aldosteronism due to an adrenal adenoma (confirmed by aldosterone measurements, magnetic resonance imaging, and subsequent adrenalectomy).
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METHODS |
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Study subjects. All volunteers were between the ages of 19 and 70 yr and were recruited at the University of Alabama at Birmingham (UAB) Hypertension Clinic (D. A. Calhoun). Normotension was defined as having an unmedicated blood pressure of <140/90 mmHg with no family history of hypertension. Controlled hypertension was defined as having a blood pressure of <140/90 mmHg while taking a single antihypertensive medication. Refractory hypertension was defined as having a blood pressure >140/90 mmHg despite ongoing therapy with three or more antihypertensive agents. Because all of the refractory hypertensive subjects were taking multiple antihypertensive medications, the diagnosis of hypertension was based on multiple evaluations before subjects entered this study. For this study, blood pressure measurements were made with a standard mercury sphygmomanometer according to American Heart Association guidelines. Values were calculated as the means of three measurements after the volunteer sat quietly for 5 min. Subjects were excluded if there was clinical or laboratory evidence of secondary causes of hypertension. The study was reviewed and approved by the UAB Institutional Review Board.
After providing informed consent, volunteers had blood collected (10 ml) for electrophysiological analysis. Nine volunteers (all from the refractory hypertensive group) were prescribed amiloride for 4 wk. At the qualifying visit, amiloride (5 mg daily) was added to their antihypertensive regimen in open-label fashion. Subjects were reassessed 2 wk later, and the dose of amiloride was increased to 5 mg twice daily. After an additional 2 wk of therapy, subjects returned for final blood pressure measurements. For these nine patients, the prescribing physician remained blinded to Na+ conductance analysis throughout the 4-wk treatment period.Whole cell patch clamp. Micropipettes were constructed by using a Narashigi pp-83 two-stage micropipette puller. Micropipettes had an inside diameter of 0.3-0.5 µm and an outside diameter of 0.7-0.9 µm. They were filled with an electrolyte solution containing (in mM) 100 K-gluconate, 30 KCl, 10 NaCl, 20 HEPES, 0.5 EGTA, <10 nM free Ca2+, and 4 ATP at a pH of 7.2. The bath solution was serum-free RPMI-1640 cell culture medium. The solutions accurately approximate the ionic gradients across the cell membrane in vivo. To our knowledge, these conditions do not inhibit any ionic currents but, rather, promote current through all active ion channels. These conditions closely approximate the conditions under which the cell membrane normally functions. Because ENaC current is easily distinguished (and confirmed by specific block with amiloride), these conditions provide the best environment for assessing the function and regulation of this current as it exists in situ.
Pipettes were mounted in a holder and connected to the head stage of an Axon 200A patch-clamp amplifier affixed to a three-dimensional micromanipulator system attached to the microscope. The pipettes were abutted to the cells, and slight suction was applied. Seal resistance was continuously monitored (Nicolet model 300 oscilloscope) by using 0.1-mV electrical pulses from an electrical pulse generator. After formation of seals with resistances in excess of 1 GDetermination of ENaC status.
Lymphocytes were separated and stored by using standard techniques.
They were divided into four 1.5-ml aliquots and frozen at 84°C
until they were tested electrophysiologically. One aliquot from each
sample was thawed. The cells were washed in serum-free RPMI and placed
in a chamber mounted on an inverted microscope. Once the cells settled,
they were subjected to whole cell patch clamp. The determination of
normal or abnormal ENaC regulation was based on the basal channel
activity (activated or quiescent) and the response to cAMP. Cells with
an abnormally high basal Na+ conductance were superfused
with 2 µM amiloride to determine whether amiloride inhibited the
channels in vitro.
Statistical analysis. The blood pressure changes before and after amiloride therapy were also compared statistically using a Student's t-test. All results are expressed as means ± SE.
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RESULTS |
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Prevalence of activated Na+ channels. Cells from blood samples obtained from 106 volunteers were examined by using whole cell patch clamp. The samples were obtained from 34 normotensive individuals, 28 individuals with controlled hypertension, and 44 individuals with refractory hypertension. A total of 817 cells were whole cell patch clamped. Of these, 319 cells were found to have hyperactive ENaC and 239 cells were indeterminate. There were various reasons why ENaC function could not be determined on these cells. For example, the gigaohm seal between the pipette and the cell may have broken during perfusion, and the cell may have swollen or shrunk, making it impossible to accurately measure the currents. The remaining 259 cells had normal ENaC function and regulation.
