Abstract 227: Molecular Mechanism of Arhgef11 via the Rho-Rock Pathway in Promoting Kidney Injury in the Dahl S Rat
We have previously implicated genetic variants in Arhgef11, a Rho guanine nucleotide exchange factor, in kidney injury exhibited in the Dahl salt-sensitive (S) rat, a widely studied model of hypertension and chronic kidney disease. We have demonstrated that genetic knockdown (lentiviral shRNA) of Arhgef11 in HEK293 cells results in reduced RhoA activity (~20%), decreased activation of Rho-ROCK pathway (40-60%), and less stress fiber formation vs. scrambled control. S rat derived primary proximal tubule cells (p-PTC) exhibit increased expression of Arhgef11, activation of Rho-ROCK, and when stimulated with the profibrotic cytokine, TGFβ1 are also more prone (vs. control) to epithelial-mesenchymal transition (EMT). These data (along with animal studies) suggest that dysregulation of Rho-ROCK in tubular cells likely play an important role in EMT and promote fibrosis; however, chronic stimulation of this pathway in other kidney cell types including vascular smooth muscle cells (VSMC) may also play a role. Accordingly, S rat primary cultures of VSMC isolated from kidney microvessels (k-VSMC) were studied for differences in the RhoA-ROCK, proliferation, migration, apoptosis, and response to cyclical stretch compared to control k-VSMC (minimal congenic). S rat k-VSMC demonstrated higher expression of Arhgef11 and activation of Rho-ROCK pathway, similar to p-PTC studies. Cell migration was significantly enhanced (scratch assay) in k-VSMC from S rats (485.0±26.2 nm) compared to 296.2±37.6 nm in cells from control. Apoptosis, evaluated by flow cytometry, was significantly increased in k-VSMC from S rats stimulated with TGFβ1 (10ng/ml, 24hrs), but not Ang II, at both early (9.9%±0.9%) and late apoptosis (22.0%±3.5%) compared to control (7.71%±0.75% and 15.5%±1.3%, respectively). S rat derived k-VSMC also demonstrated up-regulation and activation of Rho-ROCK pathway after cyclical stretch, which models in vivo stimuli such as shear stress. In summary, increased expression of Arhgef11 (RhoA-ROCK) exhibited by PTC and VSMC cell types in S rat kidney appear to promote epithelial-mesenchymal transition and enhance proliferation, migration, and apoptosis under baseline and profibrotic conditions, which likely contributes to kidney injury.
Author Disclosures: Z. Jia: None. A.C. Johnson: None. Z. Guo: None. M.R. Garrett: None.
- © 2014 by American Heart Association, Inc.