A Renin-ssance in Primary Aldosteronism Testing: Obstacles and Opportunities for Screening, Diagnosis, and Management

Primary aldosteronism (PA)9 is a group of adrenal disorders characterized by autonomous production of aldosterone independent of angiotensin II (AngII) stimulation. Idiopathic adrenal hyperplasia is the most common cause followed by aldosterone-producing adenomas (APA or Conn syndrome), unilateral adrenal hyperplasia, adrenal carcinoma, and rare familial forms. Aldosterone excess results in sodium and water retention and potassium excretion, leading to volume expansion, concomitant hypertension, and variable degrees of hypokalemia. As the most common form of secondary hypertension, PA is recognized as an important public health concern. The diagnosis is infrequently considered despite widely available screening procedures. In addition to the hypertension, the negative effects of excess aldosterone are thought to be related to inflammation and fibrosis of various target organs. As a result, patients with PA are at increased risk of cardiovascular and chronic kidney disease compared with age-matched and blood pressure–matched patients with essential hypertension. For these reasons early identification and treatment are necessary to prevent morbidity and mortality associated with this curable form of chronic hypertension. The diagnosis of PA relies on biochemical evidence of relative aldosterone excess and confirmation of abnormal aldosterone production using suppression testing. The identification of the PA subtype is primarily accomplished with adrenal venous sampling and imaging studies. The aldosterone-to-renin ratio (ARR) is generally considered the best first-line screening test for hypertensive patients in whom there is clinical suspicion of PA. Excessive and autonomous aldosterone secretion is characteristically accompanied by low or undetectable renin due to feedback inhibition from sodium excess and increased blood pressure. However, no consensus exists for an internationally recognized ARR cutoff and many challenges, from preanalytical to analytical to postanalytical, have hindered the development of definitive guidance on how to interpret the ARR in clinical practice. In this Q&A, 4 experts from around the world discuss the state-of-the-art in screening and diagnosis of PA and the many challenges associated with laboratory testing. Estimates of prevalence suggest that PA is more common than once thought. What is known about the current prevalence, and why do you think historical estimates were misleading? Are certain populations at increased risk? Daniel Holmes: The largest studies have consistently determined the prevalence to lie between 5% and 15% in hypertensive populations. Historical estimates were misleading because it was assumed that hypokalemia was a sine qua non of the diagnosis. However, about half of patients with APA, and only 17% of those with idiopathic adrenal hyperplasia, are hypokalemic. The other contributing factor may be that many patients with PA do not have increases of aldosterone above the reference interval. Rather, they have increases relative to AngII stimulation, the surrogate measure of which is plasma renin activity (PRA). The Endocrine Society Guideline, in the US, recommends a targeted screening approach in subpopulations with hypertension having clinical features suggestive of PA, which are: 1) those with moderate to severe or resistant hypertension; 2) hypertension with spontaneous or diuretic-induced hypokalemia; 3) hypertension presenting with an adrenal incidentaloma; and 4) hypertension in the context of a family history of hypertension or early cerebrovascular accident. There is also evidence that PA is more likely to be found among those with obesity, diabetes, and obstructive sleep apnea. Michael Stowasser: Throughout the 1970s to early 1990s, PA was considered a rare cause of hypertension and unworthy of consideration unless patients were hypokalemic. The two main factors that led to the demonstration that PA was much more common were: 1) the introduction of the ARR as a screening test, which proved to be more sensitive than aldosterone assessed separately with renin, and 2) the application of the ARR to a wider population to include normokalemic patients and those with milder forms of hypertension. Estimates of prevalence among all-comer hypertensive patients in most recent studies have been 5%–13%. Although PA is undoubtedly more common among hypokalemic hypertensive patients, this group represents only the “tip of the iceberg” (approximately 20% of patients), and only selecting this population for screening will miss most of the other patients with this condition. Other populations that appear to be at increased risk include hypertensive patients with obstructive sleep apnea, diabetic patients, and those with incidentally discovered adrenal lesions. A strong argument can also be made for early screening in all hypertensive patients. This claim is based on the fact that 1) PA is highly prevalent among hypertensive patients, 2) the ARR test is reasonably inexpensive, 3) surgical outcomes are better than nonspecific antihypertensive treatment, 4) early detection improves those outcomes, and 5) the ARR is most reliable if measured before patients go on antihypertensives. Richard Auchus: This is a trick question, because it depends on how PA is defined, particularly when not caused by an adrenal tumor. If strict criteria are used, such as a 24-h urine aldosterone >14 μg/day (>38.8 nmol/day) during the salt-loading confirmatory test or serum aldosterone >10 ng/dL (>277 pmol/L, using older RIA assays) after the saline infusion test, then the prevalence among hypertensive patients is approximately 5%–8%. The prevalence is up to 20% among those with resistant hypertension. Development of PA is a gradual process, and it is quite likely that lower amounts of autonomous aldosterone production can contribute to low-renin hypertension in some healthy individuals, depending on their genetic makeup and the sodium content of their diet. The ARR is established as the best front-line test to screen for PA. What makes the ARR superior to testing either serum aldosterone or renin separately? Daniel Holmes: When we think about endocrine disorders caused by autonomous hormone production, we often think of the end hormone concentration as being high-normal or overtly high while the stimulating hormone is low or undetectable. Graves disease is a good example of this: thyroxine is often overtly high and thyroid-stimulating hormone is low or undetectable. In the case of PA, this is often not the case. There are 2 reasons for this. First, the potassium wasting that frequently occurs with PA causes hypokalemia or low-normal potassium concentrations, which decreases aldosterone production. Second, patients are often receiving antihypertensives that lower aldosterone—specifically, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, β blockers, and dihydropyridine calcium channel blockers. For this reason, aldosterone in isolation is not a very useful tool, since plasma concentrations are often well within the normal range and sometimes as low as 9 ng/dL (250 pmol/L) even in the absence of hypokalemia and antihypertensive use. Renin alone is not specific because it would fail to distinguish PA from other forms of low-renin hypertension such as severe Cushing syndrome, Liddle syndrome, and the hypertensive forms of congenital adrenal hyperplasia. Michael Stowasser: Isolated measurement of plasma aldosterone lacks sensitivity for PA, since many patients exhibit concentrations that are within the wide normal range, even those with unilateral APA. Such “normal” concentrations could be viewed as “inappropriately normal” in the face of suppression of the renin-angiotensin-aldosterone system. While highly sensitive for PA, suppressed renin concentrations lack specificity. Suppressed renin can occur in treatment with β-blocking agents, clonidine, α-methyldopa, or nonsteroidal antiinflammatory agents, or with consumption of a high-salt diet, advancing age, chronic renal impairment, and a large list of other salt-dependent and low-renin forms of hypertension. In contrast to PA, aldosterone concentrations in low-renin hypertension are chronically suppressed as a result of chronic suppression of renin/AngII. Richard Auchus: Renin is critical for screening because the diagnosis can only be made when renin is below the normal range or undetectable. When renin is normal, aldosterone production can be considered physiologic. If renin is low, then any excess aldosterone is abnormal; the higher the serum aldosterone, the more likely the patient has PA. In fact, I never calculate an absolute ARR. I always interpret the 2 tests separately, especially in the context of serum potassium. This is because hypokalemia suppresses aldosterone production and often leads to falsely low serum aldosterone and, in turn, false-negative screens. There is considerable variability in cutoff values for the ARR. What has contributed to such variability? How could serum aldosterone thresholds be used to help improve interpretation of the ARR? Etienne Cavalier: This variability is the consequence of a lack of standardization between the analytical methods and between clinical laboratories. The ratio combines 2 analytical errors, which could confound the interpretation of the ARR. Furthermore, a cutoff value relies on the specific methods that were used to establish it, and, unfortunately, cutoff values are often derived without consideration of the effect of analytical methodologies and standardization. Until recently, aldosterone measurement was achieved via cumbersome RIA methods. Since then, automated immunoassays and LC-MS/MS methods have become available. While LC-MS/MS is undoubtedly more specific than immunoassay, a higher-order reference method as well as an International Standard Reference Material for aldosterone is still needed. Michael Stowasser: Disparity between laboratory approaches is a major issue, with for example, some groups retaining RIAs for determining PRA, while many others are moving to LC-MS/MS or other automated immunometric methods of measuring direct renin concentration (DRC) using mass assays. The reporting of both aldosterone and renin in different units between laboratories has added to the complexity. Hopefully, this problem will diminish with time as more laboratories adopt the Système Internationale method of reporting aldosterone concentrations (as pmol/L), follow more uniform approaches to screening that are now widely circulated in published reviews and guidelines, and change to more reliable assay methodology. It has been shown that in the presence of extremely low renin concentrations (i.e., PRA ≤0.1 ng/mL · h or DRC ≤2 mU/L), the ARR may be increased even when plasma aldosterone is also very low (i.e., 110 pmol/L or 4 ng/dL) and clearly not consistent with PA. Some investigators have therefore suggested the