UPMC Univ Paris 06, Univ Paris Descartes and INSERM, UMRS 872, Centre de recherche des Cordeliers, Paris, France.
Physiol Genomics. 2011 Feb 11;43(3):161-73. doi: 10.1152/physiolgenomics.00093.2010. Epub 2010 Nov 16.
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.
为了深入了解肾脏功能的分子机制，我们采用基因表达序列分析（SAGE）技术，对从小鼠肾脏中分离得到的肾小球和 9 种不同肾单位节段进行了高分辨率的定量基因表达分析。我们还开发了专用的生物信息学工具和数据库，用于注释 mRNA 标签作为转录本。测序得到了超过 80 万个 SAGE mRNA 标签，对应于至少在一个文库中出现两次以上的 >20,000 个不同的 mRNA 标签。标签的层次聚类分析表明，近端小管的三个解剖亚段之间、Henle 袢升支粗段的皮质和髓质段之间以及构成醛固酮敏感的远端肾单位段的三个段之间存在相似性，而肾小球和远端卷曲小管则独立聚类。我们还鉴定了每个肾单位节段亚群和大多数肾单位节段的高度特异性 mRNA 标记物。标签注释还鉴定了许多假定的反义 mRNA。该数据库构成了一个参考资源，其中给定基因的定量表达可以与同一肾单位节段中的其他基因的表达进行比较，或者在肾单位的不同节段之间进行比较。为了说明该数据库的可能应用，我们对肾小球转录组进行了更深入的分析，出乎意料地发现了几种离子和水载体的表达；在肾小球中，发现它们优先在壁层表达。它还揭示了锌指转录因子 Wt1 在肾小球基因表达特异性中的主要作用。最后，肾小球特异性转录本的功能注释表明肾小球细胞具有高增殖活性。PCNA 的免疫标记证实了壁层肾小球细胞中有高比例的增殖细胞。
