Project description:Primary aldosteronism (PA) is a common form of endocrine hypertension suspected in the presence of arterial hypertension (HT), hypokalemia and low plasma renin levels. While for a long time the genetic causes of PA have remained mysterious, recent findings have revealed an important role for an altered function of potassium channels in the pathogenesis of this disease. The basis for the unique sensitivity of adrenal zona glomerulosa (ZG) cells for plasma potassium concentration is a very high background potassium conductance, which is dependent on expression at high levels of two 2-pore domain (K2P) potassium channels, Task1 (KCNK3) and Task3 (KCNK9. Recent studies have shown that mice lacking Kcnk3 (Task1) or both Kcnk3 and Kcnk9 have PA. In Kcnk3 null mice, hyperaldosteronism is present in young animals of either sex, but is corrected in males after puberty. Strikingly, in these animals hyperaldosteronism associates with abnormal adrenal cortex functional zonation, since Cyp11b2, the rate-limiting enzyme for aldosterone production, is expressed in the inner region of the adrenal cortex and not in the glomerulosa. Additionally, this phenotype is under the control of sex hormones, as shown by the fact that castration of male Kcnk3 -/- animals prevents normal Cyp11b2 distribution, while testosterone injection in female Kcnk3 null mice restores expression of Cyp11b2 in ZG cells. We took advantage of the unique characteristics of the Kcnk3 null mouse model to search for genes that can modify their phenotype of PA and adrenocortical functional zonation. Using gene expression profiling in the adrenal glands of Kcnk3 null mice and exploiting the possibility of modulation of their phenotype by sexual hormones, we identified a cluster of genes closely associated with hyperaldosteronism in a sex- and hormone-dependent dynamic fashion. Among these genes, we focused our attention on Dkk3 (dickkopf3), encoding a peculiar member of the dickkopf family of Wnt signalling modulators because of its close association with aldosterone-producing cells in humans (11). Inactivation of Dkk3 in the Kcnk3 null background causes extension of the hyperaldosteronemic phenotype and increased expression of Cyp11b2 in the adrenal gland to the male sex, without affecting functional zonation. These data indicate that Dkk3 is a component of the genetic circuitry regulating expression of Cyp11b2 and suggest that it may be implicated in the pathogenesis of low-renin hyperaldosteronism in humans. One color -experiment with 2 strains of mice: C57BL/6 WT or Kcnk3 null (KO): male and female in basal conditions or following hormonal treatment (castration for males and testosterone injection for females), corresponding to 8 groups of animals. Total of 24 samples (n=3).

Project description:In order to analyze the transcriptome characteristics of aldosterone producing cell clusters (APCC) we compared transcript abundances of APCC, zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors. The frozen adrenal glands in O.C.T. compound were cut into 7um sections, and every 10-th section immunostained for aldosterone synthase (CYP11B2). The remaining sections were stained with cresyl violet and used for laser-capture microdissection of tissue to use in the array assays. APCC and ZG samples were captured from CYP11B2 positive regions based on the CYP11B2-stained sections. ZF and ZR were captured from lipid-rich cells in the middle layer and compact cells outside of the medulla, respectively. RNA was isolated using PicoPure RNA isolation kits (Molecular Devices, Sunnyvale, CA). 1-10 ng total RNA was reverse-transcribed and amplified with the Ovation Pico WTA System V2 (NuGEN Technologies, San Carlos, CA). cDNA was purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and biotin-labeled using Encore Biotin Module (NuGEN Technologies), followed by hybridization to GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). Expression values were calculated using the robust multi-array average method (RMA). This resulted in base-2 log-transformed data for each of the 4 tissues from each of the 4 people. In addition to the raw and processed data we also supply a supplementary Excel file holding the data and some statistical analysis, which has features to make simple graphs, and holds probe-set annotation that we used at that time (users may wish to obtain new annotation though). We fit two-way ANOVA models with terms for 4 tissues and 4 people, and compared each probe-set between every pair of tissues using F-tests for pairwise contrasts. We modeled people effects since they were not negligible. The supplement shows how to calculate the tests. Aldosterone producing cell clusters (APCC), zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors, were individually assayed on 16 Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Expression values were estimated with the Robust Multi-Arry average (RMA) algorithm, which resulted in log-2 transformed values for each of 54675 probe sets.

