title: Translational profiles of medullary myofibroblasts during kidney fibrosis.
abstract: 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.
authors.affiliation: Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; Department of Internal Medicine and Nephrology, Philipps-University, Marburg, Germany;
authors.forename: Ivica
authors.initials: I
authors.lastname: Grgic
authors.affiliation: Department of Stem Cell Biology and Regenerative Medicine, W.M. Keck School of Medicine of the University of Southern California, Los Angeles, California; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Los Angeles, California;
authors.forename: A Michaela
authors.initials: AM
authors.lastname: Krautzberger
authors.affiliation: Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; Department of Internal Medicine and Nephrology, Philipps-University, Marburg, Germany;
authors.forename: Andreas
authors.initials: A
authors.lastname: Hofmeister
authors.affiliation: Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts;
authors.forename: Matthew
authors.initials: M
authors.lastname: Lalli
authors.affiliation: Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts;
authors.forename: Derek P
authors.initials: DP
authors.lastname: DiRocco
authors.affiliation: Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; Division of Nephrology, Hannover Medical School, Hannover, Germany;
authors.forename: Susanne V
authors.initials: SV
authors.lastname: Fleig
authors.affiliation: Department of Stem Cell Biology and Regenerative Medicine, W.M. Keck School of Medicine of the University of Southern California, Los Angeles, California;
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Division of Nephrology and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington;
authors.forename: Jeremy S
authors.initials: JS
authors.lastname: Duffield
authors.affiliation: Department of Stem Cell Biology and Regenerative Medicine, W.M. Keck School of Medicine of the University of Southern California, Los Angeles, California; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Los Angeles, California;
authors.forename: Andrew P
authors.initials: AP
authors.lastname: McMahon
authors.affiliation: University of Cincinnati Department of Pediatrics, Cincinnati, Ohio; and.
authors.forename: Bruce
authors.initials: B
authors.lastname: Aronow
authors.affiliation: Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; Kidney Group, Harvard Stem Cell Institute, Cambridge, Massachusetts bhumphreys@partners.org.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Humphreys
mesh_terms: D000818:Animals; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D004195:Disease Models, Animal; D005355:Fibrosis; D020869:Gene Expression Profiling / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000293:injuries / Q000378:metabolism* / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008822:Mice, Transgenic; D058628:Myofibroblasts / Q000378:metabolism* / Q000473:pathology*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation; D014517:Ureteral Obstruction / Q000235:genetics / Q000378:metabolism / Q000473:pathology
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C000630690:Rpl10 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins