Unknown,Transcriptomics,Genomics,Proteomics

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Atlas of gene expression in the mouse kidney

ABSTRACT: To gain molecular insight into kidney function, we performed a high-resolution quantitative analysis of gene expression in glomeruli and nine different nephron seg-ments dissected from mouse kidney using the long-SAGE method. We also developed dedicated bioinformatics tools and databases to annotate mRNA tags as transcripts. Over 800,000 mRNA SAGE tags were sequenced corresponding to >20,000 different mRNA tags present at least twice in at least one library. Hierarchical clustering analysis of tags demonstrated similarities between the three anatomical sub-segments of the proximal tubule, between the cortical and medullary segments of the thick ascending limb of Henle’s loop, and between the three segments constituting the aldosterone sensitive distal nephron segments, whereas the glomerulus and distal convoluted tubule clusterized independently. We also identified highly specific mRNA markers of each subgroup of nephron segments and of most nephron segments. Tag annotation also identified numbers of putative antisense mRNAs. This database constitutes a reference resource in which the quantitative expression of a given gene can be compared with that of other genes in the same nephron segment, or between different segments of the nephron. To illustrate possible applications of this database, we performed a deeper analysis of the glomerulus transcriptome which unexpectedly revealed expression of several ion and water carriers; within the glomerulus, they were found to be preferentially expressed in the parietal sheet. It also revealed the major role of the zinc finger transcription factor Wt1 in the specificity of gene expression in the glomerulus. Finally, functional annotation of glomerulus-specific transcripts showed the high proliferation activity of glomerular cells. Immunolabelling with anti-PCNA antibodies confirmed a high percentage of proliferating glomerular parietal cells. Approximately 1000 tubules from each different nephron segments were microdissected from 6-8 male CD1 mice. Over 800,000 mRNA SAGE tags were sequenced corresponding to >20,000 different mRNA tags present at least twice in at least one library.

ORGANISM(S): Mus musculus

SUBMITTER: Lydie Cheval

PROVIDER: E-GEOD-25223 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. This functional periodicity is believed to result, at least in part, from circadian changes in secretion/reabsorption capacities of the distal nephron and collecting ducts. Here, we studied the molecular mechanisms underlying circadian rhythms in the distal nephron segments, i.e. distal convoluted tubule (DCT) and connecting tubule (CNT) and, the cortical collecting duct (CCD). Temporal expression analysis performed on microdissected mouse DCT/CNT or CCD revealed a marked circadian rhythmicity in the expression of a large number of genes crucially involved in various homeostatic functions of the kidney. This analysis also revealed that both DCT/CNT and CCD possess an intrinsic circadian timing system characterized by robust oscillations in the expression of circadian core clock genes (clock, bma11, npas2, per, cry, nr1d1) and clock-controlled Par bZip transcriptional factors dbp, hlf and tef. The clock knockout mice or mice devoid of dbp/hlf/tef (triple knockout) exhibit significant changes in renal expression of several key regulators of water or sodium balance (vasopressin V2 receptor, aquaporin-2, aquaporin-4, M-oM-^AM-!ENaC). Functionally, the loss of clock leads to a complex phenotype characterized by partial diabetes insipidus, dysregulation of sodium excretion rhythms and a significant decrease in blood pressure. Collectively, this study uncovers a major role of molecular clock in renal function. Experiment Overall Design: We examined the temporal profiles of gene expression in mouse distal nephron segments and collecting ducts. The RNA was extracted from microdissected distal convoluted tubules and connecting tubules (DCT/CNT samples) or, cortical collecting ducts (CCD samples). Animals were sacrificed for microdissection every 4 hours, i.e. at ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20 (ZT M-bM-^@M-^S Zeitgeber (circadian) time, indicates time of light-on as ZT0 and time of light-off as ZT12). The microarray hybridization was performed in duplicates on two pools of RNA composed of equivalent amounts of RNA prepared from five animals at each ZT time-point.

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Atlas of gene expression in the mouse kidney

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