Translational profiles of medullary myofibroblasts during kidney fibrosis

J Am Soc Nephrol. 2014 Sep;25(9):1979-90. doi: 10.1681/ASN.2013101143. Epub 2014 Mar 20.

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.

Publication types

  • Research Support, American Recovery and Reinvestment Act
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Collagen Type I / genetics
  • Disease Models, Animal
  • Fibrosis
  • Gene Expression Profiling / methods
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Kidney / injuries
  • Kidney / metabolism*
  • Kidney / pathology*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Inbred DBA
  • Mice, Transgenic
  • Myofibroblasts / metabolism*
  • Myofibroblasts / pathology*
  • Oligonucleotide Array Sequence Analysis
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Ribosomal Protein L10
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / metabolism
  • Up-Regulation
  • Ureteral Obstruction / genetics
  • Ureteral Obstruction / metabolism
  • Ureteral Obstruction / pathology

Substances

  • Collagen Type I
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Ribosomal Proteins
  • Rpl10 protein, mouse
  • collagen type I, alpha 1 chain
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins