Koshiro Nishimoto

Adrenocortical Zonation in Humans under Normal and Pathological Conditions

CONTEXT Aldosterone synthase (CYP11B2) and steroid 11 beta-hydroxylase (CYP11B1) catalyze the terminal steps for aldosterone and cortisol syntheses, respectively, thereby determining the functional differentiation of human adrenocortical cells. Little is known, however, about how the cells expressing the enzymes are actually distributed in the adrenals under normal and pathological conditions. OBJECTIVE The objective of the study was to determine the localization of CYP11B2 and -B1 in human adrenal specimens by using developed antibodies capable of distinguishing the two enzymes from each other. RESULTS Under normal conditions, CYP11B2 was sporadically detected in the zona glomerulosa, whereas CYP11B1 was entirely detected in the zonae fasciculata-reticularis. Adrenocortical cells lacking both enzymes were observed in the outer cortical regions. In addition to conventional zonation, we found a variegated zonation consisting of a subcapsular cell cluster expressing CYP11B2, which we termed aldosterone-producing cell cluster, and a CYP11B1-expressing area. Aldosterone-producing adenomas differed in cell populations expressing CYP11B2 from one another, whereas CYP11B1-expressing and double-negative cells were also intermingled. Adenomas from patients with Cushings syndrome expressed CYP11B1 entirely but not CYP11B2, resulting in atrophic nontumor glands. The nontumor portions of both types of adenomas bore frequently one or more aldosterone-producing cell clusters, which sustained CYP11B2 expression markedly under the conditions of the suppressed renin-angiotensin system. CONCLUSION Immunohistochemistry of the human normal adrenal cortex for CYP11B2 and CYP11B1 revealed a variegated zonation with cell clusters constitutively expressing CYP11B2. This technique may provide a pathological confirmatory diagnosis of adrenocortical adenomas.

Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands

Significance Primary aldosteronism (PA) represents the most common adrenal disease and cause of secondary hypertension. However, little is known regarding adrenal cellular origins. Recently, subcapsular aldosterone-producing cell clusters (APCCs) were observed in normal adrenals. We hypothesize that APCCs are a contributor to PA. Here, we characterized the APCC transcriptome and show that CYP11B2 expression is increased compared with the rest of the adrenal cortex. We also show that many APCCs harbor known aldosterone-producing adenoma (APA)-related ion channels/pumps (ATPase, Na+/K+ transporting, α1-polypeptide and calcium channel, voltage-dependent, L-type, α1D-subunit) mutations that stimulate CYP11B2 expression and aldosterone production. Importantly, the mutation spectrum seen in APCCs differs from that reported for APA. These results provide molecular support for APCC as a precursor of PA. Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na+/K+ transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.

Histopathological Diagnosis of Primary Aldosteronism Using CYP11B2 Immunohistochemistry

CONTEXT Although primary aldosteronism (PA) is the most common cause of endocrine hypertension, histopathological methods to reveal the presence and sites of aldosterone overproduction remain to be established. OBJECTIVE The objective of the study was to investigate the significance of immunohistochemical staining to detect CYP11B2 and CYP11B1 in adrenal tissue of patients with PA. DESIGN AND PATIENTS Thirty-two patients with PA who underwent unilateral adrenalectomy were studied. Immunohistochemical staining was performed using anti-CYP11B2 and anti-CYP11B1 antibodies on paraffin-embedded sections. We analyzed the expression of each enzyme semiquantitatively by scoring staining intensity and correlating it with clinical findings. RESULTS Twenty-two patients showed positive CYP11B2 immunostaining in their tumors (aldosterone producing adenoma, APA). Four patients with CYP11B2-negative unilateral adenomas and 4 patients without tumors on computed tomography showed aldosterone-producing cell clusters (APCCs) with CYP11B2 immunostaining in the zona glomerulosa (multiple APCCs). The remaining 2 patients had unilateral multiple adrenocortical micronodules and diffuse adrenocortical hyperplasia, respectively. In APA, CYP11B2 score adjusted for tumor volume was positively correlated with plasma aldosterone and negatively correlated with serum potassium. The APA group was divided into 3 subgroups based on relative CYP11B2 and CYP11B1 immunostaining levels. The CYP11B2/CYP11B1-equivalent and CYP11B1-dominant APA groups showed significantly higher serum cortisol after 1 mg dexamethasone and larger tumor size than the CYP11B2-dominant APA group. CONCLUSIONS The present study clearly demonstrates that CYP11B2 immunostaining is a powerful tool for histopathological diagnosis of aldosterone overproduction in PA and for subtype classification of APA, multiple APCCs, unilateral multiple adrenocortical micronodules, and diffuse hyperplasia.

