Male-dominant activation of rat renal organic anion transporter 1 (Oat1) and 3 (Oat3) expression by transcription factor BCL6
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ABSTRACT: Organic anion transporters 1 (Oat1) and 3 (Oat3) play a critical role in transport of organic anions, including frequently prescribed drugs, across cell membranes in proximal tubules of kidneys. In rats, these transporters are known to be male-predominant and testosterone dependently expressed. The molecular mechanisms that are involved in the sex-dependent expression are unknown. Our aim was to identify genes that show a sex-dependent expression and could be involved in sex-dependent regulation of Oat1 and Oat3. Comparison of gene expression in cortical kidney slices between 4 male and 4 female RCCHanTMWIST rats. Each animal were analyzed as a separate sample.
Project description:Organic anion transporters 1 (Oat1) and 3 (Oat3) play a critical role in transport of organic anions, including frequently prescribed drugs, across cell membranes in proximal tubules of kidneys. In rats, these transporters are known to be male-predominant and testosterone dependently expressed. The molecular mechanisms that are involved in the sex-dependent expression are unknown. Our aim was to identify genes that show a sex-dependent expression and could be involved in sex-dependent regulation of Oat1 and Oat3.
Project description:To identify the detailed molecular causes of the mitochondrial dysfunction, oxidative burden and more vulnerable redox balance in Rett mouse hippocampus, we screened for differential gene expression in the hippocampal CA1 subfield of adult male mice. A whole mouse genome microarray was performed to assess, whether key enzymes of the mitochondrial respiratory chain or major cellular radical scavenging enzymes are affected in this MeCP2-deficient mouse model of Rett syndrome. Comparison of gene expression between male wildtype mice (Mecp2+/y) and mice lacking the MECP2 gene (Mecp2-/y) [B6.129P2(C)-Mecp2tm-1-1Bird (Guy et al. 2001, Nat. Genet. 27: 322-326)]. In total, 9 mice of each genotype were analyzed. The mRNA of groups of 3 animals was pooled to generate 3 independent biological samples, and finally three microarrays were run for each genotype.
Project description:The transcriptional regulation of drug-metabolizing enzymes and transporters (here collectively referred to as DMEs) in the developing proximal tubule is not well understood. As in the liver, DME regulation in the PT may be mediated through nuclear receptors which are thought to “sense” deviations from homeostasis by being activated by ligands, some of which are handled by DMEs, including drug transporters. Systems analysis of transcriptomic data during kidney development predicted a set of upstream transcription factors, including Hnf4a and Hnf1a, as well as Nr3c1 (Gr), Nfe2l2 (Nrf2), Ppara, and Tp53. Motif analysis of cis-regulatory further suggested that Hnf4a and Hnf1a are the main transcriptional regulators in the PT. Available expression data from tissue-specific Hnf4a KO tissues revealed that distinct subsets of DMEs were regulated by Hnf4a in a tissue-specific manner. ChIP-seq was performed to characterize the PT-specific binding sites of Hnf4a in rat kidneys at three developmental stages (prenatal, immature, adult), which further supported a major role for Hnf4a in regulating PT gene expression, including DMEs. In ex vivo kidney organ culture, an antagonist of Hnf4a (but not a similar inactive compound) led to predicted changes in DME expression, including among others Fmo1, Cyp2d2, Cyp2d4, Nqo2, as well as organic cation transporters and organic anion transporters Slc22a1(Oct1), Slc22a2 (Oct2), Slc22a6 (Oat1), Slc22a8(Oat3), and Slc47a1(Mate1). Conversely, overexpression of Hnf1a and Hnf4a in primary mouse embryonic fibroblasts (MEFs), sometimes considered a surrogate for mesenchymal stem cells, induced expression of several of these proximal tubule DMEs, as well as epithelial markers and a PT-specific brush border marker Ggt1. These cells had organic anion transporter function. Taken together, the data strongly supports a critical role for HNF4a and Hnf1a in the tissue-specific regulation of drug handling and differentiation toward a PT cellular identity. Hnf4a binding was examined in rat kidneys at three timepoints (E20, P13 and Adult) and p300 binding was examined in adult rat kidney cortex tissue using ChIP-seq. Four corresponding input DNA samples were used as controls for peak calling.
