20-Hydroxyeicosatetraenoic Acid (20-HETE) Modulates Canonical Transient Receptor Potential-6 (TRPC6) Channels in Podocytes

Abstract

The arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) regulates renal function, including changes in glomerular function evoked during tubuloglomerular feedback (TGF). This study describes the cellular actions of 20-HETE on cultured podocytes, assessed by whole-cell recordings from cultured podocytes combined with pharmacological and cell-biological manipulations of cells. Bath superfusion of 20-HETE activates cationic currents that are blocked by the pan-TRP blocker SKF-96365 and by 50 μM La3+, and which are attenuated after siRNA knockdown of TRPC6 subunits. Similar currents are evoked by a membrane-permeable analog of diacylgycerol (OAG), but OAG does not occlude responses to maximally-activating concentrations of 20-HETE (20 μM). Exposure to 20-HETE also increased steady-state surface abundance of TRPC6 subunits in podocytes as assessed by cell-surface biotinylation assays, and increased cytosolic concentrations of reactive oxygen species (ROS). TRPC6 activation by 20-HETE was eliminated in cells pretreated with TEMPOL, a membrane-permeable superoxide dismutase mimic. Activation of TRPC6 by 20-HETE was also blocked when whole-cell recording pipettes contained GDP-βS, indicating a role for either small or heterotrimeric G proteins in the transduction cascade. Responses to 20-HETE were eliminated by siRNA knockdown of podocin, a protein that organizes NADPH oxidase complexes with TRPC6 subunits in this cell type. In summary, modulation of ionic channels in podocytes may contribute to glomerular actions of 20-HETE.

Changes in podocyte TRPC channels evoked by plasma and sera from patients with recurrent FSGS and by putative glomerular permeability factors

“…Most of the macroscopic ionic current in podocytes that is activated by native G protein signaling or other stimuli can be attributed to TRPC6-containing channels on the basis of sensitivity to TRPC6 knockdown, La 3+ and SKF-96365 [36,38,39] and SAR7334 (the present study). However, TRPC5 channels are also expressed in podocytes [48,51] and have been proposed to play a role in the progression of kidney disease [69], although the factors that normally cause them to become active are not known.…”

“…Filters were probed using primary antibodies, washed, incubated with horseradish peroxidase-conjugated secondary antibodies, and visualized using chemiluminescence. Methods for cell surface biotinylation assays to measure steady-state surface abundance of TRPC6 channels were also described previously [37,39,47–49]. Rabbit antibodies against TRPC6 (ACC-017) and TRPC5 (ACC-020) were obtained from Alomone Labs (Jerusalem, Israel), antibodies against podocin (sc-21009) and β3-integrin (sc-14009) were obtained from Santa Cruz (Santa Cruz, CA).…”

“…We have previously reported that podocin differentially regulates the sensitivity of podocyte TRPC6 channels to various activating stimuli [36]. Specifically, podocin enhances activation of TRPC6 induced by G protein signaling pathways or by diacylglycerol [36–38] or eicosanoids [39]. Conversely, podocin suppresses activation of podocyte TRPC6 channels induced by membrane stretch, a process that occurs by poorly understood transduction mechanisms [36].…”

Effect of Cytochrome P450 Metabolites of Arachidonic Acid in Nephrology

“…136 Thus, 20-HETE has been suggested to contribute to diabetic nephropathy. 138,[140][141][142] Roshanravan et al 143 recently reported that 20-HETE increases TRPC6 activity in podocytes secondary to activation of ROS production. Activation of TRPC6 causes foot process effacement, [144][145][146][147] but paradoxically, effacement prevents podocyte detachment.…”

Role of Transient Receptor Potential Canonical Channel 6 (TRPC6) in Diabetic Kidney Disease by Regulating Podocyte Actin Cytoskeleton Rearrangement

“…TRPC6 is confirmed to be a nonselective cation channel. A large body of studies [23,24] strongly support that TRPC6 is an essential functional component of TRPCs in vascular smooth muscle cells. It has been shown that activated TRPC6 regulates its contractile function by permeating cation influx and depolarization of vascular smooth muscle cells [25].…”