Histological analysis of adrenal glands removed from PA patients indicates that the subdivision of APA and BAH, useful from a clinical and operative perspective, oversimplifies the complex alterations that they undergo: in fact, the adrenals of patients with PA exhibit heterogeneous histological alterations. For example, APAs are rarely composed of a single cell type, but rather, exhibit different cell types in varying proportions comprising: zona fasciculata-like cells, clear cells with large vacuolated lipid-laden cytoplasm, and round central nuclei; lipid-poor zona ­glomerulosa-like cells; zona reticularis-like cells, compact eosinophilic cells; cells with cytological features of both zona fasciculata (ZF) and zona glomerulosa (ZG) cells showing variable contents of clear lipid micro-vacuoles and granular eosinophilic cytoplasm, also called “hybrid” or “intermediate” cells [13, 14].

Consequently, APA have been classified by some authors according to the prevalent cell type and have attempted to relate the histological findings to some clinical phenotype. For example, the prevalence of ZG-like cells corresponded to responsiveness to angiotensin II infusion and so these APA were denominated, AngII-responsive APA or AR-APA; in contrast to the presence of a prevalence of ZF/reticularis or hybrid cells that correlated to adrenocorticotropic hormone (ACTH) responsiveness (AngII-unresponsive APA, AU-APA) [16]. However, a correlation between cell type and response to posture, and thus to angiotensin II was not observed in another study [17]. Interestingly, most APA patients display either diffuse hyperplasia of the surrounding ZG in the rest of the adrenal or hyperplasia of the ZG with multiple nodules of different sizes, ranging from micro- to macro-nodules [13]. A few reports have studied the contralateral adrenal gland and described similar findings of nodular hyperplasia thereby suggesting that PA may begin, at least in some cases, with one or more unknown stimuli that trigger the development of multiple nodules throughout the adrenal cortex, and a subsequent event stimulates the formation of a single dominant APA that becomes autonomous [14, 15].

In situ hybridization (ISH) studies of CYP11B2 mRNA have demonstrated that in most cases CYP11B2 is overexpressed in the APA; however, in some cases the gene is overexpressed not only in the APA, but also in the surrounding adrenal ZG; whilst in others, CYP11B2 gene expression is not observed in the dominant nodule, but rather, is overexpressed in one or more smaller nodules; in the latter two cases, adrenalectomy did not successfully treat the PA [18, 19].

The recent development of specific antibodies to CYP11B1 (11-beta-­hydroxylase) and CYP11B2 (aldosterone synthase) enabled immunohistochemical studies to define the alternative expression patterns of these enzymes from normal adrenals (NA) compared to adrenal glands from PA patients [20].

Normal adrenals display two patterns of expression: (1) a conventional distribution, with CYP11B2 sporadically expressed in the ZG, whereas CYP11B1 is ­diffusely expressed in the ZF; (2) a variegated pattern with cell clusters strongly expressing CYP11B2 and the remaining areas expressing CYP11B1 [20]. The examination of APA tissues showed that tumors mainly display CYP11B2 positive cells whilst others have a variable proportion of cells expressing the enzyme. APA can consist of three cell types: (a) CYP11B2-positive/CYP11B1-negative cells; (b) CYP11B2-negative/CYP11B1-positive cells; (c) double-negative cells. 3 beta-hydroxysteroid dehydrogenase (3betaHSD) was detected throughout the tumors, irrespective of the expression of CYP11B2 or CYP11B1; whereas CYP17 (17-alpha-hydroxylase) exhibited a similar expression profile to CYP11B1, consistent with its function in cortisol synthesis [20]. In the zone of the adrenal cortex surrounding the APA, two expression patterns have been described: (1) a conventional zonation; and (2) cell clusters expressing CYP11B2 and 3betaHSD, but not CYP11B1, and CYP17, with weak or no expression of CYP11B1 outside the cell clusters [20].

The biochemical and genetic alterations underlying the abnormal cell growth in the zona glomerulosa (ZG) cells of the adrenals of PA patients are still largely unknown. A recent study showed that in rats fed with a high salt diet, although the width of the ZG cells was decreased and the expression of CYP11B2 encoding aldosterone synthase was suppressed, there were nonetheless a number of cells still expressing high levels of CYP11B2 [21]; it is possible to hypothesize that a mutation increased the growth of these cells, thereby resulting in the formation of a ­nodule of aldosterone-producing cells, not regulated by the increased sodium reabsorption [14].

