A panoramic view of gene expression in the human kidney

Abstract

To gain a molecular understanding of kidney functions, we established a high-resolution map of gene expression patterns in the human kidney. The glomerulus and seven different nephron segments were isolated by microdissection from fresh tissue specimens, and their transcriptome was characterized by using the serial analysis of gene expression (SAGE) method. More than 400,000 mRNA SAGE tags were sequenced, making it possible to detect in each structure transcripts present at 18 copies per cell with a 95% confidence level. Expression of genes responsible for nephron transport and permeability properties was evidenced through transcripts for 119 solute carriers, 84 channels, 43 ion-transport ATPases, and 12 claudins. Searching for differences between the transcriptomes, we found 998 transcripts greatly varying in abundance from one nephron portion to another. Clustering analysis of these transcripts evidenced different extents of similarity between the nephron portions. Approximately 75% of the differentially distributed transcripts corresponded to cDNAs of known or unknown function that are accurately mapped in the human genome. This systematic large-scale analysis of individual structures of a complex human tissue reveals sets of genes underlying the function of well-defined nephron portions. It also provides quantitative expression data for a variety of genes mutated in hereditary diseases and helps in sorting candidate genes for renal diseases that affect specific portions of the human nephron.

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.…”

A large-scale screening of the normalized mammalian mitochondrial gene expression profiles

“…Numerous SNPs or genetic variations were detected in human and rat reference mitochondrial sequences, and average allele distribution could be estimated from the pooled datasets. For example, the G-allele of human mt-Nd6 gene CTCCCGAAT C / G tag (Sau3A I anchoring enzyme) was not detected in either 11 SAGE libraries constructed using the pooled RNA from 9 individuals (Chabardes-Garonne et al, 2003) or human U937 monoblast/early monocyte cell line, while the A-allele of human mt-Nd1 gene G / A CCAACCTCC tag (NlaIII) was detected in 7 SAGE libraries of 239 tested, with the average allele frequency being only 0 . 004 ( Fig.…”

Toward Quantitative Proteomics of Organ Substructures: Implications for Renal Physiology

“…A similar problem is often encountered in preparing subcellular fractions such as brush border or basolateral membranes. Hand dissection of nephron segments has been performed for transcriptome analysis of human and rodent nephron segments 2,[19][20][21] . It often requires the perfusion and incubation of the tissue with collagenases before dissection 22,23 .…”