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 collagen1a1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1a1-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.

Cellular and molecular mechanisms in kidney fibrosis

“…Myofibroblast precursors all generate COL1 protein and other collagen proteins in healthy kidney, but the mechanism of turnover is obscure (16). Recent studies have identified ADAMTS-2 as a highly upregulated gene in FOXD1-lineage cells when they become myofibroblasts, while its endogenous inhibitor TIMP-3 is highly downregulated (42,99,100). ADAMTS-2 cleaves procollagen-I to facilitate fibril formation (101) and may therefore be an important endogenous regulator of matrix turnover in health and disease, whereas TIMP-3 deficiency results in increased matrix deposition in the kidney (42).…”

Renal Allograft Fibrosis: Biology and Therapeutic Targets

“…Using transgenic mice for example for the collagen1a1 promoter coupled with the reporter for translating ribosome affinity purification (TRAP), translational profiling of medullary pericytes was performed in healthy and fibrotic kidneys. This approach identified several previously unappreciated and highly regulated genes in fibrosis which are mostly involved in proliferation, cytoskeleton, cell-cell connections and motility (169). Similar genetic approaches allow profiling of various renal cells including fibroblasts, tubular cells, endothelium and monocytes and macrophages (using Foxd1, Six2, Cdh5, or Lyz2 promoters, respectively) and will likely lead to the identification of novel targets in renal fibrosis (38).…”

“…So far, myofibroblasts have been characterized by their prominent expression of a-smooth muscle actin (aSMA). With the development of novel markers, targeting tools, transgenic mice and cell isolation protocols, the functional and phenotypical heterogeneity and plasticity of these cells is becoming apparent (24,166,167,169). Using transgenic mice for example for the collagen1a1 promoter coupled with the reporter for translating ribosome affinity purification (TRAP), translational profiling of medullary pericytes was performed in healthy and fibrotic kidneys.…”

Kidney Pericytes: Roles in Regeneration and Fibrosis

“…70 To overcome the challenges in isolating pericyte and myofibroblast-specific RNA, we applied a unique approach called translating ribosome affinity purification. 71 This involves the cell-specific expression of the ribosomal subunit protein L10a fused in-frame to enhanced green fluorescent protein (eGFP).…”

“…A number of novel secreted proteins may be useful as noninvasive biomarkers of renal fibrosis. 70 The signaling pathways most notably up-regulated include integrin PDGF signaling, and up-regulated gene families include snail homologs as well as the Wnt modulating gene family, secreted frizzled related proteins. These pathways comprise known important regulators of myofibroblast function and kidney fibrosis, providing further internal validation of this approach.…”