RAS-challenge as a first-look test for detection of primary aldosteronism in patients with treatment-resistant hypertension

Abstract Purpose Primary aldosteronism (PA), characterised by low-renin hypertension, confers a high cardiovascular risk and is the most common cause of secondary hypertension, with an increased prevalence in patients with treatment-resistant hypertension. However, it is estimated that only a small percentage of affected patients are identified in routine clinical practice. Inhibitors of the renin-angiotensin system cause an increase in renin levels in patients with intact aldosterone regulation, and inadequate low renin with concurrent RAS inhibition (RASi) may therefore indicate PA, which could serve as a first look screening test for selection for formal work-up. Methods We analysed patients between 2016–2018 with treatment-resistant hypertension who had inadequate low renin in the presence of RASi (i. e. at risk for PA) and who were offered systematic work-up with adrenal vein sampling (AVS). Results A total of 26 pts were included in the study (age 54.8 ± 11, male 65%). Mean office blood pressure (BP) was 154/95 mmHg on 4.5 antihypertensive drug classes. AVS had a high technical success rate (96%) and demonstrated unilateral disease in the majority of patients (57%), most of which (77%) were undetected by cross-sectional imaging. Conclusion In patients with resistant hypertension, low renin in the presence of RASi is a strong indicator for autonomous aldosterone secretion. It may serve as an on-medication screening test for PA to select for formal PA work up. PLAIN LANGUAGE SUMMARY What is the context? Primary aldosteronism (PA) is associated with an uncontrolled secretion of the hormone aldosterone and often causes severe forms of high blood pressure. PA is considered the most common cause of high blood pressure which is caused by another medical condition. Medical societies have issued precise recommendations for the screening of this disease, which includes the determination of aldosterone and its main regulator renin. However, it is estimated that only a small percentage of affected patients are identified in routine clinical practice. What is the problem? In clinical studies, the determination of renin, aldosterone and its ratio (ARR) proved to be a valid screening tool. Nevertheless, in everyday life assessing and interpreting these results can be challenging for the clinician. The ARR is influenced by all first-line antihypertensives and in case of doubt, an extensive change in medication is recommended. Especially patients with resistant hypertension may require intensive medical care when medication is changed. What is important? In this study, we analysed patients at risk for PA who had inadequate low renin in presence of RASi (ACE inhibitors, Angiotensin receptor blockers). This study suggests that in patients with severe hypertension, the determination of renin in presence of RASi can provide further information on the presence of autonomic aldosterone secretion at a glance. However, this approach cannot and should not replace the algorithm proposed by current guidelines. In contrast, this approach should be an easy-to-implement concept that should prime the initiation of further appropriate diagnostics.


Introduction
Primary aldosteronism (PA), a form of low-renin hypertension, is the most common cause of secondary hypertension, affecting at least 5% of all hypertensives [1,2].Patients have increased cardiovascular mortality and morbidity, so early diagnosis and adequate therapy are critical [3].However, various studies indicate that PA is significantly underdiagnosed [4,5].
The aldosterone-renin ratio (ARR) is the basis for initial case detection.In clinical studies, the ARR proved to be a valid screening tool [6].Nevertheless, in everyday life assessing and interpreting the ARR can be challenging for the clinician [6,7].The ARR is influenced by all first-line antihypertensives and in case of doubt, an extensive change in medication is recommended [6,8].Especially patients with resistant hypertension may require intensive medical care when medication is changed.In view of the high prevalence, this time-consuming procedure is often difficult to implement.In fact, although the guidelines recommend generous and low-threshold screening [6,9], experts assume that only about 1% of all patients with hypertension are screened for a PA [10].
In everyday clinical practice, it would be useful to assess at a glance whether an in-depth screening (including ARR under appropriate medication) and a detailed diagnostic workup are worthwhile.Since low renin is a hallmark of PA, renin-guided approaches have been discussed in the past [7,10].However, low renin is usually a non-specific finding as PA with autonomous, inadequate aldosterone secretion is just one of several possible differential diagnoses [11,12].
Current hypertension-guidelines recommend firstline combination drug treatment based on reninangiotensin-system inhibitors (RASi), e.g.ACE inhibitors (ACEi) or AT1 receptor blockers (ARB), in combination with diuretics and/or calcium channel blockers (CCB) [13].ACEi/ARB disrupt the negative feedback-inhibition of renin mediated by Angiotensin II and aldosterone.Therefore, this treatment is inevitably expected to raise on-treatment renin levels, unless there is some degree of autonomous aldosterone secretion, which suppresses renin levels despite the presence of RASi [14].
The PATHWAY-2 trial demonstrated the superior effect of spironolactone in patients with resistant hypertension.The lower the renin value (in presence of an ACEi/ARB, CCB and a diuretic), the stronger the observed effect, which suggests an autonomous aldosterone secretion.In fact, in this trial, up to 38% of patients with resistant hypertension and RASi displayed a biochemical profile consistent with PA (low renin despite the presence of RASi) [15,16].
Based on these results, we hypothesised that measurement of renin in the presence of ACEi/ARB could provide initial information on the presence of autonomic aldosterone secretion.Such a renin measurement could be suitable as a first-look test for patient stratification, even before a guideline-compliant screening is initiated.Following the publication of the PATHWAY-2 trial, we began to offer a systematic examination including adrenal vein sampling (AVS) for the detection of unilateral PA to our patients at risk of PA, who had a low renin value in the presence of RASi (<18 lIU/ml).AVS was only offered to patients who were able and willing to undergo surgery.We here present an analysis of the presentation and findings.