inclusion of a minimum plasma aldosterone concentration of 15 ng/dL (415 pmol/L) within the screening criteria, but this approach would miss many cases because plasma aldosterone concentrations often fall below this cutoff. For this reason, our approach is to proceed with diagnostic workup for PA in all patients with increased ARR, except in those whose plasma aldosterone concentration is below the level used to define normal suppression during confirmatory fludrocortisone suppression testing (6 ng/dL or 165 pmol/L). In those patients, we will periodically repeat the ARR and consider further diagnostic workup. Daniel Holmes: Published ARR screening thresholds vary up to 10-fold. While the variability in the ARR is attributable in part to cohort selection, screening protocols, confirmatory testing, and disease definition, the most underappreciated problem in clinical studies is an analytical one—both for aldosterone and renin. Considering renin in isolation, PRA results are affected by assay design (incubation time or buffering pH) and the analytical components (choice of antibody, analytical methodology, and calibrator). DRC assays have challenges, particularly in the low-renin state, due to both poor precision and correlation with the more established PRA. Cross-reactivity of DRC with catalytically active conformations of prorenin is also a concern because the concentration of prorenin, which is normally 10-fold higher than renin, goes up to 100-fold higher in low-renin states such as PA. Regarding aldosterone cutoff values, about one third of PA cases may have ambulatory aldosterone concentrations <15 ng/dL (415 pmol/L). Therefore, it is prudent to avoid definitive statements about the exact concentration below which PA will never be observed and to always interpret a result with clinical presentation in mind. Richard Auchus: That is why I never calculate an ARR! At an ARR of 20 ng/dL:ng/mL · h (550 pmol/L:ng/mL · h using PRA) the sensitivity and specificity are both approximately 80%. As the cutoff value is set higher, the specificity improves little, but the sensitivity plummets. My approach is to interpret renin and aldosterone separately. If renin is low (<1 ng/mL · h) and aldosterone relatively high [≥10 ng/dL (277 pmol/L)], I proceed to confirmatory testing. In fact, I will do confirmatory testing if aldosterone is 5–10 ng/dL (139–277 pmol/L) and my index of suspicion is high, such as in a young patient with hypokalemia. It is important to realize that these cutoff values are set for patients on no medications. When a patient is taking vasodilators and diuretics, a nonsuppressed PRA (i.e., 2–3 ng/mL · h) with an aldosterone >20 ng/dL (>550 pmol/L) is highly suspicious and warrants rescreening after withdrawing them. There are many preanalytical and analytical challenges to the measurement of aldosterone and renin. Briefly discuss which interferences you think are most important to consider, and what approaches should be used to minimize inaccurate results. Michael Stowasser: The major controllable confounders are posture, time of day, dietary sodium intake, plasma potassium, and medications, which all impact the renin angiotensin aldosterone system. Medications causing false positives include β-blockers, clonidine, methyldopa, and nonsteroidal antiinflammatory drugs. False negatives can occur with angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, diuretics, and dihydropyridine calcium blockers. Selective serotonin reuptake inhibitors lower the ARR, with the potential for false negatives. Renin inhibitors lower PRA but raise DRC, and so their effect on the ARR is method dependent. ARR values are higher in premenopausal women than in age-matched males, especially during the luteal phase of the menstrual cycle when false positives can occur if renin is measured by DRC (but not PRA). Estrogen-containing oral contraceptive agents may cause false-positive ARRs with DRC assays (rather than PRA). False positives can also occur in patients of advanced age and in patients with chronic kidney disease. To minimize inaccurate results, patients should be encouraged to liberalize sodium intake. Medications that significantly affect the ARR should be withdrawn at least 4 weeks for both potassium-sparing and potassium-wasting diuretics, and at least 2 weeks for other potentially interfering drugs. Where necessary to maintain hypertension control, other antihypertensive medications that have less significant effects on the ARR (α blockers or nondihydropyridine calcium channel blocker) are recommended. Blood collection timing is important and should be undertaken midmorning after the patient has been ambulant for at least 2 h and seated for 5–15 min. Daniel Holmes: Aldosterone results produced by different immunoassays can vary 2-fold on the same samples. The origins of this bias are multifactorial. First, there is no international standardized reference material for aldosterone, meaning that all assays, whether commercial or lab developed, rely on gravimetric assignment. Second, though homogenous RIAs and automated homogenous chemiluminescent immunoassays have greatly simplified the analysis, they have introduced a problem of metabolite cross-reactivity previously mitigated by extraction steps. We have observed this to be particularly marked in the context of chronic renal impairment, where I would recommend that homogenous immunoassays for aldosterone not be used at all in favor of more specific LC-MS/MS methods. Furthermore, both PRA