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  • Antisense knockdown of Kras inhibits fibrosis in a rat model of unilateral ureteric obstruction.

Antisense knockdown of Kras inhibits fibrosis in a rat model of unilateral ureteric obstruction.

The American journal of pathology (2011-11-15)
Jia-Hui Wang, Lucy J Newbury, A S Knisely, Brett Monia, Bruce M Hendry, Claire C Sharpe
ABSTRACT

Tubulointerstitial fibrosis is the hallmark of chronic kidney disease and is characterized by an increase in the number and activity of interstitial fibroblasts and by excessive matrix deposition. Ras is an intracellular signaling molecule involved in cell proliferation and differentiation. It has recently been implicated in the pathogenesis of renal fibrosis. Of the three different isoforms of Ras (Kirsten, Harvey, and Neural), we previously demonstrated that the Kirsten isoform is key in the control of renal fibroblast proliferation in vitro. In this study, we used gene therapy in the form of antisense oligonucleotides (ASOs) specifically to silence Kras (alias Ki-ras) expression in a rat model of renal fibrosis caused by unilateral ureteric obstruction. We demonstrate that renal Kras expression increases by 70% in this model compared with sham-operated animals and that treatment with ASOs can reduce total renal Kras by >90% to levels well below basal. This silencing is associated with a dramatic inhibition of interstitial fibrosis, a fivefold reduction in α-smooth muscle actin expression, and a 2.4-fold reduction in collagen I deposition. This inhibition was observed despite histologic evidence of marked interstitial inflammation. These findings demonstrate that silencing Kras expression can markedly inhibit renal fibrosis. This strategy should be considered as a new potential therapeutic avenue.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-Actin, α-Smooth Muscle, clone 1A4, ascites fluid
Sigma-Aldrich
Anti-Rat Collagen Type I Antibody, Chemicon®, from rabbit
Sigma-Aldrich
Anti-Aquaporin 1 Antibody, 1.0 mg/mL, Chemicon®
Sigma-Aldrich
Anti-Pan-Ras (Ab-3) Mouse mAb (RAS 10), liquid, clone RAS 10, Calbiochem®
Sigma-Aldrich
Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5, clone 6C5, Chemicon®, from mouse
Sigma-Aldrich
Anti-Aquaporin 2 Antibody, Chemicon®, from rabbit
Sigma-Aldrich
Anti-v-H-Ras (Ab-1) Rat mAb (Y13-259) Agarose Conjugate, suspension (Liquid), clone Y13-259, Calbiochem®