Table 1 summarizes the age, ethnic, sex, and blood pressure characteristics of each group, including the mean number of antihypertensive medications taken by individuals in each group. Additional clinical parameters such as plasma renin and urine aldosterone levels were measured on hypertensive individuals (as clinically indicated). However, because refractory hypertensive volunteers were all taking multidrug antihypertensive therapy [including in virtually all cases angiotensin-converting enzyme (ACE) inhibitors], measurements of these parameters during the course of this study were not included because they are often altered in response to the medications (10).
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Primary aldosteronism. Several blood samples were obtained before and after removal of the adrenal adenoma from an individual diagnosed with primary aldosteronism. The diagnosis of primary aldosteronism was based on a greater than fourfold increase over normal aldosterone excretion and magnetic resonance images that indicated a unilateral adrenal adenoma.
Figure 4 shows current records from a whole cell clamped lymphocyte known to express the Liddle's disease ENaC polymorphism (A) or from a whole cell clamped lymphocyte isolated from a blood sample obtained from an individual with primary aldosteronism before (B) or after adrenalectomy (C). Figure 4, A and B, shows that the basal ENaC activation found in Liddle's disease also is present in lymphocytes from the individual with primary aldosteronism. Figure 4C shows that once the tumor was removed and aldosterone levels abated, the lymphocyte Na+ conductance reverted to the normal pattern. Figure 4, A and B, shows that in both cases this current is completely inhibited by amiloride. Figure 4C shows that ENaC current was activated with cAMP, just as with normal cells. From these findings, it is apparent that two distinct hypertensive etiologies, Liddle's disease and primary aldosteronism, produce indistinguishable effects on lymphocyte ENaC. These findings may help to explain the high incidence of activated lymphocyte ENaC (25%) in our blinded sample of refractory hypertensives. They also demonstrate in vitro the potential efficacy of amiloride for treatment of the probable underlying cause of the hypertension in both Liddle's disease and primary aldosteronism.
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DISCUSSION |
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We found that constitutively activated ENaC was present in 25% of patients with refractory hypertension in our study population. In contrast, no determinations of constitutive Na+ channel activation were made in samples from individuals with normal blood pressure or from patients with controlled hypertension. These findings indicate that abnormally functioning ENaC is associated with a hypertensive clinical phenotype similar to that produced by confirmed ENaC polymorphisms underlying Liddle's disease. These findings suggest that hyperactive ENaC may be a more common cause of refractory hypertension than was previously thought. Recent studies indicate that primary aldosteronism may be the cause of this high prevalence of hyperactive ENaC (12).
In a subset of subjects, we also found that ENaC functional status was predictive of a favorable or unfavorable antihypertensive response to amiloride. These results imply that determination of lymphocyte ENaC function may be useful for identification of pseudohyperaldosteronism and for identifying an effective antihypertensive therapy where other therapies have failed. The assessment of lymphocyte ENaC function also may be useful in helping to determine whether individuals produce enough aldosterone to induce hypertension. Currently, the determination of primary aldosteronism is somewhat problematic in that no definitive aldosterone level has been deemed to be inappropriately high. Some investigators use a plasma level of 16 ng/dl (8); others use a plasma aldosterone-to-renin ratio of >25 or >50, assuming that the aldosterone suppresses renin secretion. These assessments are subject to great variability because of fluctuations based on dietary consumption and on the fact that many of the individuals being tested are already receiving antihypertensive medication such as ACE inhibitors and other diuretics, which alter the normal secretion of these hormones (9).
Liddle's disease has been considered a rare genetic cause of low-renin
hypertension. This now well-characterized hypertensive etiology appears
to be prototypical for the clinical characteristics of the subjects of
the present study. It is now known that the genetic basis for Liddle's
disease is caused by polymorphisms in either the - or
-subunits
of ENaC. Four distinct polymorphisms have been localized to, and
truncate, the "normal" predicted carboxy-terminal region of the
-subunit of human ENaC (
-hENaC subunit) (17). Another polymorphism truncates the
-hENaC subunit (10).
