Atlas of gene expression in the mouse kidney: new features of glomerular parietal cells

Abstract

To gain molecular insight into kidney function, we performed a high-resolution quantitative analysis of gene expression in glomeruli and nine different nephron segments dissected from mouse kidney using Serial Analysis of Gene Expression (SAGE). 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 subsegments 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 that 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 suggested a high proliferation activity of glomerular cells. Immunolabeling for PCNA confirmed a high percentage of proliferating cells in the glomerulus parietal sheet.

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

“…RNA-seq offers important advantages over other approaches to kidney transcriptomics, such as DNA microarrays using biochemically isolated segments 32,33 or SAGE of microdissected renal tubule segments. 2,3 Compared with biochemical isolation techniques, [34][35][36] manual dissection virtually eliminates contamination from neighboring segments. RNA-seq does not depend on hybridization and therefore, eliminates false positivity caused by cross-hybridization in microarray studies.…”

“…A broader goal, identification of all genes expressed in each cell type, has been pursued with serial analysis of gene expression (SAGE) to identify mRNA transcripts in microdissected tubules. 2,3 This method, however, has limited sensitivity, requiring very large numbers of tubules per sample and limiting the transcriptomic depth (i.e., the number of genes identified per sample). The advent of deep-sequencing (next generation sequencing) technology has provided a quantum leap in sensitivity.…”

Protein Phosphatase 1 Inhibitor-1 Deficiency Reduces Phosphorylation of Renal NaCl Cotransporter and Causes Arterial Hypotension

“…Using freehand microdissection, previous studies established transcriptome data for the renal distal convolution, 21,22 but the gradual transition from the DCT to the connecting tubule makes it almost impossible to precisely define the border between these functionally different segments. Moreover, the low yield of microdissection requires amplification of obtained cDNAs, which bears the risk of increased false-positive and false-negative results.…”

“…Therefore, we hypothesized that the DCT may express a particular set of DCT-enriched gene products, of which at least some participate in the regulation of NCC-mediated sodium absorption. Encouraged by previous gene expression analysis on freehand-isolated renal tubules in the works by Chabardès-Garonne et al, 20 Cheval et al, 21 and Pradervand et al, 22 we aimed at identifying DCT-enriched gene products using a transcriptomic approach. We used complex object parametric analysis and sorting (COPAS) of renal tubules, which was developed by Miller et al, 23 to isolate DCTs on a large scale.…”

Collectrin and ACE2 in renal and intestinal amino acid transport

“…30 In the kidneys, it was shown to be highly expressed at the mRNA level in the medullary and cortical thick ascending limb and more distal nephron segments. 33 The function of this transporter is still not clear and a knockout mouse model is not available yet.…”