Assays of the renin-angiotensin-aldosterone system in adrenal disease

INTRODUCTION — Assessment of the renin-angiotensin-aldosterone system has assumed a much greater role in clinical practice, particularly in the evaluation of patients with hypertension. This is mainly attributable to the growing appreciation that primary aldosteronism is a much more common cause of hypertension than previously thought, accounting for as many as 5 to 10 percent of cases, and that most patients lack hypokalemia as a clinical clue. As a result, guidelines advocate screening for primary aldosteronism by measurement of plasma aldosterone and renin rather than that of plasma potassium (which is much less sensitive) and among a much wider population of individuals with hypertension than previously [1].

In addition, the renin-angiotensin-aldosterone system is often evaluated in patients with:

●Hypokalemia or hyperkalemia who may have hyperaldosteronism (or other forms of real or apparent mineralocorticoid excess) or hypoaldosteronism, respectively

●Adrenal insufficiency (to distinguish primary from secondary)

The use of renin, angiotensin, and aldosterone measurements will be reviewed here. The approach to the patient with potential mineralocorticoid excess or hypoaldosteronism is discussed separately. (See "Diagnosis of primary aldosteronism" and "Causes and evaluation of hyperkalemia in adults".)

RENIN — Renin can be measured in terms of its enzymatic activity (plasma renin activity [PRA]), or its mass (active renin concentration). (See "Diagnosis of primary aldosteronism", section on 'Initial testing'.)

Plasma renin activity — PRA is measured by incubating plasma at 37°C without addition of angiotensinogen (renin substrate), relying instead on endogenous angiotensinogen in the plasma [2]. Renin cleaves angiotensinogen to produce a decapeptide, angiotensin I, which can be measured by radioimmunoassay [3].

PRA is expressed as the amount of angiotensin I generated per unit of time. The recommended incubation time, 90 minutes, should be extended up to 18 hours for samples with levels less than 1 ng/mL/h, in order to permit enough generation of angiotensin I to ensure assay reproducibility at the lower end of the scale [4]. Subtracting the amount of preformed angiotensin I in a control aliquot incubated at 4°C is suggested, but this may reduce the reproducibility of the test [4].

Plasma renin concentration — The plasma concentration of the active (cleaved) form of renin can be measured by radioimmunoassay but is usually measured by automated immunometric assay [5]. Since renin does not affect blood pressure directly, but only through angiotensin, PRA is a better reflection of angiotensin II concentrations. Active renin concentration can be influenced by substrate concentrations. As an example, with estrogen administration, substrate increases, and renin concentration falls to maintain angiotensin II concentrations in the normal range [5].

Automated immunometric assays for measuring active renin have been adopted in many laboratories because they are faster and more convenient than PRA or renin concentration radioimmunoassays. However, these methods are not yet sufficiently accurate to supplant PRA as a diagnostic test.

Which is the preferred assay? — If available, PRA, although more time-consuming, is preferred over the direct active renin concentration for at least two reasons:

●PRA, unlike direct renin concentration, takes into account endogenous renin substrate (angiotensinogen) levels. This is important in the context of higher circulating estrogen (eg, women taking exogenous estrogen), which stimulates production of renin substrate. The resulting rise in angiotensin II (via angiotensin I) suppresses renal production of renin enzyme through a negative feedback mechanism, and direct renin concentration falls. In the face of raised substrate but reduced enzyme concentrations, PRA (angiotensin I generated by unit of time) remains relatively constant. As a result, when screening women receiving estrogen-containing preparations for the presence of primary aldosteronism by aldosterone/renin ratio testing, false-positive ratios can occur when renin is measured as direct renin concentration, but not when it is measured as PRA [6,7].

A sustained effect of the pre-ovulatory surge in estrogen is also thought to be responsible for the reported occurrence of false-positive aldosterone/renin ratios using direct renin concentration (but not PRA) during the luteal phase of the menstrual cycle [8].

●Concerns also exist about the reliability of the now widely used automated immunometric, chemiluminescent methods of measuring direct active renin concentration. This is particularly at the lower end of the clinical range, which is important when screening for primary aldosteronism using the aldosterone/renin ratio, a parameter that is dependent especially on the renin level. This may, in part, relate to the fact that these automated systems are calibrated by only a two-point recalibration against a stored master curve, contrasting with the multiple calibration points (permitting better curve resolution) used in each PRA assay.

Normal values — Normal, morning PRA values for seated individuals range from approximately 1 to 4 ng/mL per hour (0.8 to 3.0 nmol/L per hour). Corresponding active renin concentrations are 8 to 35 mU/L.

Factors that affect renin levels include [9,10]:

●Sodium intake – Renin may be stimulated by dietary salt restriction and suppressed by consumption of a high-salt diet.

●Age – Renin levels gradually fall as renal function declines.

●Sex, menstrual phase, and pregnancy – Renin levels are higher in the luteal phase of the menstrual cycle and during pregnancy [11,12], in part because of the mineralocorticoid antagonist activity of progesterone (high concentrations of progesterone antagonize aldosterone action at the mineralocorticoid receptor). The rise in PRA during the luteal phase appears to be greater than that for direct renin concentration, probably because the late follicular surge in estrogen leads to a sustained rise in renin substrate (angiotensinogen) persisting into the luteal phase, resulting in higher angiotensin I and II, which in turn results in reduced renin enzyme production by negative feedback [8]. Menstruating women have lower renin levels during menses and the follicular (but not luteal) phases of the menstrual cycle when compared with age-matched men [8].

●Time of day – Renin levels show a diurnal rhythm, being highest in the early morning upon awakening and falling during the day [13].

●Posture – Following assumption of upright posture, the translocation of blood into the lower limbs is associated with an increase in renin, released from juxtaglomerular cells in response to a fall in renal perfusion pressure and an increase in sympathetic output and beta-adrenergic receptor stimulation [14].

●Medication use – Renin is increased with diuretics (including spironolactone), dihydropyridine calcium channel blockers, angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists. Levels are decreased by beta blockers, clonidine, or alpha-methyldopa (all of which reduce beta-sympathetic stimulation of renin release), or nonsteroidal antiinflammatory agents (NSAIDs) (which promote salt retention and also inhibit renal prostaglandin production). Direct renin inhibitors lower PRA but raise direct renin concentration. In order to facilitate interpretation of aldosterone and renin levels, these agents should, when possible, be ceased for at least two and preferably four weeks (longer, eg, >6 weeks, in the case of diuretics) before sample collection. During this period, antihypertensive medications that have minimal effects on renin (eg, verapamil slow release, to which can be added hydralazine and/or alpha blockers such as prazosin and/or moxonidine) can be used in place of the above interfering agents in order to maintain control of hypertension [10,15,16].

●Chronic kidney disease – In chronic kidney disease, renal renin-producing capacity is reduced, and salt retention contributes to renin suppression.

●Race – Renin levels are lower in Black than White individuals [17].

Interpretation in adrenal disease — Adrenal diseases have a variable effect on renin levels, being determined primarily by whether the disease results in an increase or a decrease in the secretion of mineralocorticoids (which induce volume expansion, resulting in suppression of renin release).

Renin levels are usually low in:

●Primary aldosteronism, in which aldosterone production is excessive (relative to body sodium status), and independent of its normal regulator, renin/angiotensin [18].

Primary aldosteronism may be due to an aldosterone-producing adrenocortical tumor (adenoma or, rarely, carcinoma), bilateral adrenal hyperplasia, or glucocorticoid-remediable aldosteronism (familial hyperaldosteronism type I). (See "Pathophysiology and clinical features of primary aldosteronism" and "Familial hyperaldosteronism".)

●Patients with 11-beta-hydroxylase or 17-alpha-hydroxylase deficiency (due to mutations in CYP11B1 and CYP17, respectively), which are hypertensive forms of congenital adrenal hyperplasia associated with excessive production of the mineralocorticoid deoxycorticosterone (DOC), driven by corticotropin (ACTH, levels of which are high as a result of deficient cortisol production). (See "Uncommon congenital adrenal hyperplasias", section on 'Lipoid congenital adrenal hyperplasia' and "Uncommon congenital adrenal hyperplasias", section on '11-beta-hydroxylase deficiency'.)

●Primary glucocorticoid resistance [19], which is again associated with ACTH simulation and excessive DOC production, causing hypertension. (See "Uncommon congenital adrenal hyperplasias", section on 'Lipoid congenital adrenal hyperplasia'.)

●Patients with DOC-producing adrenal tumors [20]. (See "Diagnosis of primary aldosteronism".)

●Ectopic ACTH syndrome [21], in which cortisol levels may be high enough to overwhelm the 11-beta-hydroxysteroid dehydrogenase type 2 enzyme (which normally, by converting cortisol to cortisone, prevents excessive stimulation of the mineralocorticoid receptor) [21]. In addition, the high levels of ACTH may lead to excessive production of DOC. (See "Causes and pathophysiology of Cushing syndrome", section on 'Ectopic ACTH syndrome'.)

They are usually normal in:

●Secondary adrenal insufficiency (ie, hypopituitarism or isolated ACTH deficiency). (See "Causes of hypopituitarism".)

●Cushing syndrome, but they can be low when there is a marked degree of hypercortisolism. (See "Epidemiology and clinical manifestations of Cushing syndrome".)

They are usually high in:

●Primary adrenal insufficiency, including Addison disease. (See "Clinical manifestations of adrenal insufficiency in adults".)

●Patients with congenital adrenal hyperplasia due to deficiencies in either steroidogenic acute regulatory protein (StAR), side-chain cleavage enzyme (CYP11A1), 3-beta-hydroxysteroid dehydrogenase (HSD3B2), 21-hydroxylase (CYP21A2) [22] or aldosterone synthase (CYP11B2) [23] in which deficient production of steroids with mineralocorticoid activity leads to salt wasting. (See "Genetics and clinical manifestations of classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency" and "Uncommon congenital adrenal hyperplasias", section on 'Lipoid congenital adrenal hyperplasia'.)

Interpretation in nonadrenal conditions — Renin levels are low in:

●Liddle syndrome, in which gain-of-function mutations in the epithelial sodium channel lead to sodium retention, hypertension, and usually hypokalemia [24]. (See "Genetic disorders of the collecting tubule sodium channel: Liddle syndrome and pseudohypoaldosteronism type 1".)

●Congenital or acquired (eg, through ingestion of licorice) deficiency of 11-beta-hydroxysteroid dehydrogenase type 2, resulting in activation of the mineralocorticoid receptor by cortisol [25,26]. (See "Apparent mineralocorticoid excess syndromes (including chronic licorice ingestion)".)

●Mutations of the mineralocorticoid receptor gene, which cause a modest constitutive activation of the receptor, and permit progesterone and spironolactone to act as agonists rather than antagonists [27].

●Familial hyperkalemic hypertension (also known as pseudohypoaldosteronism type 2 or Gordon syndrome), which is associated with sodium and potassium retention due to defects in genes encoding serine-threonine kinases (WNK1 and WNK4) [28,29] or ubiquitinating enzymes (CUL3 and KLCH3) [30] expressed in the distal nephron and altering expression of the sodium-potassium cotransporter. (See "Etiology, diagnosis, and treatment of hypoaldosteronism (type 4 RTA)", section on 'Pseudohypoaldosteronism type 2 (Gordon's syndrome)'.)

Unlike primary aldosteronism, aldosterone concentrations are chronically suppressed (as a result of chronic suppression of renin secretion) in all of these salt-dependent, low-renin forms of hypertension, with the exception of familial hyperkalemic hypertension, in which chronically elevated plasma potassium concentrations limit suppression of aldosterone.

Renin levels are also often low in patients with chronic kidney disease, in which reduced renin-producing capacity and salt retention contribute to renin suppression [31].

Renin levels are high:

●In patients with "renovascular" forms of hypertension and reninoma (levels may also be normal).

●In malignant hypertension.

●In pseudohypoaldosteronism type 1, in which salt wasting occurs due to resistance to the actions of aldosterone, resulting from mutations in the gene encoding the epithelial sodium channel (autosomal recessive) or mineralocorticoid receptor (autosomal dominant or sporadic). (See "Genetic disorders of the collecting tubule sodium channel: Liddle syndrome and pseudohypoaldosteronism type 1", section on 'Pseudohypoaldosteronism type 1' and "Etiology, diagnosis, and treatment of hypoaldosteronism (type 4 RTA)".)

●In Bartter and Gitelman syndromes, in which mutations in renal tubular ion channels also lead to salt wasting. (See "Inherited hypokalemic salt-losing tubulopathies: Pathophysiology and overview of clinical manifestations".)

●In heart failure, hepatic cirrhosis, and nephrotic syndrome, in which edema is associated with relative intravascular hypovolemia [18].

ALDOSTERONE — Serum aldosterone is measured by radioimmunoassay [32]. The assay requires a high-affinity, highly specific antibody because aldosterone concentrations in serum are less than 1 percent those of cortisol. Faster, more convenient methods of directly measuring aldosterone are available, using immunometric techniques and automated machinery [33].

