Angiotensin II Activation of TRPC6 Channels in Rat Podocytes Requires Generation of Reactive Oxygen Species

Abstract

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(2+) 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(3+) , and by siRNA knockdown of TRPC6, indicating that TRPC6 is the primary source of Ca(2+) 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(2+) overload-converge on common pathways relevant to disease.

Angiotensin II modulates mouse skeletal muscle resting conductance to chloride and potassium ions and calcium homeostasis via the AT1 receptor and NADPH oxidase

“…7. Importantly, the effective concentrations of ANG II in our study were similar to those found by others and slightly higher with respect to those calculated in binding studies (1,16,32). Therefore, the effects observed are in line with pathophysiological concentration of ANG II in vivo, especially for autocrine/paracrine actions.…”

“…The observed plateau effect on gCl by ANG II is also in line with a physiological and reversible activation of a G protein-coupled phosphorylation pathway. In addition, the present results add clear evidence that the production of ROS via NOX works as fundamental part of the pathway leading to the observed reduction of gCl by ANG II in myofibers as in other experimental conditions (1,41,43,73). This is emphasized by the observation that effects of ANG II are completely antagonized by both the classical antioxidant NAC and by the NOX inhibitor apocynin, as well as by the PKC inhibitor, although the occurrence of a parallel ROSmediated signaling cannot be fully ruled out.…”

“…As a classical paradigm, such a transduction signal produces diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP 3 ). While recent evidence demonstrated that IP 3 plays a marginal, if any, role in releasing Ca 2ϩ from muscle SR stores (3), DAG can directly activate PKC and also reinforce the signaling by stimulating receptor-mediated calcium entry via activation of transient receptor potential (TRP) channels, such as TRPC5 and 6 (1,38). In support of this view we found that ANG II caused, via the AT 1 receptor, a "two-step shape" increase of calcium transient, which was completely abolished by the removal of external calcium.…”

“…Importantly, ANG II via AT 1 receptor activates canonical G q protein PLC/PKC signaling, which also leads to activation of NADPH-oxidase (NOX) in most of the tissues where AT 1 receptors are expressed. This pathway accounts for production of reactive oxygen species (ROS) and activation of redoxsensitive cellular process, including the regulation of ionic homeostasis, as in renal podocytes (1,41,43,63). Activation of NOX in skeletal muscle by systemic ANG II has also been observed, and overexpression of NOX is responsible of oxidative stress occurring in dystrophic muscle (41,43,50,73,74).…”

Gq signaling causes glomerular injury by activating TRPC6

“…In this regard, PAN stimulates ROS generation in podocytes (45), and ROS potently activate TRPC6 (46,47). Moreover, Gq-coupled GPCRs stimulate TRPC6 activation, both directly through the second messengers DAG and possibly IP3 (1), as well as indirectly by stimulating ROS generation (48).…”