Project description:In order to analyze the transcriptome characteristics of aldosterone producing cell clusters (APCC) we compared transcript abundances of APCC, zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors. The frozen adrenal glands in O.C.T. compound were cut into 7um sections, and every 10-th section immunostained for aldosterone synthase (CYP11B2). The remaining sections were stained with cresyl violet and used for laser-capture microdissection of tissue to use in the array assays. APCC and ZG samples were captured from CYP11B2 positive regions based on the CYP11B2-stained sections. ZF and ZR were captured from lipid-rich cells in the middle layer and compact cells outside of the medulla, respectively. RNA was isolated using PicoPure RNA isolation kits (Molecular Devices, Sunnyvale, CA). 1-10 ng total RNA was reverse-transcribed and amplified with the Ovation Pico WTA System V2 (NuGEN Technologies, San Carlos, CA). cDNA was purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and biotin-labeled using Encore Biotin Module (NuGEN Technologies), followed by hybridization to GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). Expression values were calculated using the robust multi-array average method (RMA). This resulted in base-2 log-transformed data for each of the 4 tissues from each of the 4 people. In addition to the raw and processed data we also supply a supplementary Excel file holding the data and some statistical analysis, which has features to make simple graphs, and holds probe-set annotation that we used at that time (users may wish to obtain new annotation though). We fit two-way ANOVA models with terms for 4 tissues and 4 people, and compared each probe-set between every pair of tissues using F-tests for pairwise contrasts. We modeled people effects since they were not negligible. The supplement shows how to calculate the tests.

Project description:Primary aldosteronism (PA) is the most common endocrine hypertension comprising 10% of hypertensive patients. A recent series of transcriptome analyses using DNA microarray has shown that neumerous genes are differentially expressed between aldosterone-producing adenoma (APA) and its adjacent adrenal gland (AAG) tissue from the same patient. However, the molecular mechanism(s) of pathogenesis of APA has not yet been fully clarified. Although growing body of evidence has shown that epigenetic abnormalities including DNA methylation play a key role in tumorigenesis, there are few studies with regard to DNA methylation in APA. In the present study, to elucidate the pathogenic relationship between gene expression and DNA methylation in PA, we conducted an integrated analysis of transcriptome and methylome data for paired APA-AAG samples from the same patient. Adrenal specimens were obtained from 7 Japanese patients with APA, who underwent adrenalectomy at Tokyo Medical and Dental University. RNA and DNA samples were extracted from those 7 paired APA and AAG tissues. Gene expression and genome-wide DNA methylation profiles were obtained using SurePrint G3 Human GE 8x60K Microarray (Agilent) and Infinium HumanMethylation450 Beadchip (Illumina), respectively. Transcriptome anlaysis identified 244 significantly (2 fold<) upregulated genes in APA compared to AAG. The upregulated genes include the previously studied genes such as PCP4, ALDH1A2, and CYP11B2, and other genes that have not been previously studied, such as HOXA9, HOXA11, and HOXB9. Gene ontology (GO) analysis for these upregulated genes identified the calcium signaling pathway to be most significantly enriched with the upregulated genes (8 genes). Methylome analysis revealed that APA was globally hypomethylated compared to AAG regardless of gene feature groups, namely, TSS1500, TSS200, 5’UTR, 1stExon, gene body, and 3’UTR. GO analysis for the genes showing hypomethylation at TSS1500/TSS200 regions in APA identified the term “cytokine-cytokine receptor interaction” to be most significantly enriched with hypomethylated genes (37 genes). Integrated analysis of gene expression and genome-wide DNA methylation profiles identified 18 genes that are upregulated and whose TSS1500/TSS200 regions are hypomethylated in APA compared to AAG. These genes include CYP11B2 and MC2R (ACTH receptor). In conclusion, this is the first genome-wide study for PA that integrated transcriptome and methylome data. Global DNA hypomethylation in APA and concordant transcriptional up-regulation of some key genes, such as CYP11B2 and MC2R, may play crucial roles in the pathophysiological significance in PA. Adrenal specimens were obtained from 7 Japanese patients with APA, who underwent adrenalectomy at Tokyo Medical and Dental University. RNA and DNA samples were extracted from those 7 paired APA and AAG tissues. Gene expression and genome-wide DNA methylation profiles were obtained using SurePrint G3 Human GE 8x60K Microarray (Agilent) and Infinium HumanMethylation450 Beadchip (Illumina), respectively.

Project description:The source of aldosterone in 30 to 40 % of patients with primary hyperaldosteronism (PA) is unilateral aldosterone-producing adenoma (APA). The mechanisms causing elevated aldosterone production in APA are unknown. Herein, we examined expression of G-protein coupled receptors (GPCR) in APA and demonstrate that compared to normal adrenals there is a general elevation of certain GPCR in many APA and/or ectopic expression of GPCR in others. RNA samples from normal adrenals (n = 5), APAs (n = 10), and cortisol-producing adenomas (CPAs) (n=13) were used on 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least one out of 15 of the independent microarrays. The array results were confirmed using real-time RT-PCR (qPCR). Four GPCR transcripts exhibited a statistically significant increase that was greater than 3-fold compared to normal adrenals, suggesting a general increase in expression compared to normal adrenal glands. Four GPCR transcripts exhibited a greater than 15-fold increase of expression in one or more of the APA samples compared to normal adrenals. qPCR analysis confirmed array data and found the receptors with the highest fold increase in APA expression to be luteinizing hormone receptor (LH-R), serotonin receptor 4 (HTR4), gonadotropin-releasing hormone receptor (GnRHR), glutamate receptor metabotropic 3 (GRM3), endothelin receptor type B-like protein (GPR37), and ACTH receptor (MC2R). There are also sporadic increased expressions of these genes in the CPAs. Together, these findings suggest a potential role of altered GPCR expression in many cases of PA and provide candidate GPCR for further study. Keywords: disease state analysis Normal human adult adrenals were obtained through the Cooperative Human Tissue Network (Philadelphia, PA) and Clontech (Palo Alto, CA). All the normal adrenal samples came from patients that underwent adrenalectomy along with a renalectomy due to renal carcinoma. The adrenal samples acquired from autopsies were obtained no more than six hours after death, and it was confirmed that the causes of death were unrelated to adrenal function. Real-time quantitative RT-PCR was used to check the legitimacy of the normal control adrenal samples. Examination of tissue sections from each adrenal gland also suggested normal histology. The APA samples were obtained from the Departments of Medicine at UT Southwestern and Division of Endocrinology at the University of Padua. All APA samples were from Conn’s Syndrome patients with significantly elevated circulating aldosterone that was lowered to the normal range after surgical removal of the tumor. Thirty-two adenomas were collected from patients with primary hyperaldosteronism. Twenty-eight of these adenomas had levels of CYP11B2 that were two standard deviations (SD) greater than seen in normal adrenal glands and these samples were used for analyses. It is currently not clear if the adenomas with low CYP11B2 expression are the source of aldosterone in these patients. Other studies suggest that subcapsular micronodules have varying expression of steroidogenic enzymes-some with aldosterone synthase and some without (Shigematsu et al., 2006). Future studies are required to determine if some tumor-bearing adrenals from primary hyperaldosteronsim patients also have small CYP11B2 positive micronodules as the source of aldosterone production. The cortisol-producing adenoma (CPA) tissues were obtained from Division of Endocrinology at the University of Padua from patients with Cushing’s syndrome. Each of the patients was cured following surgical removal of the adenoma. The use of these tissues was approved by the Institutional Review Boards of the University of Texas Southwestern Medical Center (Dallas, TX), the Medical College of Georgia (Augusta, GA), and University of Padua (Italy). In addition, this study was approved by the ethical committee at the University of Padua, and informed consent was obtained from every patient. Microarray Analysis Total RNA isolated from normal adult adrenal glands (n = 5) and APA (n = 10) were used on 15 genomic expression arrays. In brief, RNA was hybridized to an Affymetrix human HG-U133+2 oligonucleotide microarray set containing 54,675 probe sets representing approximately 40,500 independent human genes. The arrays were scanned at high resolution using an Affymetrix GeneChip Scanner 3000 located at Medical College of Georgia Microarray Core Facility (Augusta, GA). Results were analyzed using GeneSpring GX 7.3.1 software (Silicon Genetics, Redwood City, CA) to identify differences in expression of GPCR between normal adult adrenal and APA. RNA Extraction The tissue was pulverized in liquid nitrogen to a powder. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA). The purity and integrity of the RNA were assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and its quantity was determined by NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA Synthesis 2 µg of total RNA was reverse transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) following manufacturer recommendations and incubated at 25 ºC for 10 min, and 37 ºC for 2 h. The synthesized cDNA was subjected to 1:10 dilution and stored at -20ºC. Real-Time Quantitative PCR Primers and probes for the amplification of the selected GPCR sequences were performed using the TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) based on published sequences for genes encoding the respective human GPCR. The gene symbols and AB assay numbers are listed in Table 1. The primer and probe set for human CYP11B2 was designed using Primer Express 3.0 (Applied Biosystems, Foster City, CA) and purchased from IDT (Integrated DNA Technologies, Inc, IA) as published previously (Saner-Amigh et al., 2006). In brief: Forward: 5’-GGCAGAGGCAGAGATGCTG-3’, Reverse: 5’- CTTGAGTTAGTGTCTCCACCAGGA-3’, Probe: 5’-CTGCACCACGTGCTGAAGCACT-3’. PCR reactions were performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) with a total volume of 20 μl per reaction following the reaction parameters recommended by the manufacturer, which includes denaturation at 95ºC for 20 s followed by amplification for 40 cycles (95ºC at 3 s, 60ºC at 30 s, fluorescence measurement). For each GPCR the 20 μl total volume consisted of 10 µl TaqMan Fast Universal PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 900 nM of each primer, 400 nM of the probe and 5 μl of each first-strand cDNA sample. CYP11B2 reaction mix consisted of 10µl TaqMan PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 100 nM of primer/probe mix and 5 μl of each first-strand cDNA sample. 18s rRNA was detected and quantified using the TaqMan Ribosomal RNA Control Reagents (Vic Probe) (Applied Biosystems, Foster City, CA). Each reaction included 10 µl of TaqMan PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 100 nM probe and 50 nM primers. Negative controls contained water instead of first-strand cDNA. Quantitative normalization of cDNA in each tissue-derived sample was performed using expression of 18s rRNA as an internal control. The generated Ct value of each gene was normalized by its respected Ct value of 18s rRNA (∆Ct). Then each gene was further normalized using the average ∆Ct value of the normal adult adrenal (∆∆Ct). The final fold-expression changes were calculated using the equation 2-∆∆Ct (Livak & Schmittgen, 2001). Statistical Analysis Data were analyzed and compared to control values (mean of normal adrenal samples) using the Mann-Whitney Rank Sum test with the SigmaStat 3.0 software package (SPSS, Chicago, IL). Results were considered significantly different when p value was  0.05.

