Visualizing adrenal steroids in primary aldosteronism: the expected and the unexplained

Primary aldosteronism (PA) is the most common secondary cause of hypertension with an incidence between 5–20% depending on the severity of hypertension. The basis of the disorder is the overproduction of the mineralocorticoid aldosterone. In the normal adrenal, aldosterone is synthesized in the outermost layer of the cortex called the zona glomerulosa (zG). Most enzymes involved in aldosterone synthesis are expressed in both the zG and the underlying zona fasciculata (zF)¹. Aldosterone synthase (CYP11B2) is an exception and is exclusively expressed in the zG. CYP11B2 is a partially processing enzyme that successively converts deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and finally aldosterone¹ (Figure 1). In this issue of Hypertension, Sugiura et al ² address if the areas of the adrenal that express CYP11B2 are those where aldosterone and the hybrid steroid 18-oxocortisol are synthesized.

The figure shows steroidogenesis in the zona glomerulosa (zG) and zona fasciculata (zF) of the human adrenal cortex. Cholesterol, pregnenolone and progesterone are synthesized in both cortical layers. Aldosterone is exclusively produced in the zG because in the normal adrenal, CYP11B2 is expressed only in this zone. In some patients with primary aldosteronism, 18-oxocortisol production is elevated and this would most likely require co-expression of CYP11B2 (shown in red) and CYP17A1 in the same cell: CYP17A1 for the provision of precursors and CYP11B2 for product formation. Alternatively, zF cells could provide cortisol to neighboring zG cells via a paracrine mechanism.

An aldosterone-producing adenoma (APA) is often the source of the aldosterone excess and somatic driver mutations in either an ion channel, ion transporter (KCNJ5, CACNA1D, ATP1A1, ATP2B2) or in CTNNB1 have been identified ³. Combined, somatic mutations that potentially account for aldosterone overproduction in APAs are found in close to 90% of tumors when using a targeted approach to areas of the tumor that express CYP11B2⁴. There is a distinct sex difference in APA mutation prevalence with KCNJ5 mutations more frequent in females than males (70% versus 24%). Conversely, mutations in CACNA1D are more prevalent in males than females (33% versus 3%) as are ATP1A1 mutations (22% in males and 10% in females) and mutations in ATP2B3 (7% in males and 0% in females) ⁴. The reasons for these distinct patterns of mutation frequencies between males and females remain unknown.

The development of adrenal vein sampling, to identify unilateral forms of hyperaldosteronism, and of monoclonal antibodies against CYP11B2 for use in immunostaining of the resected gland, have helped uncover the much greater complexity of unilateral PA than previously recognized ^{3, 5}. CYP11B2 immunohistochemistry has demonstrated the considerable heterogeneity of CYP11B2 expression within tumors and revealed that the source of aldosterone production may not always correspond with macroscopic adenomas if they do not stain for CYP11B2⁵. Immunohistochemistry of the human adrenal has shown scattered CYP11B2-expressing cells throughout the zG in the young, whereas in older individuals CYP11B2 expression is concentrated in tight clusters of cells called aldosterone-producing cell clusters (APCC)⁶. APCCs display intense CYP11B2 immunostaining but are negative for CYP17A1 and CYP11B1. APCC in normal individuals often carry mutations in CACNA1D, less often in ATP1A1 or ATP2B3, very rarely (if at all) in KCNJ5 and accumulate with age and have been suggested as potential precursors of APA⁶.

18-Oxocortisol is produced by 17α-hydroxylation of a precursor by CYP11B2. In patients with APAs there are cells that co-express CYP11B2 and CYP17A1 which would supply the required precursors, such as 11-deoxycortisol, for the formation of 18-oxocortisol and aldosterone within the same cell and this is the likely mechanism of 18-oxocortisol biosynthesis⁵ (Figure 1). Cells expressing CYP11B2 (zG cells) are also in close contact with cells that express CYP11B1 and CYP17A1 (zF cells)⁵. Cortisol is the main product from zF cells and could act as a paracrine substrate for the biosynthesis of 18-oxocortisol.

Sugiura et al ² study the co-localization of CYP11B2 and 18-oxocortisol in different adrenals which would not only require CYP11B2, but also the StAR protein to move cholesterol to the mitochondria plus all other enzymes for deoxycorticosterone formation (the main substrate of CYP11B2)¹. The co-existence of these enzymes and factors is expected and has been assumed, but not proven. Sugiura et al ² address an important issue and they do this using the novel technique of matrix-assisted laser desorption/ionization mass spectrometry (imaging mass spectrometry), and in some cases with chemical derivatization to visualize specific steroids in adrenal tissue sections.