The molecular basis for the deregulated cell growth and the autonomous hyperproduction of aldosterone observed in PA remains to be fully elucidated. Transcriptosome analysis employing either microarrays or serial analysis of gene expression (SAGE) together with technologies to validate the resultant genetic profiles, such as ­quantitative real-time PCR (qRT-PCR), have led to several studies attempting to determine the molecular fingerprints of adrenal adenomas. Strict patient selection is of primary importance for such studies and, in fact, insufficiently stringent criteria for APA classification may have caused the discrepancy between the reported differentially expressed genes in APA. Because aldosterone-producing cells are able to store only minimal quantities of the hormone, the synthesis of aldosterone is finely regulated and coupled to its secretion. It is unsurprising therefore, to find aldosterone synthase, up-regulated in APA compared to normal adrenal cortex [22] and all transcriptosome analyses to date have identified the up-regulation of the aldosterone synthase gene (CYP11B2) [23–26]; in contrast, one microarray study identified two sub-groups of APA classified on the basis of a paradoxical up- or down-regulation of a set of gene transcripts that included CYP11B2 [27].

Transcripts that code for other steroid metabolizing enzymes, namely HSD3B2 (encoding 3 beta-hydroxysteroid dehydrogenase type II) and CYP21 (encoding 21-hydroxylase) have also been identified [24]; the latter gene was also found to be overexpressed in APA in a SAGE analysis and validated by ISH [23].

The LHR (luteinizing hormone receptor gene) transcript has been identified as up-regulated by microarray analysis [25, 28] and exogenous expression of the receptor in the aldosterone-secreting adrenocortical carcinoma cell line, NCI H295R [29], and subsequent stimulation with LH resulted in an increased transcription of a luciferase gene reporter construct harboring the CYP11B2 gene promoter.

A second G protein coupled receptor gene, the serotonin receptor 4 (hydro­xytryptamine receptor 4, HTR4), that stimulates cAMP release in response to serotonin, has been identified as up-regulated in APA [26, 30, 31]. The aberrant expression of G protein coupled receptors such as LHR and HTR4, among others has been proposed as a putative mechanism for the deregulated steroid production in APA [25, 28].

A recent genomic analysis study by Williams et al. [26] used adenoma tissues from a homogeneously selected group of patients following rigorous diagnostic procedures, including strict criteria for adrenal venous sampling (AVS) interpretation and postadrenalectomy evaluation. Microarray analysis and validation by qRT-PCR identified the epidermal growth factor-like teratocarcinoma-derived growth factor-1 (TDGF1) as the most up-regulated gene in APA compared with NA (21.4-fold). The functional role of TDGF1 was also studied using the H295R cell model: the exogenously expressed growth factor resulted in the activation of phosphatidylinositol 3-kinase (PI3Kinase)/Akt signaling and mediated both an increase in aldosterone secretion (3.8-fold) as well as an inhibition of apoptosis. Both functional effects were blocked by PI3Kinase inhibitors [26].

Interestingly, a sixfold up-regulation of the TDGF1 gene transcript in APA had been identified previously in another study by SAGE [23]. Therefore, two independent methods of transcriptosome analysis have identified TDGF1 as an up-regulated gene transcript in APA compared to normal tissue [23, 26] that, considered together with the functional data, indicate that this gene may represent a key player in the development and pathophysiology of PA.

Traditionally, aldosterone has been considered the main regulator of water and ­electrolyte homeostasis due to its effects on epithelial cells, particularly in the collecting ducts of the kidney and distal colon. The physiological actions of aldosterone in non-epithelial tissues are much less evident, but over the last 15 years a wealth of studies both in humans and animal models have allowed new insights on its pathophysiological effects, that are mainly targeted in the heart, blood vessels, kidney, and central nervous system (CNS).

The Guidelines for the diagnosis and treatment of PA recently published the different categories of hypertensive patients that display a relatively high prevalence of PA; it is recommended that patients belonging to these categories undergo a screening test. The screening test should be performed in all patients with: (1) resistant hypertension; (2) hypertension grade 2 or 3; (3) hypertension and spontaneous or diuretic-induced hypokalemia; (4) hypertension with adrenal incidentaloma; (5) hypertension and a family history of early-onset hypertension or cerebrovascular accident at a young age (<40 year); (6) all hypertensive first-degree relatives of patients with PA [4] (Fig. 1.1).

