Michael A. Edwards

Effect of KCNJ5 Mutations on Gene Expression in Aldosterone-Producing Adenomas and Adrenocortical Cells

CONTEXT Primary aldosteronism is a heterogeneous disease that includes both sporadic and familial forms. A point mutation in the KCNJ5 gene is responsible for familial hyperaldosteronism type III. Somatic mutations in KCNJ5 also occur in sporadic aldosterone producing adenomas (APA). OBJECTIVE The objective of the study was to define the effect of the KCNJ5 mutations on gene expression and aldosterone production using APA tissue and human adrenocortical cells. METHODS A microarray analysis was used to compare the transcriptome profiles of female-derived APA samples with and without KCNJ5 mutations and HAC15 adrenal cells overexpressing either mutated or wild-type KCNJ5. Real-time PCR validated a set of differentially expressed genes. Immunohistochemical staining localized the KCNJ5 expression in normal adrenals and APA. RESULTS We report a 38% (18 of 47) prevalence of KCNJ5 mutations in APA. KCNJ5 immunostaining was highest in the zona glomerulosa of NA and heterogeneous in APA tissue, and KCNJ5 mRNA was 4-fold higher in APA compared with normal adrenals (P < 0.05). APA with and without KCNJ5 mutations displayed slightly different gene expression patterns, notably the aldosterone synthase gene (CYP11B2) was more highly expressed in APA with KCNJ5 mutations. Overexpression of KCNJ5 mutations in HAC15 increased aldosterone production and altered expression of 36 genes by greater than 2.5-fold (P < 0.05). Real-time PCR confirmed increases in CYP11B2 and its transcriptional regulator, NR4A2. CONCLUSIONS KCNJ5 mutations are prevalent in APA, and our data suggest that these mutations increase expression of CYP11B2 and NR4A2, thus increasing aldosterone production.

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.

a Novel Y152C KCNJ5 mutation responsible for familial hyperaldosteronism type III.

CONTEXT Primary aldosteronism is a heterogeneous group of disorders comprising both sporadic and familial forms. Mutations in the KCNJ5 gene, which encodes the inward rectifier K(+) channel 4 (G protein-activated inward rectifier K(+) channel 4, Kir3.4), cause familial hyperaldosteronism type III (FH-III) and are involved in the pathogenesis of sporadic aldosterone-producing adenomas. OBJECTIVE The objective of the study was to characterize the effects of a newly described KCNJ5 mutation in vitro. PATIENTS AND METHODS The index case is a 62-year-old woman affected by primary aldosteronism, who underwent left adrenalectomy after workup for adrenal adenoma. Exon 1 of KCNJ5 was PCR amplified from adrenal tissue and peripheral blood and sequenced. Electrophysiological and gene expression studies were performed to establish the functional effects of the new mutation on the membrane potential and adrenal cell CYP11B2 expression. RESULTS KCNJ5 sequencing in the index case revealed a new p.Y152C germline mutation; interestingly, the phenotype of the patient was milder than most of the previously described FH-III families. The tyrosine-to-cysteine substitution resulted in pathological Na(+) permeability, cell membrane depolarization, and disturbed intracellular Ca(2+) homeostasis, effects similar, albeit smaller, to the ones demonstrated for other KCNJ5 mutations. Gene expression studies revealed an increased expression of CYP11B2 and its transcriptional regulator NR4A2 in HAC15 adrenal cells overexpressing KCNJ5(Y152C) compared to the wild-type channel. The effect was clearly Ca(2+)-dependent, because it was abolished by the calcium channel blocker nifedipine. CONCLUSIONS Herein we describe a new germline mutation in KCNJ5 responsible for FH-III.

ACTH is a potent regulator of gene expression in human adrenal cells

The adrenal glands are the primary source of minerocorticoids, glucocorticoids, and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by ACTH. Although it has been established that ACTH can stimulate steroidogenesis, the effects of ACTH on overall gene expression in human adrenal cells have not been established. In this study, we defined the effects of chronic ACTH treatment on global gene expression in primary cultures of both adult adrenal (AA) and fetal adrenal (FA) cells. Microarray analysis indicated that 48 h of ACTH treatment caused 30 AA genes and 84 FA genes to increase by greater than fourfold, with 20 genes common in both cell cultures. Among these genes were six encoding enzymes involved in steroid biosynthesis, the ACTH receptor and its accessory protein, melanocortin 2 receptor accessory protein (ACTH receptor accessory protein). Real-time quantitative PCR confirmed the eight most upregulated and one downregulated common genes between two cell types. These data provide a group of ACTH-regulated genes including many that have not been previously studied with regard to adrenal function. These genes represent candidates for regulation of adrenal differentiation and steroid hormone biosynthesis.