Like the automated active renin concentration assay, considerable work is required to validate and improve these methods before they can be considered accurate enough to use for diagnostic purposes (eg, evaluating patients for possible primary aldosteronism).

Concerns with the automated aldosterone assay include:

●Potential for false-negative suppression tests. In an analysis, aldosterone levels were below the assay's limit of detection for over half the samples collected from normal subjects and nearly half those from patients with primary hypertension (formerly called "essential" hypertension) [34].

●Nonspecific interference, possibly due to the brevity of the "wash" immediately prior to chemiluminescence, leading to an unacceptably high "blank" value in bilaterally adrenalectomized and Addisonian patients.

●Weaknesses of the assay system: It is calibrated by only a two-point recalibration against a stored master curve, and the two calibrators are reconstituted lyophilized aldosterone. Inclusion of blanks using plasma from bilaterally adrenalectomized or Addisonian patients, as well as calibration using plasma pools from persons with known low, medium or high values, would permit very valuable, in-house, ongoing evaluation of the system.

Highly accurate and reproducible methods of measuring aldosterone using high-performance liquid chromatography and tandem mass spectrometry have been developed [35] and are now in clinical use. The incorporation of new semi-automated technology has meant that these methods can generate results rapidly and with relatively high throughput.

Normal values — Morning serum (and plasma) aldosterone concentrations range from 5 to 30 ng/dL (140 to 830 pmol/L) in seated, healthy individuals with unrestricted salt intakes [36]. Concentrations are lower in Black than in White individuals [37]. Other aspects that affect aldosterone concentrations include:

●Diurnal variation – There is a diurnal variation in serum aldosterone concentrations, with highest concentrations at approximately the time of awakening and lowest in the evening [38].

●Extracellular fluid volume – Serum aldosterone concentrations are normally related to extracellular fluid volume, being increased by dehydration, dietary sodium restriction, or sodium diuresis, and decreased by oral or intravenous sodium loading.

●Plasma potassium concentration – Aldosterone concentrations are also related to plasma potassium concentrations, being increased by hyperkalemia and decreased by hypokalemia.

●Menstrual phase and pregnancy – Aldosterone concentrations tend to be increased in the luteal phase of the menstrual cycle (during which they are significantly higher than in age-matched males) [8] and are increased up to 10 times normal by the third trimester of pregnancy [11,12].

●Posture – Following assumption of upright posture, the translocation of blood into the lower limbs is associated with a rise in plasma aldosterone [39]. This results partly from an increase in renin [14]. In addition, reduced metabolic clearance of aldosterone occurs due to reduced hepatic blood flow [14].

In practice, for most situations where aldosterone and renin levels are to be assessed, most centers draw a morning, ambulatory, upright sample.

Interpretation in adrenal disease — Serum aldosterone levels are usually low:

●In patients with 11-beta-hydroxylase or 17-alpha-hydroxylase deficiencies [40,41], which are associated with excessive production of deoxycorticosterone (DOC) (and possibly corticosterone in the latter condition).

●In some patients with Cushing syndrome, with the extent of suppression depending upon the degree of hypersecretion of cortisol and other salt-retaining steroids.

Serum aldosterone levels are usually low or undetectable:

●In primary adrenal insufficiency and may be normal or low in patients with chronic corticotroph deficiency.

●In patients with deficiencies of either steroidogenic acute regulatory protein (StAR), side-chain cleavage enzyme, 3-beta-hydroxysteroid dehydrogenase, and aldosterone synthase since aldosterone synthesis is directly impaired [23]. (See "Genetics and clinical manifestations of classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency" and "Uncommon congenital adrenal hyperplasias", section on 'Lipoid congenital adrenal hyperplasia'.)

Levels are high in:

●Many patients with primary aldosteronism but are often normal (inappropriately, in the face of renin suppression) [10,14].

Interpretation in other (nonadrenal) conditions — Aldosterone levels may be low:

●In patients with Liddle syndrome, 11-beta-hydroxysteroid dehydrogenase deficiency, primary glucocorticoid resistance, 11-beta-hydroxylase and 17-alpha-hydroxylase deficiencies, ectopic corticotropin (ACTH) syndrome, or activating mutations of the mineralocorticoid receptor. (See 'Interpretation in nonadrenal conditions' above.)

●In hyporeninemic hypoaldosteronism associated with diabetic nephropathy and some other renal disorders. (See "Etiology, diagnosis, and treatment of hypoaldosteronism (type 4 RTA)".)

