Abstract 255: Allelic Variants in Arhgef11 via the Rho-Rock Pathway Promote Epithelial-Mesenchymal Transition in Proximal Tubule Cells and Contributes to Kidney Injury

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The Dahl salt-sensitive (S) rat is a widely studied model of hypertension that develops progressive kidney injury characterized by proteinuria, glomerulosclerosis and tubulointerstitial fibrosis, culminating in a significant decline in kidney function. Through positional cloning approaches, Arhgef11, a Rho guanine nucleotide exchange factor for the Rho-ROCK pathway was identified as a strong candidate gene. The most likely pathophysiological mechanism (based on animal studies) relates to the development of tubulointerstitial fibrosis, suggesting that chronic activation of Rho-ROCK in tubular cells may contribute to kidney injury observed in the S rat. Arhgef11 knockdown cell lines (up to 60% knockdown) as well as primary cultures of proximal tubule cells (PTC) were studied. Knockdown of Arhgef11 resulted in reduced RhoA activity (30% decrease, 0.57 ± 0.05 vs 0.82 ± 0.06 ng/mg protein), decreased activation of Rho-ROCK pathway (40-60%) and less stress fiber formation (2-fold reduction, 2.28 ± 0.18 vs 4.39 ± 0.64 in length/width ratio) versus control. S rat PTCs exhibited increased expression of Arhgef11, activation of Rho-ROCK, and decreased uptake of FITC-albumin (5.85 ± 0.71 vs 8.25 ± 0.64 mg/g protein) compared to control (minimal congenic). Both Arhgef11 knockdown cell-lines and PTCs stimulated with the profibrotic cytokine TGFβ1 were more prone (versus control) to epithelial-mesenchymal transition as demonstrated by increased expression of N-Cadherin, Vimentin, Col1a1 and spindle shape morphology. While dysregulation of the Rho-ROCK pathway in tubular cells appear to play an important role in EMT and promote fibrosis, chronic stimulation in other kidney cell types including vascular smooth muscle cells (VSMC) and glomerular epithelial cell (GEC) could also contribute to S rat kidney injury. Thus, we are currently investigating Arhgef11 knockdown in VSMC and GEC cell-lines as well as primary cells derived from the S rat. In summary, these studies suggest that the functional significance of genetic variants in Arhgef11 may be of major importance in promoting changes in cell morphology (Rho-Rock and/or EMT), function of tubule cells, and/or act in concert with dysregulation in VMSC and GEC to promote kidney injury.

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