Project description:The E3 ubiquitin -protein ligases (E3s) plays a role as regulators of protein trafficking and degradation. We aimed to identify E3s in rat kidney which are associated with dDAVP-induced urine concentration. Kidney inner medulla collected from vehicle treated control (n=2), dDAVP infusion for 5 d (D5d, n=2) and 3 h-withdrawal of dDAVP after 5 d-infusion (D5d-3h, n=2) groups were subjected to a transcriptome analysis.
Project description:Murine inner medullary collecting duct cells were treated for 1 hour with vehicle (control) or aldosterone. Total RNA was isolated and used as template to generate the eventual cRNA target. The experiment was repeated a total of three times. Six cRNA samples, three control and three treated, were generated and used in a total of six hybridizations. The mineralocorticoid aldosterone is a major regulator of Na+ and acid-base balance and control of blood pressure. Although the long-term effects of aldosterone have been extensively studied, the early aldosterone-responsive genes remain largely unknown. Using DNA array technology, we have characterized changes in gene expression after 1 h of exposure to aldosterone in a mouse inner medullary collecting duct cell line, mIMCD-3. Results from three independent microarray experiments revealed that the expression of many transcripts was affected by aldosterone treatment. Northern blot analysis confirmed the upregulation of four distinct transcripts identified by the microarray analysis, namely, the serum and glucose-regulated kinase sgk, connective tissue growth factor, period homolog, and preproendothelin. Immunoblot analysis for preproendothelin demonstrated increased protein expression. Following the levels of the four transcripts over time showed that each had a unique pattern of expression, suggesting that the cellular response to aldosterone is complex. The results presented here represent a novel list of early aldosterone-responsive transcripts and provide new avenues for elucidating the mechanism of acute aldosterone action in the kidney.
Project description:The mineralocorticoid hormone aldosterone controls sodium reabsorption and blood pressure largely by regulating the cell-surface expression and function of the epithelial sodium channel ENaC in target kidney tubules. Part of the stimulatory effect of aldosterone on ENaC is mediated by the induction of SGK1, a kinase that interferes with the ubiquitylation of ENaC by ubiquitin-protein ligase Nedd4-2. We performed a microarray study in order to investigate which other gene products are rapidly regulated by aldosterone in target cells that participate to the control of Na+ reabsorption and K+ secretion.
Project description:Aldosterone, the main mineralocorticoid hormone in Humans, has a major role in maintaining the hemo-electrolytique homeostasis, by acting in the distal part of nephron. This steroid hormone mainly acts through its binding to a ligand-induced transcription factor, the mineralocorticoid receptor (MR). MR binds to specific genomic sequences, where it recruits transcriptional coregulators, to activate or repress target gene transcription. The aim of this work was to access the whole aldosterone-dependand MR target genes, by comparing sequenced data from aldosterone or vehicle-treated samples. Anti-MR ChIP-seq in Human Kidney GFP-hMR cells treated with vehicle or Aldosterone.
Project description:The affect of aldosterone on the miRNA landscape in mIMCD-3 cells was determined In the study presented here, the affect of aldosterone was examined on the miRNA content in a murine inner medullary collecting duct cell line
Project description:Activation of the renal urine concentrating mechanism by vasopressin requires the coordinated regulation of multiple gene products including ion transporters, and water channels as well as regulatory proteins like protein kinases and phosphatases or enzymes involved in the energy-metabolism of the cells. We used microarray analysis of AVP-regulated gene products in a rat model of central diabetes insipidus (DI) to generate a comprehensive database documenting these changes. For microarray studies young (8 weeks) adult male Brattleboro rats were randomly divided into 2 groups (n = 3 per group) and treated for 3 days with either 1-desamino-8-D-Arg vasopressin (dDAVP; 5ng/h; Sigma Aldrich, Germany) or vehicle via osmotic minipump (ALZET minipump model 2001, Charles River, Sulzfeld, Germany). At the end of the treatment period animals were sacrificed and the kidneys removed. The outer medulla was isolated and used for cDNA generation and subsequent microarray analysis using [Rat230_2] Affymetrix Rat Genome 230 2.0 Array.
Project description:Target gene of mineralocorticoid receptor (MR) is comparatively unknown, although distal convoluted tubule (DCT) expresses MR in in vivo. We used microarray and immortalized murine DCT cell-line overexpressing human MR with treatment of aldosterone to elucidate target genes of MR in DCT. mDCT overexpresses human MR by lipofection and is treated for 3 hours with ethanol (g1), or 10^-9 M aldosterone (g2), or 10^-7 M aldosterone (g3), or 10^-7 M aldosterone with pretreatment of 5 x 10^-6 M spironolactone (g4) for 2 hours.
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. A total of 13 samples were analyzed (8 KCNJ5 mutant APAs and 5 CACNA1D/ATP1A1 mutant APAs). 43 genes had a false discovery rate (FDR) <0.5% and were >2-fold different between KCNJ5 mutant APAs and 5 CACNA1D/ATP1A1 mutant APAs. The two sets of genotypes were separated on unsupervised hierarchical clustering of 1475 genes correlating >0.6 with CYP11B2.
Project description:To validate the predicted Sh2b3 derived gene regulatory subnetwork using integrative network approach in human population study, we examined the gene expression levels of whole blood in WT (wild-type) and Sh2b3-/- mice by RNA sequencing, and identified the differentially expressed genes. RNA sequencing whole blood samples from 4 WT and 4 Sh2b3-/- mice.