Project description:Learn about the transcriptome profiling of zona glomerulosa (ZG), zona fasciculata (ZF) and aldosterone-producing adenomas (APA) in human adrenals
Project description:Learn about the transcriptome profiling of zona glomerulosa (ZG), zona fasciculata (ZF) and aldosterone-producing adenomas (APA) in human adrenals 21 pairs of zona fasciculata (ZF) and zona glomerulosa (ZG), and 14 paired aldosterone-producing adenomas (APAs) from 14 Conn’s syndrome patients and 7 phaeochromocytoma patients were assayed on the Affymetrix Human Genome U133 Plus 2.0 Array. Laser capture microdissection was used to acquire samples of ZF, ZG and APA as previously described (Azizan EA, et al. J Clin Endocrinol Metab. 2012;97:E819-E829). For differentiation of ZG from ZF, sections were stained with cresyl violet using the LCM Staining Kit (AM1935, Ambion, USA). Data processing and analysis was performed using AffymetrixGeneChip Command Console Software and PartekGenomicSuite 6.5 (Partek Inc., St. Louis, MO). Gene expressions were portrayed as the summarized log-signal of the Robust Multichip Average (RMA) with quantilenormalisation and median polish for probe set summarisation. Validation by qPCR was performed on genes >10 fold up-regulated in zona glomerulosa (compared to zona fasciculata) and >10 fold up-regulated in aldosterone-producing adenomas (compared to zona glomerulosa).
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:Aldosterone-producing adenomas (APA) are heterogeneous. The objectives of this study were to compare the transcriptional profiles in APA and normal adjacent adrenal gland (AAG). This was done through the Illumina beadchip analysis of RNA from eight paired APA-AAG. Other tissues (phaeochromocytoma, cushing, and hyperplastic adrenals) were included as controls.
Project description:The human adrenal gland consists of concentrically organized functionally distinct regions responsible for hormone production. Dysregulation of adrenocortical cell differentiation alters the proportion and organization of the functional zones of the adrenal cortex leading to disease. Current models of adrenocortical cell differentiation are based on mouse studies, but there are known organizational and functional differences between human and mouse adrenal glands. This study aimed to investigate the centripetal differentiation model in the human adrenal cortex and characterize aldosterone-producing micronodules (APMs) to better understand adrenal diseases such as primary aldosteronism. We applied spatially resolved in situ transcriptomics to human adrenal tissue sections from two individuals and identified distinct cell populations and their positional relationships. The results supported the centripetal differentiation model in humans, with cells progressing from the outer capsule to the zona glomerulosa, zona fasciculata, and zona reticularis. Additionally, we characterized two APMs in a 72-year-old female. Comparison with earlier APM transcriptomes indicated a subset of core genes, but also heterogeneity between APMs. The findings contribute to our understanding of normal and pathological cellular differentiation in the human adrenal cortex.
Project description:Analysis of aldosterone-producing adenoma (APA) samples from patients with primary hyperaldosteronism. These APAs have a somatic mutation in either KCNJ5, CACNA1D, or ATP1A1. Results provide insight into the different mechanisms each mutation may cause leading to elevated aldosterone production in APA. In this dataset, we include expression data from aldosterone-producing adenomas (APAs) with a somatic mutation in either KCNJ5, CACNA1D, or ATP1A1. These data are used to obtain differentially expressed genes between KCNJ5 mutant APAs and CACNA1D/ATP1A1 mutant APAs.
Project description:The mineralocorticoid hormone, aldosterone, is secreted by the adrenal zona glomerulosa (ZG) in response to high plasma K+ and hypovolemia and promotes renal Na+ reabsorption and K+ secretion. Hence, the regulation of aldosterone secretion is critical for the control of ion homeostasis and blood pressure. While the kinase pathways regulating aldosterone production are well studied, little is known about the involved phosphatases. Using the human adrenocortical carcinoma cell line NCI-H295R, we found that the mRNA expression of the aldosterone synthase increases significantly within 6 hours after K+ exposure. This increase was inhibited in a dose-dependent manner by the calcineurin inhibitors tacrolimus and cyclosporine A. Calcineurin (Cn) is a serine-threonine-specific, Ca2+ and CaM-activated protein phosphatase essential for lymphocyte, neuronal and cardiac function. The physiologic role of Cn in the ZG cells and the molecular pathways by which Cn regulates the K+-stimulated secretion of aldosterone are unknown. To answer these questions, we stimulated NCI-H295R cells with K+ with or without Tacrolimus and studied the phosphorylation pattern of cytoplasmic proteins by phospho-proteomics. We generated a map of the changes in the Ser/Thr phosphorylation in adrenocortical cells upon stimulation with K+ and identified Cn-regulated phosphoproteins.