Aldosterone and 18-oxocortisol were visualized in APCCs of 4 patients, in the adrenal of a patient with what they referred to as an APCC-to-APA transitional lesion (characterized by subcapsular APCC features with inner clear cells like an APA) and in 3 cases of APA. They demonstrated that in most cases aldosterone and 18-oxocortisol corresponded to the areas of CYP11B2 expression. Paradoxical findings were described in case 8 where the APA exhibited irregular but strong CYP11B2 staining with aldosterone and 18-oxocortisol visualization limited to the border of the APA². It is unclear if in this case aldosterone and 18-oxocortisol were in fact synthesized in the central areas of the adenoma but in amounts that were below detection limits as the technique appears to be relatively insensitive. This is indicated by their study with rat adrenals where aldosterone was only detected in the zG of adrenals from rats on a low sodium diet².

A concern is the origin of the substrate for 18-oxocortisol especially in APCC that do not express CYP17A1. The authors postulated that the source came from surrounding zF cells that express CYP11B1 and CYP17A1 with the substrate acting in a paracrine manner. One unanswered issue is that the adrenal has high levels of expression of the multiple drug resistance P-glycoprotein which actively pumps polar steroids from the cells and is assumed to be responsible for the active movement of aldosterone from inside to outside the cell⁷. Cortisol is an important substrate for P-glycoprotein and cortisol would not be expected to penetrate APCC in sufficient amounts for conversion to 18-oxocortisol. The issue of substrate source for the synthesis of 18-oxocortisol therefore remains unclear. An additional point is that although the studies were done in single slides, the area of the APCCs were very small where aldosterone was detected and unless there are many more APCCs in other areas of the adrenal, it is unclear if there is enough tissue to produce an excess of aldosterone that can cause primary aldosteronism especially considering that previous studies in normal individuals appear to have as many or more APCCs ⁶.

Patients with APAs carrying KCNJ5 mutations produce significantly higher levels of 18-oxocortisol compared with APAs of other genotypes. ⁸ While 18-oxocortisol is a relatively weak mineralocorticoid, it is unclear if it plays a role in the development of hypertension or is just a marker of the mutation in the APAs. Histologically, these tumors tend to be larger and comprise predominantly clear cells similar to the zF, while tumors bearing other mutations have more zG characteristics. The different histology of tumors with KCNJ5 mutations may contribute in some way to their increased production of 18-oxocortisol ⁸. Many APAs exhibit a more-or-less uniformly high expression of CYP11B2 in contrast to the irregular expression evident in other adenomas ⁵. The zG adjacent to the adenoma often displays APCCs which is contrary to expectations if the tumor was producing enough aldosterone to downregulate CYP11B2 expression in the surrounding zG. The technique of Sugiura et al ² should be able to answer this vexing question.

The classical histological description of the adrenal defines the medulla and the three zones of the adult cortex (glomerulosa, fasciculata and reticularis). Using a combined approach of MALDI (Matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI) metabolomic imaging and immunohistochemistry, Sun et al ⁹ described a new molecular definition of the adrenal anatomy comprising 10 distinct zones including a highly structured corticomedullary interphase. In the metabolomics study of Sun et al,⁹ a wide range of small metabolites was analyzed (nucleoside phosphates, sterol and steroid metabolites, intermediates of glycolysis and the tricarboxylic acid cycle, lipids and fatty acids) with a minimal analysis of steroids. The study of Sugiura et al ² addressed a selected number of steroids. It would be of interest to combine the approaches of both studies and correlate the various metabolic clusters with steroid production and histopathologic structure especially APCCs.

Elegant studies in patients with APA where the affected gland was identified by adrenal vein sampling and then surgically removed by unilateral adrenalectomy have demonstrated the presence of somatic mutations in almost all adenomas or in glands with APCCs⁴. It has now been established that these lesions indeed produce aldosterone and in most cases are capable of 18-oxocortisol synthesis ². Unfortunately, removal of the affected gland with the mutations results in the cure of hypertension in less than half of patients, with a further 30–40% showing an improvement and while most patients are biochemically cured; up to 15% are not. The resected glands from patients who are not biochemically cured appear to display a higher prevalence of cortical hyperplasia suggesting that these cases correspond to the dominant gland from patients with asymmetrical bilateral hyperaldosteronism ¹⁰.

In conclusion, state-of-the art technology is increasing our knowledge of steroidogenesis in the normal and pathological adrenal and has confirmed the production of aldosterone in areas of CYP11B2 expression but despite this expected finding many questions remain unexplained.