An Alternative Method for Adrenal Venous Sampling in Cases in Which Right Adrenal Vein Sampling is Difficult

PurposeCatheterization of the right adrenal vein (rt.AdV) to obtain blood samples can often be difficult. The aim of the present study was to investigate whether blood sampling from the inferior vena cava (IVC) at its juncture with the rt.AdV can be an alternative to sampling of blood directly from the rt.AdV.Materials and MethodsThis study included 44 patients diagnosed with primary aldosteronism (PA) in whom AVS with adrenocorticotropic hormone (ACTH) was performed, resulting in a diagnosis of idiopathic hyperaldosteronism (IHA) (n=24), and patients diagnosed with unilateral aldosterone-producing adenoma (APA) (n=20; rt.APA=8, lt.APA=12). In addition to regular blood sampling, blood was also sampled from the IVC, as the substitute rt.AdV [S-rt.AdV]. Diagnostic performance with the conventional lateralized index (LI) and the modified LI using the S-rt.AdV was compared to examine the utility of the modified LI.ResultsThe modified LI of the rt.APA (0.4±0.4) was significantly lower than those of the IHA (1.4±0.7) (p<0.001) and the lt.APA (3.5±2.0) (p<0.001). The modified LI of the lt.APA was significantly higher than those of the IHA (p<0.001) and rt.APA (p<0.001). Sensitivity and specificity to diagnose rt.APA and lt.APA using the modified LI with threshold values of 0.7 and 2.2, respectively, they were 87% and 75%, respectively, and 94% and 94%, respectively.ConclusionThe modified LI has the potential to be an alternative method for rt.AdV sampling in cases in which rt.AdV sampling is difficult. Obtaining the modified LI is extremely simple, which might complement conventional AVS.

ATP1A1 Mutant in Aldosterone-Producing Adenoma Leads to Cell Proliferation

The molecular mechanisms by which ATP1A1 mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated ATP1A1 L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the ATP1A1 mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with ATP1A1 mutation. ATP1A1 L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in ATP1A1 mutated APA were more abundant than those in non-functioning adrenocortical adenoma or KCNJ5 mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in ATP1A1 mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an ATP1A1 mutant APA.

In Silico Analysis of the Gene Expression Patterns between Aldosterone-Producing Adenoma and Nonfunctional Adrenocortical Adenoma

Primary aldosteronism is the most common form of secondary hypertension, and aldosteronoma makes up a significant proportion of primary aldosteronism cases. Aldosteronoma is also called aldosterone-producing adenoma (APA). Although there have been many studies about APA, the pathogenesis of this disease is not yet fully understood. In this study, we aimed to find out the difference of gene expression patterns between APA and nonfunctional adrenocortical adenoma (NFAA) using a weighted gene coexpression network (WGCNA) and differentially expressed gene (DEG) analysis; only the genes that meet the corresponding standards of both methods were defined as real hub genes and then used for further analysis. Twenty-nine real hub genes were found out, most of which were enriched in the phospholipid metabolic process. WISP2, S100A10, SSTR5-AS1, SLC29A1, APOC1, and SLITRK4 are six real hub genes with the same gene expression pattern between the combined and validation datasets, three of which indirectly or directly participate in lipid metabolism including WISP2, S100A10, and APOC1. According to the gene expression pattern of DEGs, we speculated five candidate drugs with potential therapeutic value for APA, one of which is cycloheximide, an inhibitor for phospholipid biosynthesis. All the evidence suggests that phospholipid metabolism may be an important pathophysiological mechanism for APA. Our study provides a new perspective regarding the pathophysiological mechanism of APA and offers some small molecules that may possibly be effective drugs against APA.

