TRANSLATIONAL PHYSIOLOGY
Hyperactive ENaC identifies hypertensive individuals amenable to amiloride therapy

Artensie R. Carter1, Zhen Hong Zhou2, David A. Calhoun1, and James K. Bubien2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha , beta , and gamma , of ENaC have been cloned (6, 7, 13, 14). Subsequently, it was found that polymorphisms in beta -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 beta -ENaC subunit gene underlies the disorder in the proband (17). Additional polymorphisms in the beta - and gamma -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 alpha - and beta -ENaC subunits (5). Also, immunofluorescence analysis with anti-alpha -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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 GOmega , another suction pulse was applied to form the whole cell configuration by rupturing the membrane within the seal but leaving the seal intact. Successful completion of this procedure produced a sudden increase in capacitance with no change in seal resistance. The magnitude of the capacitance is a direct function of the membrane available to be voltage clamped (i.e., the membrane area and, hence, cell size). Typically, this capacitance was between 5 and 10 pF for peripheral blood lymphocytes. The cells were then held at a membrane potential of -60 mV to approximate the normal membrane potential. The cells were then voltage clamped sequentially for 0.8 s each to membrane potentials of -160, -140, -120, -100, -80, -60, -40, -20, 0, 20, and 40 mV, returning to the holding potential of -60 mV for 0.8 s between each test voltage. This procedure provided voltages sufficient to measure inward Na+ (at more hyperpolarized potentials) and outward K+ currents (at more depolarized potentials). The currents were recorded digitally and filed in real time. The entire procedure was performed with the use of a DOS Pentium computer modified for analog-to-digital (A/D) signals with pCLAMP 6 software, with an A/D interface controlled by pCLAMP (Axon Instruments, Sunnyvale, CA).

Determination 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.

Initially, three cells were examined from each sample. This number proved to be insufficient, because when the blind was broken, an excessive number of false positive determinations were discovered (i.e., activated Na+ channels in cells from normotensive and controlled hypertensive individuals). Subsequently, the samples were reblinded and reexamined electrophysiologically by using six additional cells from each original blood sample.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Ethnic/sex makeup of the study population

Table 2 shows the average number of cells for each sample and the average number of activated, indeterminate, and normal cells for each sample. What is not shown by this descriptive statistical summary is that in 11 samples from the refractory hypertensive group, there were zero normal cells. It was the failure to find any normal cells that was the determining factor that classified a sample as abnormal. All the samples from the other groups had at least one normal cell and, therefore, were classified as normal.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Average number of cells per blood sample that were tested for each clinical subset of study subjects

We have demonstrated previously that lymphocytes from individuals with confirmed Liddle's disease have constitutively activated ENaC (2, 5, 11). Figure 1 shows whole cell clamp recordings from a transformed lymphocyte expressing the ENaC beta -subunit polymorphism responsible for Liddle's disease in the proband. In these cells the Na+ currents are constitutively activated, and all of the activated current is inhibited by amiloride.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   Electrophysiological records from a transformed lymphocyte obtained from an individual with genetically confirmed Liddle's disease (the proband). These current records show hyperactive epithelial Na+ channels (ENaC) (A; downward deflections) and the ability of amiloride to completely inhibit the hyperactive ENaC current in vitro (B).

Figure 2 shows whole cell current records from lymphocytes isolated from volunteer blood samples. Figure 2A shows records from a lymphocyte isolated from a blood sample donated by an individual with normal blood pressure. These current records show that there is virtually no basal Na+ current. The current is activated by treatment with cAMP. In Fig. 2A, left, the downward deflections are compressed, indicating that the Na+ channels were closed under nonstimulated conditions. In Fig. 2A, right, the downward deflections (inward Na+ currents) are much larger, in response to treatment with cAMP. The "ragged" appearance of the inward currents results from channels opening and closing in groups, rather than independently, and is a direct consequence of the single-channel activity (5). The activating effect of cAMP reflects normal ENaC function (1, 3-5). In contrast, cells with activated ENaC (Fig. 2B, left) are inhibited by cAMP treatment (Fig. 2B, right). This phenomenon has been described in detail previously (3).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2.   Peripheral blood lymphocyte whole cell currents. A: currents from a cell obtained from a blood sample donated by a normotensive volunteer. The relatively small basal inward currents (left) are activated by treatment with cAMP (right). This is normal ENaC function and regulation. B: currents obtained from a cell isolated from a blood sample given by a volunteer with refractory hypertension. The inward currents are constitutively activated (left). These currents are inhibited by cAMP treatment (right). These currents are also inhibited completely by amiloride (see Fig. 1). In this cell, ENaC function and regulation is abnormal. 8-CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate.