Adrenal nodularity and somatic mutations in primary aldosteronism: One node is the culprit?

CONTEXT Somatic mutations in genes that influence cell entry of calcium have been identified in aldosterone-producing adenomas (APAs) of adrenal cortex in primary aldosteronism (PA). Many adrenal glands removed for suspicion of APA do not contain a single adenoma but nodular hyperplasia. OBJECTIVE The objective of the study was to assess multinodularity and phenotypic and genotypic characteristics of adrenals removed because of the suspicion of APAs. DESIGN AND METHODS We assessed the adrenals of 53 PA patients for histopathological characteristics and immunohistochemistry for aldosterone (P450C18) and cortisol (P450C11) synthesis and for KCNJ5, ATP1A1, ATP2B3, and CACNA1D mutations in microdissected nodi. RESULTS Glands contained a solitary adenoma in 43% and nodular hyperplasia in 53% of cases. Most adrenal glands contained only one nodule positive for P450C18 expression, with all other nodules negative. KCNJ5 mutations were present in 22 of 53 adrenals (13 adenoma and nine multinodular adrenals). An ATP1A1 and a CACNA1D mutation were found in one multinodular gland each and an ATP2B3 mutation in five APA-containing glands. Mutations were always located in the P450C18-positive nodule. In one gland two nodules containing two different KCNJ5 mutations were present. Zona fasciculata-like cells were more typical for KCNJ5 mutation-containing nodules and zona glomerulosa-like cells for the other three genes. CONCLUSIONS Somatic mutations in KCNJ5, ATP1A1, or CACNA1D genes are not limited to APAs but are also found in the more frequent multinodular adrenals. In multinodular glands, only one nodule harbors a mutation. This suggests that the occurrence of a mutation and nodule formation are independent processes. The implications for clinical management remain to be determined.

Case report: Nodule development from subcapsular aldosterone-producing cell clusters causes hyperaldosteronism

CONTEXT We previously reported that the human adrenal cortex remodels to form subcapsular aldosterone-producing cell clusters (APCCs). Some APCCs were recently found to carry aldosterone-producing adenoma (APA)-associated somatic mutations in ion channel/pump genes, which implied that APCCs produce aldosterone autonomously and are an origin of APA. However, there has been no report describing an APCC-to-APA transitional lesion. CASE DESCRIPTION A histological examination revealed unilateral multiple adrenocortical micronodules in the adrenals of two patients with primary aldosteronism (PA). Based on immunohistochemistry for aldosterone synthase, some of the micronodules were identified as possible APCC-to-APA transitional lesions (pAATLs; a tentative term used in this manuscript), which consisted of a subcapsular APCC-like portion and an inner micro-APA-like (mAPA-like) portion without an apparent histological border. Genomic DNA samples prepared from pAATL histological sections were analyzed by next-generation sequencing for the known APA-associated mutations. The mAPA-like portions from two of the three large pAATLs examined harbored mutations (KCNJ5 [p.G151R] in pAATL 3 and ATP1A1 [p.L337M] in pAATL 7), whereas their corresponding APCC-like portions did not, suggesting their role in the formation of mAPA. Another lesion carried novel mutations in ATP1A1 (p.Ile322_Ile325del and p.Ile327Ser) in both the mAPA-like and APCC-like portions, thereby supporting these portions having a clonal origin. CONCLUSION A novel aldosterone-producing pathology, pAATL that causes unilateral PA, was detected in the adrenals of two patients. Next-generation sequencing analyses of the large pAATLs suggested that the introduction of APA-associated mutations in the ion channel/pump genes may be involved in the development of mAPA from existing APCCs.

Potassium channels related to primary aldosteronism: Expression similarities and differences between human and rat adrenals

Three potassium channels have been associated with primary aldosteronism (PA) in rodents and humans: KCNK3 (TASK-1), KCNK9 (TASK-3), and KCNJ5 (Kir3.4). Mice with deficiency in Kcnk3 and Kcnk9 have elevated aldosterone production and blood pressure. In humans, adrenal tumors with somatic mutations in KCNJ5 cause PA. However, there are very few reports on the expression patterns of these genes in humans versus rodents. Herein, we compared human and rat mRNA expression (by quantitative real-time polymerase chain reaction (qPCR) and protein levels (by immunohistochemistry) across three tissues (adrenal, brain, heart) and two laser-captured adrenal zones (zona glomerulosa, zona fasciculata). Our findings show that expression patterns of KCNK3, KCNK9, and KCNJ5 are inconsistent between rats and humans across both tissues and adrenal zones. Thus, species variation in the expression of PA-related potassium channels indicates an evolutionary divergence in their role in regulating adrenal aldosterone production.