Colocalization of Wnt/β-catenin and ACTH signaling pathways and paracrine regulation in aldosterone producing adenoma

Context Aldosterone-producing adenomas (APA) are a common cause of primary aldosteronism. Despite the discovery of somatic mutations in APA and characterization of multiple factors regulating adrenal differentiation and function, the sequence of events leading to APA formation remains to be determined. Objective We investigated the role of Wnt/β-catenin and ACTH signaling, as well as elements of paracrine regulation of aldosterone biosynthesis in adrenals with APA and their relationship to intratumoral heterogeneity and mutational status. Design We analyzed expression of CYP11B2, CYP17A1, β-catenin, MC2R, pCREB, Tryptase, S100, CD34 by multiplex immunofluorescence and IHC guided RT-qPCR. Setting 11 adrenals with APA and one with micronodular hyperplasia from patients with PA were analysed. Main Outcome Measure(s) Localization of CYP11B2, CYP17A1, β-catenin, MC2R, pCREB, Tryptase, S100, CD34 in APA and aldosterone producing cell clusters (APCC). Results Immunofluorescence revealed abundant mast cells and a dense vascular network in APA, independent of mutational status. Within APA, mast cells were localized in areas expressing CYP11B2 and were rarely co-localized with nerve fibers, suggesting that their degranulation is not controlled by innervation. In these same areas, ß-catenin was activated, suggesting a zona glomerulosa cell identity. In heterogeneous APA with KCNJ5 mutations, MC2R and VEGFA expression was higher in areas expressing CYP11B2. A similar pattern was observed in APCC, with high expression of CYP11B2, activated β-catenin, and numerous mast cells. Conclusions Our results suggest that aldosterone producing structures in adrenals with APA share common molecular characteristics and cellular environment, despite different mutation status, suggesting common developmental mechanisms.

Recurrence of Primary Aldosteronism 10 Years After Left Adrenalectomy for Aldosterone-Producing Adenoma: A Case Report

A 29-year-old female patient diagnosed with primary aldosteronism (PA) in 2004 underwent complete adrenalectomy for left aldosterone-producing adenoma (APA) confirmed by hematoxylin and eosin (HE) and CYP11B2 staining. Her hypokalemia was corrected, and her blood pressure (BP) normalized and maintained without medication for 10 years. In 2014, her BP became elevated again, and a recurrence of PA with an adenoma on the right adrenal gland was discovered by computed tomography scan. She underwent partial right adrenalectomy in 2018 due to unsatisfactory BP control with medication and gradually enlarging adenoma. The resected adrenal tissue contained a CYP11B2 staining positive APA. Her BP was then controlled by two drugs. Sanger sequencing of DNA extracted from tissue slices revealed that both left and right adenomas carried the same aldosterone-driver KCNJ5 gene mutation, but with different nucleotide changes. We suggest that patients who undergo adrenalectomy for APA should be followed up for life.

Characteristics of a Novel ATP2B3 K416_F418delinsN Mutation in a Classical Aldosterone-Producing Adenoma

In patients with primary aldosteronism (PA), the prevalence of ATP2B3 mutation is rare. The aim of this study is to report a novel ATP2B3 mutation in a PA patient. Based on our tissue bank of aldosterone-producing adenomas (APA), we identified a novel somatic ATP2B3 K416_F418delinsN mutation. The affected individual was a 53 year-old man with a 4 year history of hypertension. Computed tomography (CT) showed bilateral adrenal masses of 1.6 (left) and 0.5 cm (right) in size. An adrenal venous sampling (AVS) showed a lateralization index (LI) of 2.2 and a contralateral suppression index (CLS) of 0.12; indicating left functional predominance. After a left unilateral adrenalectomy, he achieved partial biochemical and hypertension–remission. This classical adenoma harbored a novel ATP2B3 K416_F418delinsN somatic mutation, which is a deletion from nucleotides 1248 to 1253. The translated amino acid sequence from 416 to 418, reading as lysine-phenylalanine-phenylalanine, was deleted; however, an asparagine was inserted due to merging of residual nucleotide sequences. The CYP11B2 immunohistochemistry staining demonstrated strong immunoreactivity in this classical adenoma. The ATP2B3 K416_F418delinsN mutation is a functional mutation in APA, since HAC15 cells, a human adrenal cell line, transfected with the mutant gene showed increased CYP11B2 expression and aldosterone production.