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 7

Silencing SMOC2 reduces TGFβ1-induced fibrotic markers in vitro and folic acid–induced kidney fibrosis in mice.

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Silencing SMOC2 reduces TGFβ1-induced fibrotic markers in vitro and foli...
(A) Scheme of the experimental procedure for SMOC2 siRNA–transfected NIH3T3 cells. After 24 hours of treatment with SMOC2 siRNA or scrambled siRNA (ssiRNA), NIH3T3 fibroblasts were either treated with/without TGFβ1 for 24 hours. Representative Western blot (n = 3/condition; Supplemental Figure 14) was performed for SMOC2, αSMA, collagen 1α1, and fibronectin expression. (B) Scheme of the experimental procedure for SMOC2 siRNA– or ssiRNA-injected C57BL/6 mice treated with/out folic acid (FA). Mice were injected i.v. with 30 μg/200 μl of SMOC2 siRNA or ssiRNA 4 hours before and 2, 4, and 6 days after an i.p. injection of 250 mg/kg of FA. Representative Western blot (n = 5/group; Supplemental Figure 16) was performed for SMOC2, αSMA, collagen 1α1, and fibronectin. (C) Immunofluorescent αSMA staining of kidneys obtained from mice at day 7 following with/out FA either treated with ssiRNA or SMOC2 siRNA (n = 5). (D) Masson’s trichrome staining of normal and FA-treated kidneys obtained at day 7 following ssiRNA or SMOC2 siRNA administration. Confocal and light microscopy images are 20× magnification. Scale bars: 50 μM. Quantification of images is represented as a box plot (n = 5/condition, 10 visual fields/mice), which describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 (ssiRNA + vehicle) and #P < 0.05 (ssiRNA respective treatment) determined by one-way ANOVA with Tukey post-hoc analysis.