Project description:Primary aldosteronism (PA) is the most common endocrine hypertension comprising 10% of hypertensive patients. A recent series of transcriptome analyses using DNA microarray has shown that neumerous genes are differentially expressed between aldosterone-producing adenoma (APA) and its adjacent adrenal gland (AAG) tissue from the same patient. However, the molecular mechanism(s) of pathogenesis of APA has not yet been fully clarified. Although growing body of evidence has shown that epigenetic abnormalities including DNA methylation play a key role in tumorigenesis, there are few studies with regard to DNA methylation in APA. In the present study, to elucidate the pathogenic relationship between gene expression and DNA methylation in PA, we conducted an integrated analysis of transcriptome and methylome data for paired APA-AAG samples from the same patient. Adrenal specimens were obtained from 7 Japanese patients with APA, who underwent adrenalectomy at Tokyo Medical and Dental University. RNA and DNA samples were extracted from those 7 paired APA and AAG tissues. Gene expression and genome-wide DNA methylation profiles were obtained using SurePrint G3 Human GE 8x60K Microarray (Agilent) and Infinium HumanMethylation450 Beadchip (Illumina), respectively. Transcriptome anlaysis identified 244 significantly (2 fold<) upregulated genes in APA compared to AAG. The upregulated genes include the previously studied genes such as PCP4, ALDH1A2, and CYP11B2, and other genes that have not been previously studied, such as HOXA9, HOXA11, and HOXB9. Gene ontology (GO) analysis for these upregulated genes identified the calcium signaling pathway to be most significantly enriched with the upregulated genes (8 genes). Methylome analysis revealed that APA was globally hypomethylated compared to AAG regardless of gene feature groups, namely, TSS1500, TSS200, 5’UTR, 1stExon, gene body, and 3’UTR. GO analysis for the genes showing hypomethylation at TSS1500/TSS200 regions in APA identified the term “cytokine-cytokine receptor interaction” to be most significantly enriched with hypomethylated genes (37 genes). Integrated analysis of gene expression and genome-wide DNA methylation profiles identified 18 genes that are upregulated and whose TSS1500/TSS200 regions are hypomethylated in APA compared to AAG. These genes include CYP11B2 and MC2R (ACTH receptor). In conclusion, this is the first genome-wide study for PA that integrated transcriptome and methylome data. Global DNA hypomethylation in APA and concordant transcriptional up-regulation of some key genes, such as CYP11B2 and MC2R, may play crucial roles in the pathophysiological significance in PA.

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Adrenal gene expression profiles of male and female Kcnk3 null mice in basal conditions or after hormonal treatment.

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