and DRC assays are vulnerable to spurious increases from prorenin cryoactivation. About 2% of prorenin is catalytically active but this fraction can be increased by the spontaneous and reversible unfolding of the prosegment, which exposes the active site and causes consumption of endogenous angiotensinogen leading to overestimation of renin activity and concentration. The unfolding of the prosegment is facilitated by refrigeration but can also occur at room temperature. We recommend rapid of plasma from and at and rapid at the time of PRA and DRC assays) are both widely used methods for renin In what factors should laboratories consider when which method to Are there where one renin method would be better than the Daniel Holmes: the to PRA DRC is often not a of for and the they My is that the of has more than do the of That being if there is a PRA a of better analytical sensitivity with and the to LC-MS/MS no However, PRA is more and Michael Stowasser: have been about the more methods of DRC using immunometric and automated which have widely been in laboratories in recent include and poor at the low end of the reference range, which is particularly to PA. PRA, DRC assays do not concentrations of endogenous and are therefore affected by factors such as endogenous or This has been to cause false-positive ARRs among women in the luteal phase of the menstrual cycle and in those receiving oral For the PRA analytical sensitivity can be by the of the during which angiotensin I is from of endogenous angiotensinogen by renin. Etienne Cavalier: DRC and PRA assays but clinical laboratories should immunoassays on the one screening for PA be available and, on the other using immunoassays will help variability and improve PRA should be as the reference method by for renin Richard Auchus: PRA is the but DRC has primarily and In the US, the DRC assay is not widely available. of the DRC method is that it is where to set the cutoff for a renin It is also well known that DRC is more vulnerable to false increases from and renin medications such as In the US, the Endocrine Society recommend that a ARR test should be followed up with of 4 confirmatory oral sodium saline infusion fludrocortisone suppression or the one is superior and what factors the of one the What tests are used to for Daniel Holmes: The for the diagnosis of PA is the of after for or of receptor for For this testing is not diagnostic in the test has and and it would be to one as best in all it to of and test and available largest of is with the and we have become with and in the context of LC-MS/MS aldosterone the test is the one you do not to do at so it is that if the clinical presentation and screening is of PA hypertension, aldosterone above the reference and undetectable it is to confirmatory testing and proceed to adrenal venous sampling for of in patients are potential surgical Michael Stowasser: We have found to be most sensitive and but it is to and is relatively has the of only a However, we have previously found to lack with many patients being because aldosterone concentrations are lower in the In a published we that in the seated of patients with PA, while was in only The oral suppression test, by the to be more sensitive than when we a of it However, it has the potential to from the related to 24-h urine and the to which the measured aldosterone concentration the of aldosterone due to cross-reactivity to forms found in The test, while relatively and to is not as well and has been to be associated with a significant of false positives and negatives. Etienne Cavalier: The most reliable of unilateral by from with that aldosterone forms of PA is adrenal venous sampling of aldosterone and is to A laboratory should be during the It is important to be that aldosterone and values may be very high, which may to What are the major in our of the genetic for PA, and how this in diagnosis and Etienne Cavalier: PA is the consequence of a from an during causing of aldosterone in to hormone with this disease severe hypertension and at a young of PA 2 is characterized by various among the same or hyperplasia. Although is no have been to of this subtype not from PA. The recent a in a channel for a potassium channel was shown to cause familial PA As a result, leads to aldosterone production, and hyperplasia, and In these hypertension is severe with hypokalemia. is frequently necessary to hypertension. Richard Auchus: The identification of in familial PA and in a high fraction of is the most significant in the in a This then led to the of various plasma channel plasma and calcium channel in other the of idiopathic the of these and help potential for of PA. important Are the of aldosterone production and the or Are these to cause there a that can be early and to prevent the to We are likely to many these in the Michael Stowasser: The identification of these and has greatly the of these in adrenal and and has the potential to to diagnostic and However, the argument genetic testing as a clinical is that these are always and only very associated with the familial of PA. detection in not have for of patients with APA. This could change as more is discovered about those factors which patients to and their associated aldosteronism angiotensin II aldosterone aldosterone-to-renin ratio plasma renin activity direct renin concentration assay saline infusion suppression test fludrocortisone suppression test potassium channel plasma calcium channel

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