Project description:In this study, a discovery approach was designed to identify novel mRNA transcripts that may be involved in kidney aging. RNA-Seq revealed increase in the expression of Clca1 mRNA in the aged kidney. CLCA1 facilitates Cl- current operated by TMEM16A, a Ca++-dependent Cl- channel. Cl- movement accompanied by cations such as Na+ and K+ or in exchange for other anions occurs among fluid compartments in many segments of the nephron. Cl- shift among these compartments is governed by electrochemical potential and transporters and exchangers.
Project description:The aim of this study is to evaluate the transcripitomics changes during dedifferentiation up to 7 days in a transwell system of rat primary proximal tubular cell (PTC) cultures isolated from rat kidneys. Samples were taken from freshly isolated PTC (0 days) and at timeponts during PTC differentiation (1, 2, 5 and 7 days). Samples from the kidney cortex, medulla and whole kidney slices were taken as control samples.
Project description:Sex-dependent differences in kidney function have been recognized. However, the molecular mechanisms underlying these differences remain largely unexplored. Advances in genomics and proteomic technologies now allow for an extensive characterization of sex differences. In this study, the authors apply multi-omics approaches integrating RNA-seq, ATAC-seq, and proteomics to investigate gene expression, chromatin accessibility, and protein expression between male and female mouse proximal tubules. This study identifies a large number of sex-biased genes and proteins associated with various kidney functions, including metabolism and transport processes. The authors demonstrate that sex differences may also arise from differences in interaction between transcription factors and accessible chromatin regions. A comprehensive web resource is provided to the research community to advance understanding of sex differences.
Project description:Sex differences have been increasingly recognized to play an important role in kidney physiology and pathophysiology, but limited resources are available for comprehensive interrogation of sex differences. Here, we coupled renal tubule microdissection with RNA-seq and ATAC-seq with a focus on proximal tubules that exhibited the greatest sex differences and carried out whole-kidney proteomics to build a comprehensive sex difference landscape between male and female mouse kidneys. The transcriptomic data indicate that the major sex differences are in S2 and S3 of proximal tubule segments, where signaling pathways including monocarboxylic acid metabolic process, organic anion transport, and organic acid transport display the greatest differences. We develop an ATAC-seq method on microdissected tubules to capture chromatin accessibility differences between sexes. The analysis reveals that major sex differences are in autosomes instead of sex chromosomes, and many of them are in S2/S3 segments related to the signaling pathways identified by transcriptomic analysis. Motif analysis identifies several transcription factors (Tead1, Nfia/b, and Pou3f3) whose interplay with other transcription factors (e.g. Hnf1b, Hnf4a) may contribute to sex differences. Finally, whole-kidney quantitative proteomics analysis correlates with the transcriptome analysis and identifies a large number of proteins (Cyp2e1, Acsm2/3) that are sex-dependent. We develop a knowledge portal to promote our understanding of sex differences in kidneys in kidneys at https://esbl.nhlbi.nih.gov/MRECA/PT/.
Project description:The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing kidney. The central thesis is straightforward. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing kidney. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in FACS isolated components of the developing kidney. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. Tie2-GFP transgenic mice were utilized to isolate the endothelial cell population from the glomerulus of adult kidneys. The glomerular endothelial cells were isolated from kidneys using a combination of collagenase treatment, differential seiving, trypsin treatment and FACS. The RNA was isolated from purified endothelial cells and the gene expression profiles were determined by microarrays.
Project description:The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing kidney. The central thesis is straightforward. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing kidney. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in FACS isolated components of the developing kidney. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. Tie2-GFP transgenic mice were utilized to isolate the endothelial cell population from the cortical region of adult kidneys. The cortical endothelial cells were isolated from kidneys using a combination of collagenase treatment, differential seiving, trypsin treatment and FACS. The RNA was isolated from purified endothelial cells and the gene expression profiles were determined by microarrays.
Project description:The renal clearance of perfluorooctanoic acid (PFOA) increased in Abcb4 null mice compared with wild type mice, especially in male Abcb4 null mice. We evaluated the expression changes of transporters in kidney of male Abcb4 null mice by using microarray to reveal the candidate transporters of PFOA. Male of Abcb4-null mice and wild-type mice (3 mice of 8 weeks old in each group) were sacrificed, collected their kidneys. Six independent experiments were performed.