Basic ResearchTranslational Profiles of Medullary Myofibroblasts during Kidney FibrosisGrgic, Ivica*,†; Krautzberger, A. Michaela‡,§; Hofmeister, Andreas*,†; Lalli, Matthew*; DiRocco, Derek P.*; Fleig, Susanne V.*,‖; Liu, Jing‡; Duffield, Jeremy S.¶,**; McMahon, Andrew P.‡,§; Aronow, Bruce††; Humphreys, Benjamin D.*,‡‡ Author Information *Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; †Department of Internal Medicine and Nephrology, Philipps-University, Marburg, Germany; ‡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; ‖Division of Nephrology, Hannover Medical School, Hannover, Germany; ¶Division of Nephrology and **Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington; ††University of Cincinnati Department of Pediatrics, Cincinnati, Ohio; and ‡‡Kidney Group, Harvard Stem Cell Institute, Cambridge, Massachusetts Correspondence: Dr. Benjamin D. Humphreys, Harvard Institutes of Medicine, Room 536, 77 Avenue Louis Pasteur, Boston, MA 02115. Email: [email protected] Received October 31, 2013 Accepted December 31, 2013 Journal of the American Society of Nephrology 25(9):p 1979-1990, September 2014. | DOI: 10.1681/ASN.2013101143 Buy Metrics 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. Copyright © 2014 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.