Using these criteria (i.e., quiescent inward currents activated by cAMP treatment = normal; hyperactive basal inward currents inhibited by cAMP = abnormal), we characterized all study subjects as normal or activated. Finding a single normal cell in any given sample was sufficient to classify that sample as normal. This was possible because aldosterone induces irreversible activation of ENaC in lymphocytes (18). Also, ENaC polymorphisms (if present) would have to be in every lymphocyte. These facts virtually eliminate the possibility of false negative determinations.

In all samples with basal hyperactive ENaC, in vitro efficacy of amiloride was also assessed. Initially, three cells per sample were used for the analysis. When the blind was broken, 9 of 34 samples from normotensive individuals and 5 of 28 samples from subjects with controlled hypertension were categorized as abnormal, for a total of 14 of 62 (23%). Because pseudohyperaldosteronism produces severe hypertension that does not respond to conventional antihypertensive therapy, we had hypothesized that all samples from normotensive and controlled hypertensive individuals would be normal. Thus these findings strongly suggested that making a determination from three cells per sample produced an unacceptably high proportion of false positives.

Use of the patch-clamp technique to make these determinations lends itself to false positives because the cells express the channels constitutively. The mechanical forces produced during patch clamping could activate the channels, or the cells could be in a physiological state where the channels are endogenously activated. Conversely, it is improbable that normal channel regulation can be observed in cells expressing abnormal channels, or channels activated by aldosterone. It already has been demonstrated that aldosterone-mediated ENaC activation does not reverse within 4 h (18). Therefore, the possibility of false negative determinations is virtually nonexistent. We then hypothesized that the inherent sources of variability that were capable of producing produce false positive determinations could be overcome by increasing the number of cells tested from each sample.

To test this hypothesis, we reblinded and reexamined the same samples, using six additional cells from each sample as the criterion for segregating normal from abnormal. In this second blinded trial, 5 of 27 samples from normotensive individuals and 1 of 15 samples from controlled hypertensives were categorized as abnormal. The false positive percentage (14%) remained unacceptably high. However, when the results of the two sample sets were combined (producing a minimum sample size of 9 cells per determination), it was found that 0 of 34 samples from normotensive individuals and 0 of 28 samples from controlled hypertensives were abnormal, because every sample from these groups had at least one normal set of current records. Thus the hypothesis that individuals from these clinical subsets do not have constitutively activated amiloride-sensitive Na+ channels was substantiated. These findings are summarized in Fig. 3. The results show that because of the intrinsic variability inherent in the technique, the minimum number of cells that had to be examined was nine. On the basis of these findings, a whole cell patch-clamp analysis showed that nine of nine peripheral blood lymphocytes with constitutively activated ENaC resulted in a determination of hyperactive ENaC with a reliability of >99%. When this criterion (a minimum of 9 of 9 cells of hyperactive ENaC) was applied to the samples from the refractory hypertensive subset of volunteers, it was found that 11 of 44 samples (25%) had hyperactive ENaC and that no cells had normal ENaC activity. The subset with hyperactive ENaC included one African-American female, six African-American males, two Caucasian males, and two Caucasian females. Thus the sex and racial makeup of this group were not significantly different from those of the overall study population. The age and blood pressure characteristics of this group (age: 46.6 ± 3.1 yr; blood pressure: 167 ± 6.3/102 ± 3.0 mmHg) were not different from the characteristics of the entire refractory hypertensive group (Table 1). Thus individuals with this potential hypertensive etiology could not be segregated on the basis of blood pressure measurements alone.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Percent of confirmed false positive determinations. The x-axis represents the number of cells per sample used to make a normal or abnormal determination. Values are means ± SE; n = total no. of samples. When 3 cells were used, the false positive percentage was the highest. This percentage was approximately halved when the number was increased to 6 cells in an independent blinded reexamination of the same samples. Combining these 2 studies (increasing the minimum number of cells per sample that were examined to 9) resulted in no positive findings from the controlled hypertensive and normotensive groups of study subjects.

Concurrently, nine randomly selected volunteers with refractory hypertension completed 4 wk of amiloride therapy. Initially, the mean resting blood pressure of these nine subjects was 176 ± 6.0/108 ± 3 mmHg (on an average of 4 antihypertensive medications). For the first 2 wk of therapy, the volunteers took 5 mg of amiloride once a day. All of these volunteers had their daily dose of amiloride increased to 5 mg twice a day after 2 wk. Blood pressure was measured as described previously at the conclusion of the 4 wk of therapy.

When the blind was broken, the whole cell current analysis showed that three of the nine subjects had hyperactive ENaC. The remaining six individuals had normal lymphocyte Na+ channel function. These percentages approximated the overall percentage of activated individuals in the entire refractory hypertensive study population. In the subjects with activated Na+ channels, 4 wk of amiloride therapy reduced the average systolic and diastolic blood pressures by 24 ± 7.0 (P = 0.03) and 15 ± 6.0 mmHg (P = 0.04), respectively (Table 3). As a group, the average blood pressure of these individuals fell into the normal range.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Systolic and diastolic blood pressure in subjects at baseline and after 4 wk of amiloride therapy in subjects with activated lymphocyte amiloride-sensitive ENaC

In the six subjects with normal Na+ channel function, the average blood pressure was not significantly altered by Na+ channel inhibition with amiloride (systolic blood pressure: 174 ± 19 mmHg; mean diastolic blood pressure: 104 ± 11 mmHg). Table 4 shows the blood pressure measurements before and after amiloride treatment of the individuals in this group. Of interest is the reduction in blood pressure of subject 6 in this group. It is possible that this reduction in blood pressure is an example of a placebo effect. However, it is known that both insulin and catecholamines can activate ENaC. ENaC activation by these agonists is transient and washes out rapidly. Lymphocytes from an individual with chronically elevated insulin or catecholamines would appear normal in our assay. Thus it is possible that hyperactive ENaC did play a role in this individual's hypertension, but the etiology was not aldosteronism or pseudohyperaldosteronism. These findings show that specific inhibition of Na+ channels with amiloride normalized blood pressure in individuals with hyperactive ENaC but (as a group) had no significant effect on blood pressure of severely hypertensive individuals with normal ENaC function.

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Systolic and diastolic blood pressure in subjects at baseline and after 4 wk of amiloride therapy in subjects with normal lymphocyte ENaC

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.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 4.   A: whole cell clamp records from a cell known to express the prototypical ENaC polymorphism for Liddle's disease. The hallmark currents are the downward deflections seen in the basal state. The upward deflections result from K+ currents. Expression of K+ channels varies greatly from lymphocyte to lymphocyte. Some cells express multiple K+ channel types, resulting in the complex whole cell currents shown in A. Some cells express a single type of K+ channel, typified by the records in B, and some cells do not express K+ channels, as shown by the records in C. The expression of K+ channels (or lack thereof) appears to have no influence on the expression of ENaC currents and therefore does not affect the outcome of the findings reported here. B: whole cell records from a lymphocyte isolated from a peripheral blood sample taken from an individual with primary aldosteronism before surgical treatment. C: whole cell current records obtained from a peripheral blood lymphocyte isolated from the same patient after adrenalectomy.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta - or gamma -subunits of ENaC. Four distinct polymorphisms have been localized to, and truncate, the "normal" predicted carboxy-terminal region of the beta -subunit of human ENaC (beta -hENaC subunit) (17). Another polymorphism truncates the gamma -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.


    ACKNOWLEDGEMENTS

We acknowledge with great appreciation the efforts of Drs. D. J. Benos and S. Oparil for support and critical review of the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bubien, JK. Whole cell sodium conductance of principal cells freshly isolated from rat cortical collecting duct. Am J Physiol Cell Physiol 269: C791-C796, 1995[Abstract].

2.   Bubien, JK, Ismailov II, Berdiev BK, Cornwell T, Lifton RP, Fuller CM, Achard J-M, Benos DJ, and Warnock DG. Liddle's disease: abnormal regulation of amiloride-sensitive Na+ channels by beta -subunit mutation. Am J Physiol Cell Physiol 270: C208-C213, 1996[Abstract/Free Full Text].

3.   Bubien, JK, Jope RS, and Warnock DG. G-proteins modulate amiloride-sensitive sodium channels. J Biol Chem 269: 17780-17783, 1994[Abstract/Free Full Text].

4.   Bubien, JK, and Warnock DG. Amiloride-sensitive sodium conductance in human B lymphoid cells. Am J Physiol Cell Physiol 265: C1175-C1183, 1993[Abstract/Free Full Text].

5.   Bubien, JK, Watson B, Kahn MA, Langloh A-L B, Fuller CM, Berdiev B, Tousson A, and Benos DJ. Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes. J Biol Chem 276: 8557-8566, 2001[Abstract/Free Full Text].

6.   Canessa, CM, Horisberger J-D, and Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467-470, 1993[ISI][Medline].

7.   Canessa, CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger J-D, and Rossier BC. Amiloride-sensitive Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994[ISI][Medline].

8.   Fardella, CE, Mosso L, Gomez-Sanchez C, Cortes P, Soto J, Gomez L, Pinto M, Huete A, Oestreicher E, Foradori A, and Montero J. Primary hyperaldosteronism in essential hypertensives: prevalence, biochemical profile, and molecular biology. J Clin Endocrinol Metab 85: 1863-1867, 2000[Abstract/Free Full Text].

9.   Hansen, EF, Bendtsen F, and Henriksen JH. Effects on plasma angiotensin-converting enzyme activity and circulating renin of lisinopril and enalapril alone and in combination with propranolol in healthy volunteers. Pharmacol Toxicol 84: 110-114, 1999[ISI][Medline].

10.   Hansson, JI, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa CM, Iwasaki T, Rossier BC, and Lifton RP. Hypertension caused by a truncated sodium channel gamma  subunit: genetic heterogeneity of Liddle syndrome. Nat Genet 11: 76-82, 1995[ISI][Medline].

11.   Ismailov, II, Berdiev BK, Fuller CM, Bradford AL, Lifton RP, Warnock DG, Bubien JK, and Benos DJ. Peptide block of constitutively activated Na+ channels in Liddle's disease. Am J Physiol Cell Physiol 270: C214-C223, 1996[Abstract/Free Full Text].

12.   Lim, PO, Dow E, Brennan G, Jung RT, and MacDonald TM. High prevalence of primary aldosteronism in the Tayside hypertension clinic. J Hum Hypertens 14: 311-315, 2000[ISI][Medline].

13.   McDonald, FJ, Price MP, Snyder PM, and Welsh MJ. Cloning and expression of the beta - and gamma -subunits of the human epithelial sodium channel. Am J Physiol Cell Physiol 268: C1157-C1163, 1995[Abstract/Free Full Text].

14.   McDonald, FJ, Snyder PM, McCray PB, and Welsh MJ. Cloning, expression and tissue distribution of a human amiloride-sensitive Na+ channel. Am J Physiol Lung Cell Mol Physiol 268: L728-L734, 1995.

15.   Rayner, BL, Opie LH, and Davidson JS. The aldosterone/renin ratio as a screening test for primary aldosteronism. S Afr Med J 90: 394-400, 2000[ISI][Medline].

16.   Scheinman, SJ, Guay-Woodford Thakker RV, and Warnock DG. Genetic disorders of renal electrolyte transport. N Engl J Med 340: 1177-1187, 1999[Free Full Text].

17.   Shimkets, RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, Gill JR, Jr, Ulick S, Milora RV, Finding JW, Canessa CM, Rossier BC, and Lifton RP. Liddle's syndrome: heritable human hypertension caused by mutations of the beta  subunit of the epithelial sodium channel. Cell 79: 407-414, 1994[ISI][Medline].

18.   Zhou, Z-H, and Bubien JK. Nongenomic regulation of ENaC by aldosterone. Am J Physiol Cell Physiol 281: C1118-C1130, 2001[Abstract/Free Full Text].


Am J Physiol Cell Physiol 281(5):C1413-C1421
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society