The effects of ACTH on steroid metabolomic profiles in human adrenal cells

The adrenal glands are the primary source of mineralocorticoids, glucocorticoids, and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by ACTH. Although it is well established that ACTH can stimulate steroidogenesis in the human adrenal gland, the effect of ACTH on overall production of different classes of steroid hormones has not been defined. In this study, we examined the effect of ACTH on the production of 23 steroid hormones in adult adrenal primary cultures and 20 steroids in the adrenal cell line, H295R. Liquid chromatography/tandem mass spectrometry analysis revealed that, in primary adrenal cell cultures, cortisol and corticosterone were the two most abundant steroid hormones produced with or without ACTH treatment (48  h). Cortisol production responded the most to ACTH treatment, with a 64-fold increase. Interestingly, the production of two androgens, androstenedione and 11β-hydroxyandrostenedione (11OHA), that were also produced in large amounts under basal conditions significantly increased after ACTH incubation. In H295R cells, 11-deoxycortisol and androstenedione were the major products under basal conditions. Treatment with forskolin increased the percentage of 11β-hydroxylated products, including cortisol and 11OHA. This study illustrates that adrenal cells respond to ACTH through the secretion of a variety of steroid hormones, thus supporting the role of adrenal cells as a source of both corticosteroids and androgens.

Gene expression profiles in aldosterone-producing adenomas and adjacent adrenal glands

BACKGROUND Primary aldosteronism (PA) is the most common form of endocrine hypertension affecting ∼8-10% of hypertensive subjects. Aldosterone production in PA occurs under low-renin conditions, and the mechanisms that maintain the production of aldosterone in PA remain unknown. Objective This study was designed to compare the transcript profiles between aldosterone-producing adenoma (APA) and their adjacent adrenal gland (AAG) from the same adrenal. METHODS Total RNA was extracted from ten APA and ten AAG; and subsequently analyzed by microarray and real-time quantitative RT-PCR (qPCR). The microarray data were paired for each APA-AAG, and analyzed by GeneSpring GX 11 with paired t-test and fold change calculations for each transcript. Changes identified by microarray analysis were confirmed by qPCR. RESULTS Microarray analysis indicated that 14 genes had significantly up-regulated expression in APA compared to AAG. Among the elevated genes were aldosterone synthase (CYP11B2) as well as novel transcription factors, calmodulin-binding proteins, and other genes that have not been previously studied in APA. Selective analysis of 11 steroidogenic enzymes using microarray demonstrated that only CYP11B2 showed a significantly higher transcript level in APA compared to AAG (P<0.001). In contrast, AKR1C3 (17β-hydroxysteroid dehydrogenase type 5), CYP17 (17α-hydroxylase/17,20 lyase), and CYB5 (cytochrome b5) showed significantly lower transcript level in APA (P<0.05). CONCLUSION The transcriptome analysis of APA compared with AAG showed several novel genes that are associated with APA phenotype. This gene list provides new candidates for the elucidation of the molecular mechanisms leading to PA.

Intraoperative ultrasonography during planned liver resections: why are we still performing it?

BackgroundIntraoperative ultrasonography (US) is used in many centers before oncologic liver resections to detect additional tumors and to evaluate the relationship of tumors to major vascular structures. As preoperative imaging improves, it is expected that the diagnostic yield from intraoperative US will diminish. In this study we attempt to determine if fewer unrecognized tumors were being detected and whether intraoperative US is having less impact on surgical decision making.MethodsWe compared 50 consecutive cases (mean age = 57.2 ± 10 years; 27 men) who underwent laparotomy for a planned resection of primary liver malignancies or metastases between September 2003 and July 2005 with 50 consecutive cases (mean age = 56.9 ± 14 years; 25 men) between January 1999 and September 2003. Dedicated intraoperative liver US was performed or supervised by a gastrointestinal radiologist using a 5.0-MHz linear- or curvilinear-array transducer during each procedure.ResultsThe rate of detecting unrecognized tumors has not changed significantly (14% vs. 20%, p = 0.70). The use of US to establish the relationship between tumor and the vasculature has not changed (48% vs. 60%, p = 0.23). The percentage of cases where the US findings were responsible for altering surgical management was 20% for both groups. The resection rate was 72% for both groups. The negative resection margin rate has also not changed significantly (86% vs. 69%, p = 0.09).ConclusionsDespite the advances in cross-sectional imaging, the frequency of unrecognized tumors found during intraoperative liver US and its use for surgical guidance has not changed significantly. Currently routine intraoperative US alters the management of approximately one fifth of our patients undergoing attempted liver resection for primary malignancies or metastases.