The aldosterone to renin ratio (ARR) is the most reliable, currently available method for PA screening: in fact, many studies have demonstrated that the ARR is superior to measurements of both potassium and aldosterone (which are less sensitive) as well as renin alone (which is less specific) [84–86]. Presently, there is no general consensus on ARR cut-off and therefore, individual centers use different values, ranging from 7.2 to 100 ng dL⁻¹ per ng mL⁻¹ h⁻¹, with a consequent wide variation in the sensitivity (64–100%) and specificity (87–100%) of the screening test. However, in a recent study published by Rossi et al., the ARR was found to provide a good within-patient reproducibility and an accuracy of 80% for identifying APA patients [87]. The most important confounding factors affecting renin and/or aldosterone measurements thus reducing the sensitivity or specificity of the ARR are antihypertensive drugs [4] (Fig. 1.2). In particular, β-blockers, α-methyldopa and clonidine decrease the beta-adrenoreceptor mediated stimulation of renin production and sympathetic nervous system output thereby causing false-positives. Conversely, other drugs stimulate renin secretion, such as ACE-inhibitors, ATII receptor antagonists, dihydropyridine calcium antagonists, and diuretics. All these drugs (except diuretics that increase aldosterone secretion) may cause a reduction in aldosterone levels and increase in renin levels leading to false-negative results. The recently introduced renin inhibitor Aliskiren reduces plasma aldosterone levels whilst stimulating renin secretion: this results in false-positive ARR levels for renin measured as plasma renin activity (PRA) and false negatives for renin measured as direct renin concentration (DRC) [4]. Antihypertensive medications other than diuretics, that should always be withdrawn for at least 4–6 weeks (6–8 weeks for spironolactone), should be stopped at least 2–3 weeks before ARR testing, but in many patients this is clearly not feasible. Because α-antagonists do not appear to have any significant effect on plasma aldosterone and renin levels, they can be safely used to control hypertension before the screening test is performed. Non dihydropyridine calcium antagonists can cause a modest increase in renin levels and reduce aldosterone secretion, but rarely to an extent that can significantly affect the ARR and confirmatory tests and can be administered to patients whose blood pressure is not adequately controlled by an α-antagonist alone [4, 88].

Further, the effect of oral contraceptive agents has received little attention, but should be considered because estrogen-containing preparations induce angiotensinogen synthesis by the liver. Blood sampling conditions can also have an effect and should be standardized to avoid potential fluctuations in levels of the two ­hormones and subsequent difficulties in interpreting the ARR [4] (Table 1.2).

The absolute aldosterone value (>15 ng dL⁻¹) and the lowest detectable level of PRA should also be taken into account. Because the ARR is dependent on PRA, effectively anyone with suppressed PRA will have an increased ARR; this is particularly important in elderly or black populations, who often display PRA values as low as 0.1 ng mL⁻¹ h⁻¹, thereby resulting in an increase in the plasma aldosterone concentration: plasma renin activity (PAC:PRA) ratio even with a PAC of 5 ng dL⁻¹. It has been shown that all individuals with aldosterone levels of less than 9 ng dL⁻¹ demonstrate normal suppression during a fludrocortisone-suppression test [89].

In the majority of published studies, ARR is reported as a function of renin, measured as PRA, and few data are available for direct active renin (DAR). PRA and DAR are closely correlated (overall correlation coefficient 0.98), but the correlation is weaker for the low compared to the high/normal range of values (r  =  0.77 for samples with PRA <0.65 ng mL⁻¹ h⁻¹), i.e., in the range of patients potentially affected by PA [90, 91]. Even newer automated methods for renin measurements have not resolved this issue (r  =  0.14 with PRA  <  1) [92]. To date, no prospective studies have been performed that have compared the accuracy of the aldosterone to active renin ratio to that of the well-established ARR and therefore, DAR should be considered cautiously as a screening test for PA.

In light of the high prevalence of low renin hypertension [90], it is important to emphasize that an increased ARR is not itself diagnostic of PA and a confirmatory test is always required to avoid patients unnecessarily undergoing costly and potentially harmful lateralization procedures.

Although the Guidelines clearly recommend that patients with a positive ARR undergo a confirmatory test to definitively confirm or exclude the diagnosis of PA, the choice of test remains a matter of debate and there is currently insufficient direct evidence to recommend one rather than another [4] (Fig. 1.1).

Four testing procedures are approved by the Guidelines: oral sodium loading (OLT), saline infusion (SLT), fludrocortisone suppression (FST), and a captopril challenge (CCT) [4] (Table 1.3).

Briefly, FST consists of fludrocortisone acetate administration for 4 days with KCl and NaCl supplements. If PAC on the fourth day is ≥6 ng dL⁻¹, PA is confirmed (Table 1.3) [4, 16, 93].