Study design
This analysis was designed as a retrospective study including patients with uncontrolled or resistant hypertension referred to the University Hypertension Centre, Department of Nephrology and Hypertension, Hannover Medical School, Germany, in the period 2016-2018.This study was approved by the local ethics committee (7978_BO_K_2018), and patients have given their informed consent.The study was performed according to the principles of the Declaration of Helsinki.

Diagnostic procedures
The patients were selected for an initial screening for PA according to the criteria of the current guidelines.Before taking a blood sample it was ensured that each patient had been in therapy with ACEi/ARB for at least four weeks.Any therapy with a beta-blocker or alpha-2 agonist was tapered off beforehand (for at least two weeks).No patient had been prescribed NSAIDs (according to history and medication schedule).Furthermore, no clinical signs of volume overload should be present in the physical examination.Blood samples were drawn from ambulatory patients after resting and while sitting in the mid-morning.Ethylenediaminetetraacetic acid (EDTA) plasma samples were all analysed in the central laboratory of the Hannover Medical School.Active renin and aldosterone were determined based on a chemiluminescence assay (CLIA) with the DiaSorin (Saluggia, Italy) Liaison analyser.
A renin value was defined as suppressed (in the presence of ACEi/ARB and in the absence of betablocker) if it was measured <18 lIU/ml in the presence of ACEi/ARB.A sub-analysis of the PATHWAY-2 cohort later revealed that the lowest tertile of renin was below <19 mIU/L [16].
Cross-sectional imaging was carried out in all patients with PA.Patients who were potentially candidates for surgery were informed about the therapeutic options (medical/conservative therapy, surgical therapy).Patients who were potentially willing to undergo surgery were offered adrenal vein sampling (AVS) for subtype classification.AVS was performed with pointof-care cortisol verification (rapid cortisol assay) without any pharmacological stimulation during AVS.Technical success and lateralisation were assessed according to current expert recommendations [17].

Statistical analysis
The analysis was carried out using the statistics program GraphPad Prism and Microsoft Excel 2013.Data are reported as number and percent for categorical variables.For continuous variables data is shown as mean ± standard deviation (SD), or median (range).Groups were compared using the ANOVA (analysis of variance) test.

Results
During the period of January 2016 to August 2018 we identified 26 patients at risk for PA, defined as suppressed or low renin in the presence of RASi and in absence of a beta-blocker.These were offered AVS supported by collimated C-arm Computed Tomography (CACT) for subtype detection, after completion of screening [18].
24 patients agreed to AVS-CACT, which was successfully completed in 23 of 24 pts.(96% success rate) with no complications (Figure 1).57% (13/23) of patients showed clinically significant lateralisation.Of those, 77% (10/13) were classified as microadenoma, since they showed no signs of adenoma or adrenal mass on cross-sectional imaging, which was performed prior to AVS.Surgical adrenalectomy was performed in 10/13 patients with proven lateralisation.All remaining patients received conservative therapy with mineralocorticoid receptor antagonists (MRA) (Figure 1).
The various subtypes (macro-, microadenoma, idiopathic) did not principally differ in their renin levels, nor in ARR (Figure 2).
During an average follow-up time of 16.5 ± 6.1 months, office and home BP in operated and non-operated patients improved significantly (Figure 3(A)).However, pill burden and antihypertensive drugs per defined daily dose decreased significantly more in operated patients (Figure 3(B)).Furthermore, hypertension control rate (office BP < 140/90) was higher in patients after adrenalectomy, and 3/10 operated patients achieved clinical cure, defined as normal blood pressure with no treatment (Figure 3(C)).

