Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies.
Pubmed ID: 24652793 RIS Download
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Project aggregates and provides experimental gene expression data from genito-urinary system. International consortium providing molecular atlas of gene expression for developing organs of GenitoUrinary (GU) tract. Mouse strains to facilitate developmental and functional studies within GU system. Experimental protocols and standard specifications. Tutorials describing GU organogenesis and primary data via database. Data are from large-scale in situ hybridization screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of developing mouse genitourinary (GU) system.
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