Do increased training requirements in gastrointestinal endoscopy and advanced laparoscopy necessitate a paradigm shift? A survey of program directors in surgery.

BACKGROUND Many modifications to the traditional residency model contribute to the ongoing paradigm shift in surgical education; yet, the frequency and manner by which such changes occur at various institutions is less clear. To address this issue, our study examined the variability in endoscopy and laparoscopy training, the potential impact of new requirements, and opinions of Program Directors in Surgery (PDs). METHODS A 22-item online survey was sent to 251 PDs in the United States. Appropriate parametric tests determined significance. RESULTS In all, 105 (42%) PDs responded. No difference existed in response rates among university (56.2%), university-affiliated/community (30.5%), or community (13.3%) program types (p = 0.970). Surgeons alone (46.7%) conducted most endoscopy training with a trend toward multidisciplinary teams (43.8%). A combination of fellowship-trained minimally invasive surgeons and other surgeon types (66.7%) commonly provided laparoscopy training. For adequate endoscopy experience in the future, most PDs (74.3%) plan to require a formal flexible endoscopy rotation (p < 0.001). For laparoscopy, PDs intend for more minimally invasive surgery (59%) as well as colon and rectal surgery (53.4%) rotations (both p < 0.001). Respondents feel residents will perform diagnostic endoscopy (86.7%) and basic laparoscopy (100%) safely on graduation. Fewer PDs confirm graduates will safely practice therapeutic endoscopy (12.4%) and advanced laparoscopy (52.4%). PDs believe increased requirements for endoscopy and laparoscopy will improve procedural competency (79% and 92.4%, respectively) and strengthen the fields of surgical endoscopy and minimally invasive surgery (55.2% and 68.6%, respectively). Less believe new requirements necessitate redesign of cognitive and technical skills curricula (33.3% endoscopy, 28.6% laparoscopy; p = 0.018). A national surgical education curriculum should be a required component of resident training, according to 79% of PDs. CONCLUSIONS PDs employ and may implement varied tools to meet the increased requirements in endoscopy and laparoscopy. With such variability in educational methodology, establishment of a national surgical education curriculum is very important to most PDs.

Bile Acids, FXR, and Metabolic Effects of Bariatric Surgery

Overweight and obesity represent major risk factors for diabetes and related metabolic diseases. Obesity is associated with a chronic and progressive inflammatory response leading to the development of insulin resistance and type 2 diabetes (T2D) mellitus, although the precise mechanism mediating this inflammatory process remains poorly understood. The most effective intervention for the treatment of obesity, bariatric surgery, leads to glucose normalization and remission of T2D. Recent work in both clinical studies and animal models supports bile acids (BAs) as key mediators of these effects. BAs are involved in lipid and glucose homeostasis primarily via the farnesoid X receptor (FXR) transcription factor. BAs are also involved in regulating genes involved in inflammation, obesity, and lipid metabolism. Here, we review the novel role of BAs in bariatric surgery and the intersection between BAs and immune, obesity, weight loss, and lipid metabolism genes.

Imaging of Gastrointestinal Bleeding

Radiological techniques are important in evaluating patients with gastrointestinal bleeding. Scintigraphic, computed tomographic angiographic, and enterographic techniques are sensitive tools in identifying the source of bleeding and may be useful in identifying patients likely to have a benign course and in selecting patients for therapeutic intervention. Angiography plays a key role in bleeding localization, and modern embolization techniques make this a viable therapeutic option. With the refining developments in body imaging and related reconstructive techniques, it is likely that radiological interventions will play an expanding and critical role in evaluating patients with gastrointestinal hemorrhage in the future.