Angiotensin II Activation of TRPC6 Channels in Rat Podocytes Requires Generation of Reactive Oxygen Species MARC ANDERSON, 1 HILA ROSHANRAVAN, 1 JUSTIN KHINE, 1 AND STUART E. DRYER 1,2 * 1 Department of Biology and Biochemistry, University of Houston, Houston, Texas 2 Division of Nephrology, Baylor College of Medicine, Houston, Texas Angiotensin II (AII) plays a major role in the progression of chronic kidney diseases. Podocytes are essential components of the ultrafiltration apparatus, and are targets for AII signaling. AII has been shown to increase generation of reactive oxygen species (ROS) in podocytes. Canonical transient receptor potential-6 (TRPC6) channels stimulate Ca 2þ influx in podocytes, and have been implicated in glomerular disease. We observed that AII increased cationic currents in rat podocytes in an isolated glomerulus preparation in which podocytes are still attached to the underlying capillary. This effect was completely blocked by SKF-96365, by micromolar La 3þ , and by siRNA knockdown of TRPC6, indicating that TRPC6 is the primary source of Ca 2þ influx mobilized by endogenously expressed angiotensin II receptors in these cells. These responses were also blocked by the AT1R antagonist losartan, the phospholipase C inhibitor D-609, and by inhibition of G protein signaling. The pan-protein kinase C inhibitor chelerythrine had no effect. Importantly, pretreating podocytes with the ROS quencher manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) eliminated AII activation of TRPC6. Significant reductions of AII effects on podocyte TRPC6 were also observed after pretreatment with NADPH oxidase inhibitors apocynin or diphenylene iodonium (DPI). These data suggest that ROS production permits activation of TRPC6 channels by G protein and PLC- dependent cascades initiated by AII acting on AT1Rs in podocytes. This pathway also provides a basis whereby two forms of cellular stress —oxidative stress and Ca 2þ overload—converge on common pathways relevant to disease. J. Cell. Physiol. 229: 434–442, 2014. ß 2013 Wiley Periodicals, Inc. Angiotensin II (AII) is an important modulator of renal physiology and pathophysiology (Ru ¨ster and Wolf, 2006). Within the renal corpuscle, sustained increases in AII signaling lead to apoptosis (Zhang et al., 2009; Hsu et al., 2011) and autophagy (Yadav et al., 2010) of visceral epithelial cells known as podocytes, which form an essential component of the ultrafiltration apparatus (Pavensta ¨dt et al., 2003). The resulting loss of podocytes is a crucial part of pathological processes that lead to end-stage renal disease (D’Agati, 2008; Fukuda et al., 2012). AII exerts a number of direct effects on podocytes. For example, exposure to AII results in depolarization and increased Ca 2þ influx in primary podocytes (Gloy et al., 1997; Nitschke et al., 2000; Eckel et al., 2011), and in immortalized mouse podocyte cell lines (Henger et al., 1997; Tian et al., 2010; Nijenhuis et al., 2011). AII exposure also increases the generation of reactive oxygen species (ROS) in podocytes (Hsu et al., 2008; Yadav et al., 2010). We have previously shown that ROS such as H 2 O 2 induces rapid mobilization of Ca 2þ - permeable TRPC6 channels in cultured podocytes (Kim et al., 2012a,b). AII also evokes robust activation of the small GTPases Rac and Rho in immortalized mouse podocyte cell lines that over-express the type 1 receptor for AII (AT1R) (Hsu et al., 2008; Tian et al., 2010). AII signaling in these cell lines caused reorganization of the podocyte cytoskeleton (Hsu et al., 2008; Tian et al., 2010) resulting in profound effects on permselectivity across podocyte–podocyte contacts in monolayer cultures (Macconi et al., 2006). A recent report has shown that signaling through the over-expressed AT1R in these cells can lead to activation of TRPC6 and TRPC5 channels (Tian et al., 2010). Moreover, AII activation of TRPC6 increased the number of stress fibers as a result of Rho activation, whereas TRPC5 activation decreased stress fibers through activation of Rac (Tian et al., 2010). These workers have proposed that the relative amplitude of these opposing pathways determined the balance between con- tractility and motility in response to AII (Tian et al., 2010). On the other hand, studies in primary mouse podocytes indicated that AII can evoke an increase in cationic currents that is not seen when the dissociated cultures are prepared from constitutive TRPC6 knockout mice (Eckel et al., 2011). There are well-documented compensatory changes in TRP channel expression in constitutive TRPC6 knockout mice (Dietrich et al., 2005), and changes occur when cells are Contract grant sponsor: American Society of Nephrology. Contract grant sponsor: Pfizer, Inc. *Correspondence to: Stuart E. Dryer, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001. E-mail: sdryer@uh.edu Manuscript Received 27 June 2013 Manuscript Accepted 27 August 2013 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 3 September 2013. DOI: 10.1002/jcp.24461 ORGINAL RESEARCH ARTICLE 434 Journal of Journal of Cellular Physiology Cellular Physiology ß 2013 WILEY PERIODICALS, INC.