Lessons from the gene expression pattern of the rat zona glomerulosa

We recently identified hundreds of transcripts with differential expression in rat zona glomerulosa (zG) and zona fasciculata. Although the genes up-regulated in the zG may be playing important roles in aldosterone production, the relationship between most of these genes and aldosterone production has not been uncovered. Because aldosterone, in the presence of a high sodium diet, is now considered a significant cardiovascular risk factor, in this review we performed gene ontology and pathway analyses on the same microarray data to better define the genes that may influence zG function. Overall, we identified a number of genes that may be involved in aldosterone production through transforming growth factor β (TGF-β), WNT, calcium, potassium, and ACTH signaling pathways. The list of genes we present in the current report may become an important tool for researchers working on primary aldosteronism and aldosterone-related cardiovascular diseases.

Sodium deficiency regulates rat adrenal zona glomerulosa gene expression

Aldosterone is the primary adrenocortical hormone regulating sodium retention, and its production is under the control of the renin-angiotensin-aldosterone system (RAAS). In vitro, angiotensin II can induce aldosterone production in adrenocortical cells without causing cell proliferation. In vivo, a low-sodium diet activates the RAAS and aldosterone production, at least in part, through an expansion of the adrenal zona glomerulosa (zG) layer. Although these mechanisms have been investigated, RAAS effects on zG gene expression have not been fully elucidated. In this study, we took an unbiased approach to define the complete list of zG transcripts involved in RAAS activation. Adrenal glands were collected from 11-week old Sprague-Dawley rats fed either sodium-deficient (SDef), normal sodium (NS), or high-sodium (HS) diet for 72 hours, and laser-captured zG RNA was analyzed on microarrays containing 27 342 probe sets. When the SDef transcriptome was compared with NS transcriptome (SDef/NS comparison), only 79 and 10 probe sets were found to be up- and down-regulated more than two-fold in SDef, respectively. In SDef/HS comparison, 201 and 68 probe sets were up- and down-regulated in SDef, respectively. Upon gene ontology (GO) analysis of these gene sets, we identified three groups of functionally related GO terms: cell proliferation-associated (group 1), response to stimulus-associated (group 2), and cholesterol/steroid metabolism-associated (group 3) GO terms. Although genes in group 1 may play a critical role in zG layer expansion, those in groups 2 and 3 may have important functions in aldosterone production, and further investigations on these genes are warranted.

Prediction of extraprostatic extension by prostate specific antigen velocity, endorectal MRI, and biopsy Gleason score in clinically localized prostate cancer.

Objectives:  To investigate the clinical value of prostate specific antigen velocity (PSAV) in predicting the extraprostatic extension of clinically localized prostate cancer.

Human Adrenocortical Remodeling Leading to Aldosterone-Producing Cell Cluster Generation

Background. The immunohistochemical detection of aldosterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1) has enabled the identification of aldosterone-producing cell clusters (APCCs) in the subcapsular portion of the human adult adrenal cortex. We hypothesized that adrenals have layered zonation in early postnatal stages and are remodeled to possess APCCs over time. Purposes. To investigate changes in human adrenocortical zonation with age. Methods. We retrospectively analyzed adrenal tissues prepared from 33 autopsied patients aged between 0 and 50 years. They were immunostained for CYP11B2 and CYP11B1. The percentage of APCC areas over the whole adrenal area (AA/WAA, %) and the number of APCCs (NOA, APCCs/mm2) were calculated by four examiners. Average values were used in statistical analyses. Results. Adrenals under 11 years old had layered zona glomerulosa (ZG) and zona fasciculata (ZF) without apparent APCCs. Some adrenals had an unstained (CYP11B2/CYP11B1-negative) layer between ZG and ZF, resembling the rat undifferentiated cell zone. Average AA/WAA and NOA correlated with age, suggesting that APCC development is associated with aging. Possible APCC-to-APA transitional lesions were incidentally identified in two adult adrenals. Conclusions. The adrenal cortex with layered zonation remodels to possess APCCs over time. APCC generation may be associated with hypertension in adults.