Discussion
PA is a common and potentially curable cause of arterial hypertension [6,19].Despite adequate hypertension control, PA is associated with increased cardiovascular mortality and morbidity [3], which underlines the importance of an early diagnosis.Therefore, initial case detection is one of the major challenges in the diagnostic work-up.
In this retrospective study, a low renin in presence of RASi was shown to be a strong predictor of autonomic aldosterone secretion in patients with resistant hypertension.
Low or even suppressed renin is a hallmark of PA, while aldosterone levels may not only be elevated but also within the normal range [1,5].However, low renin (measured under non-standardised conditions) in itself is an unspecific finding, as it can be associated with a high sodium diet, certain medications (e.g.beta-blockers) or various other forms of hypertension [6,8,12].Thus, despite acceptable sensitivity, a single renin determination is not sufficient for PA screening due to a lack of specificitywhich is the major reason for determining the ARR [6,12].
In a study with 6,228 participants without antihypertensive medication, the renin distribution was examined.60% of subjects had renin levels within the normal range, while 20% of subjects had high normal or elevated levels.A low plasma renin concentration was found in about 20% of all cases [20], which again underlines the low specificity of this finding.However, this bell-shaped renin-distribution is altered in presence of RASi.Normally, Ang II and aldosterone limit renin secretion through negative feedback inhibition.As ACEi/ARB disrupt this mechanism, renin-levels inevitably rise in presence of RASi [14].
Thus, reduced renin levels in this constellation are a striking finding, which may be induced by reninindependent aldosterone secretion.In line with this, in a study with 25 patients and proven PA, PRA did not increase significantly after initiation of ACEi  therapy with Ramipril [21].Further evidence comes from the PATHWAY-2 trial, which demonstrates the superior effect of spironolactone in patients with resistant hypertension.The lower the renin value (in presence of an ACEi/ARB, CCB and a diuretic), the stronger the observed effect [15,16], which suggests an autonomous aldosterone secretion.In case of a likewise suppressed aldosterone, other causes (e.g.intravascular volume overload) would theoretically be conceivable, however the low renin/low aldosterone phenotype was only rarely detectable in the PATHWAY-2 trial [15,16].
The findings of low renin levels under RASi should be interpreted in the context of the EMIRA study  C) Hypertension control and cure rate (defined as normal office blood pressure (OBP) with no treatment) stratified according to therapeutic strategy at the last follow up.[22].In this study, all pts.received MRA, with or without RASi, and 9% of patients with autonomic aldosterone secretion show an increase in renin on MRA and RASi, leading to a small increase in the false negative detection rate [22].However, the extent to which these results can be applied to the setting discussed here (renin under RASi, no MRA) is not clear and would need further systematic investigation.
The consequent use of AVS for subtype evaluation had a high technical success rate and demonstrated unilateral disease in most patients (57%), most of which (77%) were undetected by cross-sectional imaging.The technical success rate depends on the experience of the centre and has probably been improved by both a rapid cortisol test [23,24] and the use of CACT-AVS, which improves selective catheterisation in patients with difficult anatomy [18].
Taken together, measurement of renin in presence of ACEi/ARB can provide further information on the presence of autonomic aldosterone secretion at a glance.However, this approach cannot and should not replace the algorithm proposed by current guidelines [6,9].In contrast, we propose an easy-to-implement concept that should prime the initiation of further appropriate diagnostics.
Various studies have shown that PA screening needs to be improved.Despite clear guideline recommendations, studies demonstrate that still too few patients are screened for PA [4,5,25].Several approaches to improve and enhance PA screening have been suggested, most of which also included a renin determination [7,10].The proposed approach adds further value, as RASi (RAS-Challenge) may reveal autonomic aldosterone secretion more clearly than non-standard renin determination.It can enable clinicians to decide at a glance whether further screening is requiredwithout any adjustment of a guideline-based therapy.
This study has weaknesses.Due to the small cohort size, the missing control group exact diagnostic sensitivity and specificity as well as the corresponding cutoff values cannot be provided.The described principle requires that patients adhere exactly to the prescribed medication.However, non-adherence is a common problem [26].In order to better understand the influence of non-adherence on this test principle, drug levels should also be determined in a further study.We describe the principle of a diagnostic approach; further characteristics need to be established in large prospective studies.However, as the RASi is expected to induce a drastic increase of renin [14], further diagnostics should be initiated if low renin is detected.

Conclusion
Measurement of renin in the presence of ACEi/ARB (without beta-blockade) in patients with refractory hypertension provides clinically useful information on the presence of autonomic aldosterone secretion at a glance without the need for prior medication change.It may therefore serve as a first screening-step.

Figure 1 .
Figure 1.Patient flow diagram of the diagnostic evaluation of patients at increased risk for PA.

Figure 2 .
Figure 2. Renin and ARR stratified according to the PA subtype.Active renin and aldosterone were measured in presence of RASi.Data are shown as median and range.Dashed lines indicate reference ranges.(A) The figures above marks indicate the maximum.(B) The figures below marks indicate the minimum.BAH Bilateral adrenal hyperplasia.

Figure 3 .
Figure 3. Treatment outcome of patients with low renin resistant hypertension with RASi.(A) Office BP and home blood pressure.Dashed lines indicate the respective hypertension threshold.Data is shown as mean and standard deviation.(B) Antihypertensive medication at initial presentation and in the further course after initiation of therapy (non-OP ¼ conservative, OP ¼ adrenalectomy).Values after 3, 6 months and from the last follow-up (LFU) are shown.Data is shown as mean and standard deviation.Ã Significant intergroup-difference (2-way-ANOVA).ÃÃ Significant intergroup-difference (2-way-ANOVA).(C) Hypertension control and cure rate (defined as normal office blood pressure (OBP) with no treatment) stratified according to therapeutic strategy at the last follow up.
Data are mean ± SD; except for lab results for which the median (range) is shown.The medication at the time of the first presentation in the outpatient clinic is shown.BMI: body mass index; DDD: defined daily dose; OBP: office blood pressure; HBPM: Home blood pressure monitoring; UACR: Urine Albumin-to-Creatinine Ratio.