●Or normal in pseudohypoaldosteronism type 2. (See 'Interpretation in nonadrenal conditions' above.)

Levels may be high in patients with secondary hyperaldosteronism in which raised aldosterone is secondary to raised renin/angiotensin levels:

●In renal artery stenosis or reninoma (causing renin-dependent hypertension).

●In malignant hypertension.

●In pseudohypoaldosteronism type 1 (resulting in resistance to aldosterone action). (See "Genetic disorders of the collecting tubule sodium channel: Liddle syndrome and pseudohypoaldosteronism type 1", section on 'Pseudohypoaldosteronism type 1'.)

●In heart failure, cirrhosis, and nephrotic syndrome, in which edema is associated with relative intravascular hypovolemia and (in the case of heart failure and hepatic cirrhosis) aldosterone metabolism may be impaired [18].

●In patients receiving diuretic medications (including spironolactone) or preparations containing progesterone or drospirenone (both of which antagonize aldosterone action at the level of the mineralocorticoid receptor) [6].

●Or normal in Bartter syndrome and Gitelman syndrome, in which hypokalemia limits the aldosterone rise that results from stimulation by raised renin levels.

Urine aldosterone excretion — Aldosterone, in the form of its 18-glucuronide, is measured by radioimmunoassay. Its excretion varies from approximately 5 to 19 mcg (14 to 53 nmol) per 24 hours. The excretion of tetrahydroaldosterone-18-glucuronide, a more abundant metabolite, ranges from approximately 12 to 65 mcg (33 to 178 nmol) per 24 hours.

As with aldosterone/renin ratio testing, diuretics (including spironolactone) should be withheld for at least six weeks before testing, and beta-blockers, methyldopa, clonidine, dihydropyridine calcium channel blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, direct renin inhibitors, and nonsteroidal antiinflammatory drugs (NSAIDs) for at least two weeks (and preferably four) before urinary aldosterone excretion is measured. Hypokalemia should be corrected with potassium chloride (KCl) supplements, and the patient should be instructed to follow a diet with a liberal sodium intake.

Urinary aldosterone excretion varies in patients with adrenal disease in a manner similar to that described above for serum aldosterone.

Aldosterone secretory rate — Aldosterone secretion or production rates can be measured by isotopic dilution methods [18,40]. Aldosterone, labeled with a radioactive or nonradioactive isotope, is injected intravenously. Either a series of blood samples or a 24-hour urine collection is then obtained. The extent of dilution of labeled aldosterone by endogenous unlabeled aldosterone (ie, the decrease in specific activity) is determined and can be used to calculate the amount of endogenous aldosterone secreted during the interval.

Because these assays are technically demanding, time-consuming, and expensive and the information required for clinical decisions can usually be obtained by other means, steroid secretion rates are rarely measured nowadays.

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Primary aldosteronism".)

SUMMARY

●Clinical uses – Assessment of the renin-angiotensin-aldosterone system has assumed a much greater role in clinical practice, particularly in the evaluation of patients with hypertension. This is mainly attributable to the growing appreciation that primary aldosteronism is a much more common cause of hypertension than previously thought, accounting for as many as 5 to 10 percent of cases, and that most patients lack hypokalemia as a clinical clue. (See 'Introduction' above and "Diagnosis of primary aldosteronism".)

In addition, the renin-angiotensin-aldosterone system is often evaluated in patients with hypokalemia or hyperkalemia who may have hyperaldosteronism (or other forms of real or apparent mineralocorticoid excess) or hypoaldosteronism, respectively, and adrenal insufficiency (to distinguish primary from secondary).

●Renin measurement – Renin can be measured in terms of its enzymatic activity (plasma renin activity [PRA]) or its mass (active renin concentration). (See 'Renin' above and "Diagnosis of primary aldosteronism", section on 'Initial testing'.)

Normal, morning PRA values for seated individuals range from approximately 1 to 4 ng/mL per hour (0.8 to 3.0 nmol/L per hour). Levels may be affected by sodium intake, age, time of day, upright posture, medication use, and endogenous progesterone. (See 'Normal values' above.)

●Aldosterone measurement – Morning serum (and plasma) aldosterone concentrations range from 5 to 30 ng/dL (140 to 830 pmol/L) in seated, healthy individuals with unrestricted salt intakes. Like PRA, aldosterone levels are affected by sodium intake, time of day, upright posture, and endogenous progesterone. (See 'Normal values' above.)