These gains of function polymorphisms result in constitutively
activated channels (2, 11). The genetic changes produce an
abnormally functioning protein complex that is responsible for the
hypertensive pathophysiology characteristic of the disease. The
findings of the present study imply that similar functional
abnormalities are the probable cause of the hypertension in 25% of our
refractory hypertensive study subjects. Whether these abnormalities are
genetically based is not known. However, because the pathological
hypertension can be controlled by amiloride therapy, identification of
specific genetic alterations underlying the phenomenon does not appear to be essential for successful clinical outcomes.
Our findings support the hypothesis that a high percentage of individuals with pseudohyperaldosteronism will have a clinical status of severe hypertension that is refractory to antihypertensive therapy exclusive of amiloride or other compounds that inhibit ENaC, such as triamterene. All of the subjects in this study who had constitutively activated lymphocyte Na+ channels had severe refractory hypertension. However, there may be a number of different genetic, metabolic, and/or regulatory alterations that can induce abnormally functioning Na+ channels. The example of Liddle's disease (where multiple gene changes already have been confirmed) implies that there may be many other polymorphisms that produce similar functional consequences at the level of the Na+ channel.
Metabolic changes such as an increase in aldosterone also may underlie abnormally activated Na+ channels. The primary aldosteronism patient described in this study had a fivefold excess in 24-h urinary aldosterone excretion and a 2 × 1.5-cm adrenal tumor identified by magnetic resonance imaging. Unmedicated, her blood pressure was 202/106 mmHg. Treated with benazepril (an ACE inhibitor; 20 mg twice daily), amlodipine, (a Ca2+ channel blocker; 10 mg twice daily), and hydrochlorothiazide (a diuretic that does not block ENaC; 25 mg) before being diagnosed of primary aldosteronism, her blood pressure remained uncontrolled at 150/100 mmHg. These clinical characteristics fall precisely within the range observed in our refractory hypertensive study group. Also, amiloride reduced her blood pressure to 133/80 mmHg. Electrophysiological analysis indicated that her lymphocyte Na+ channels were constitutively activated (Fig. 4B). Upon surgical removal of the adenoma and normalization of her urinary aldosterone excretion, her lymphocyte Na+ conductance also normalized (Fig. 4C). Thus, in this individual, primary aldosteronism produced inappropriate activation and regulation of lymphocyte ENaC and produced the same degree of hypertension seen in individuals with pseudohyperaldosteronism.
Because the underlying etiology of hyperactive ENaC may, in many cases, be genetic, assessing the lymphocyte Na+ conductance of direct genetic relatives of affected individuals also may be useful in identifying individuals at high risk, including children. Whole cell patch clamp of peripheral blood lymphocytes is capable of providing both a straightforward method of identification of individuals with pseudohyperaldosteronism at an early age and a method for in vitro testing of potential therapeutic interventions before the development of the pathophysiological consequences produced by prolonged severe hypertension.
Finally, the efficacy of amiloride appears to be substantial on the basis of the positive responses reported here. However, the efficacy appears to be all or none. Amiloride either completely controlled blood pressure or completely failed to alter blood pressure. No intermediate effects of amiloride were observed. This means that in a blinded population-based study of amiloride efficacy, the diuretic would not be very effective, because it would only reduce blood pressure in ~2.5% of the individuals. This small number would be diluted when mean population blood pressure measurements were derived. When translated into practice, this means that amiloride is not considered a first-option antihypertensive. This state of affairs means that more precise identification of individuals who can benefit from amiloride therapy is required. Assessment of lymphocyte ENaC function and regulation is one means of providing this type of specific identification.
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ACKNOWLEDGEMENTS |
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We acknowledge with great appreciation the efforts of Drs. D. J. Benos and S. Oparil for support and critical review of the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-52789-01, National Heart, Lung, and Blood Institute Grant HL-07457, and American Heart Association Grant-in-Aid 0050001N (to J. K. Bubien). J. K. Bubien is an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: J. K. Bubien, Dept. of Physiology and Biophysics, 726 MCLM, Univ. of Alabama at Birmingham, Birmingham, AL 35294 (E-mail: bubien{at}physiology.uab.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 April 2001; accepted in final form 12 July 2001.
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