The cytoprotective role of Ras in complement-mediated glomerular epithelial cell injury Carl Huynh, Guohui Ren, Joan Papillon, Julie Guillemette, Tomoko Takano, Andrey V. Cybulsky ⁎ Department of Physiology, McGill University, Montreal, Quebec, Canada Department of Medicine, McGill University, Montreal, Quebec, Canada Received 25 July 2008; accepted with revision 24 November 2008 Available online 9 January 2009 Abstract In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to loss of glomerular permselectivity and proteinuria. Incubation of cultured GEC with antibody and serially-increasing concentrations of complement induced cytotoxicity in a dose-dependent manner. Stable expression of constitutively-active Ras (V 12 Ras) in GEC attenuated injury significantly. In the V 12 Ras-expressing GEC, disruption of the F-actin cytoskeleton with latrunculin B or swinholide A, or stabilization of F-actin with jasplakinolide reversed the cytoprotective effect of V 12 Ras. GEC displayed cortical F-actin; V 12 Ras-expressing GEC showed smaller and more rounded morphology, and decreased activity of the Rho GTPase, Rac1, compared with control GEC. Thus, the protective effect of V 12 Ras is dependent on remodeling of the actin cytoskeleton, and may be associated with a reduction in Rac activity, thereby altering the equilibrium in the activities of Rho GTPases. Activation of Ras signaling is a novel pathway to consider in developing strategies for cytoprotection in complement-mediated injury. © 2008 Elsevier Inc. All rights reserved. KEYWORDS Actin; Cytoskeleton; Rho GTPases; Protein kinases Introduction Ras, a small highly-conserved GTPase that is activated upon engagement of various membrane receptors by their ligands, is responsible for a wide variety of signaling pathways that control cytoskeletal integrity, proliferation, cell adhesion, cell migration, and apoptosis. Ras signaling is dependent on many downstream effectors [1]. Among the best character- ized pathways is the Raf, mitogen-activated protein kinase (MAPK) kinase (MEK), extracellular signal-regulated kinase1/ 2 (ERK) cascade [2]. In addition to ERK, there are a number of Abbreviations: cPLA 2 , cytosolic phospholipase A 2 ; ERK, extracel- lular signal-regulated kinase; GEC, glomerular epithelial cell; GST- CRIB, glutathione S-transferase fusion protein containing the cdc42/ Rac-GTP interactive binding domain; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; PI3K, phosphatidylinositol 3-kinase. ⁎ Corresponding author. Division of Nephrology, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. Fax: +1 514 843 2815. E-mail address: andrey.cybulsky@mcgill.ca (A.V. Cybulsky). 1521-6616/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2008.11.012 available at www.sciencedirect.com www.elsevier.com/locate/yclim Clinical Immunology (2009) 131, 343–353