Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation
Casimiro Gerarduzzi, Ramya K. Kumar, Priyanka Trivedi, Amrendra K. Ajay, Ashwin Iyer, Sarah Boswell, John N. Hutchinson, Sushrut S. Waikar, Vishal S. Vaidya
Casimiro Gerarduzzi, Ramya K. Kumar, Priyanka Trivedi, Amrendra K. Ajay, Ashwin Iyer, Sarah Boswell, John N. Hutchinson, Sushrut S. Waikar, Vishal S. Vaidya
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Research Article Nephrology

Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation

Abstract

Secreted modular calcium-binding protein 2 (SMOC2) belongs to the secreted protein acidic and rich in cysteine (SPARC) family of matricellular proteins whose members are known to modulate cell-matrix interactions. We report that SMOC2 is upregulated in the kidney tubular epithelial cells of mice and humans following fibrosis. Using genetically manipulated mice with SMOC2 overexpression or knockdown, we show that SMOC2 is critically involved in the progression of kidney fibrosis. Mechanistically, we found that SMOC2 activates a fibroblast-to-myofibroblast transition (FMT) to stimulate stress fiber formation, proliferation, migration, and extracellular matrix production. Furthermore, we demonstrate that targeting SMOC2 by siRNA results in attenuation of TGFβ1-mediated FMT in vitro and an amelioration of kidney fibrosis in mice. These findings implicate that SMOC2 is a key signaling molecule in the pathological secretome of a damaged kidney and targeting SMOC2 offers a therapeutic strategy for inhibiting FMT-mediated kidney fibrosis — an unmet medical need.

Authors

Casimiro Gerarduzzi, Ramya K. Kumar, Priyanka Trivedi, Amrendra K. Ajay, Ashwin Iyer, Sarah Boswell, John N. Hutchinson, Sushrut S. Waikar, Vishal S. Vaidya

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Figure 3

SMOC2 induces a fibroblast-to-myofibroblast transition.

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SMOC2 induces a fibroblast-to-myofibroblast transition. (A) RNAseq was p...
(A) RNAseq was performed using kidneys from SMOC2 Tg and WT mice at day 7 following UUO treatment. REVIGO treemap visualizations are shown for enriched gene ontology (GO) categories. Highly similar GO terms for cellular components are grouped and visualized by different colors and sizes of the rectangles using semantic similarity and enrichment P values. Representative Western blots of αSMA, collagen 1α1, and fibronectin from serum-deprived primary human kidney fibroblasts (B, n = 3/condition; Supplemental Figure 8C) and NIH3T3 fibroblasts (C, n = 3/condition; Supplemental Figure 8D) treated with 10 ng/ml SMOC2 with/without TGFβ1. (D) After 1 hour of antibody pretreatment, SMOC2 or TGFβ1 was treated to serum-deprived NIH3T3 cells for 24 hours and then tested for conventional fibrotic markers, while integrin β1 antibody was pretreated with NIH3T3 cells and then treated with SMOC2 (n = 3/condition; Supplemental Figure 8E). (E) NIH3T3 fibroblasts were transfected with SMOC2-MYC, empty vector control, or negative control MGP-MYC and then immunoprecipitated with a MYC (above) or integrin antibody (below). Western blots are representative immunoprecipitation experiments. (F) Representative Western blot for Phospho-Focal Adhesion Kinase (P-FAK) Y925, P-Myosin Light Chain (MLC) Ser19, and P-Paxillin Tyr118 from NIH3T3 cells treated with 10 ng/ml SMOC2 or 5 ng/ml TGFβ1 for 60 minutes (n = 5/condition; Supplemental Figure 8H). (G) Phalloidin staining of F-actin after NIH3T3 cells were treated 24 hours with 10 ng/ml SMOC2 or 5 ng/ml TGFβ1 (n = 3). Top row 40×, 25 μM. Bottom row 100×, 15 μM. Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 determined by t test.