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. 2016 Aug 31:7:351.
doi: 10.3389/fphys.2016.00351. eCollection 2016.

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

Hila Roshanravan  1 , Eun Y Kim  1 , Stuart E Dryer  2
Affiliations

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

Hila Roshanravan et al. Front Physiol. .

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 La(3+), 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.

Keywords: FSGS; TRPC6; calcium; eicosanoids; ion channels; podocytes.

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Figures

Figure 1
Figure 1
Currents activated by 20-HETE in cultured podocytes. (A) Examples of currents evoked by 2.5-s ramp voltage commands (−80 mV to +80 mV). Trace to the left shows currents shortly after making whole-cell contact. Middle trace is from the same cell 3 min after exposure to 1 μM 20-HETE, and trace to the right shows complete blockade of the current by SKF-96365. (B) Similar recording from a different cell showing complete block of 20-HETE-evoked current by 50 μM La3+. (C) Summary of results of this experiment (n = 10 cells per group). Data are presented as fold-increase over initial baseline current at +80 mV. The differences in means were calculated by Tukey's HSD post hoc test following one-way ANOVA. (D) Percentage of maximal current amplitudes at +80 mV plotted vs. concentration of 20-HETE. Note saturation of the response by 20 μM 20-HETE. (E) Immunoblot analysis showing effects of non-targeting siRNA (con) and siRNA targeting TRPC6 on total abundance of TRPC6. A typical immunoblot is shown above graphical summary of experiments from three different transfections. Asterisk indicates P < 0.01 (unpaired t-test). (F) Examples of responses to 1 μM 20-HETE in cells pre-treated with non-targeting (control) or TRPC6-targeting siRNA using procedures shown in E. Note lack of response to 20-HETE after TRPC6 knockdown. (G) Summary of the results of the experiment in panel F (n = 6 cells per group). Asterisk indicates P < 0.05 (unpaired t-test). (H) Cell-surface biotinylation assays show that exposure to 1 μM 20-HETE for 24 hr increases steady-state surface abundance of TRPC6. Typical blots are shown on top of graphical summary of three repetitions of this experiment. Asterisk indicates P < 0.05 (unpaired t-test).
Figure 2
Figure 2
Effects of 20-HETE in presence and absence of a membrane-permeable analog of diacylgycerol (OAG). (A) Examples of currents from a single podocyte shortly after making whole-cell contact; after exposure to 20 μM 20-HETE; and then after 100 μM OAG was added to the superfusate. At the end of the experiment, 50 μM La3+ was added to the superfusate, which brought currents back to the original baseline. Note that OAG increased currents even in presence of a maximally active concentration of 20-HETE. (B) Summary of the results of this experiment (n = 5 cells per group). Data were analyzed by Tukey's HSD test following one-way ANOVA. (C) Similar experiment to the one shown in (A), except that cells were first exposed to 100 μM OAG and then exposed to the combination of lipids. (D) Summary of results of the experiment shown in (C). The responses to OAG and 20-HETE are approximately additive.
Figure 3
Figure 3
Role of ROS in actions of 20-HETE on TRPC6. (A) Increased bulk cytosolic ROS concentration in podocytes as measured using fluorescence assay in control cells and cells exposed to 1 μM 20-HETE for 24 h. Asterisk indicates P < 0.01 (unpaired t-test). (B) Traces showing responses to 1 μM 20-HETE. (C) Example of response to 1 μM 20-HETE in cell that had been pre-treated with 10 mM TEMPOL, an agent that quenches ROS. (D) Summary of the results of this experiment (n = 6 cells per group). Note complete inhibition of response to 20-HETE in cells pretreated with TEMPOL (asterisk indicates P < 0.05, unpaired t-test).
Figure 4
Figure 4
G protein signaling is required for 20-HETE modulation of podocyte TRPC6 channels. (A) Trace showing increased current evoked by 1 μM 20-HETE recorded with normal pipette saline. (B) Recording from different cell made with pipette containing 50 μM GDP-βS. Note that 20-HETE does not evoke a change in current under those conditions. (C) Summary of fold increase in current at +80 mV evoked by 20-HETE compared to baseline in absence or presence of GDP-βS in recording pipette. Asterisk indicates P < 0.05 (unpaired t-test, n = 15 cells per group).
Figure 5
Figure 5
Podocin is required for 20-HETE modulation of podocyte TRPC6 channels. (A) Representative immunoblots showing podocin abundance in cells treated with non-targeting siRNA and siRNA targeting podocin. Bar graph summarizing three repetitions of this experiment is shown to the right. Asterisk indicates P < 0.01 (unpaired t-test). (B) Treatment with siRNA targeting podocin does not affect TRPC6 abundance. Representative immunoblots are shown to the left, and bar graph summarizing results from three different transfections is shown to the right. (C) Representative trace showing increased currents in podocyte evoked by 1 μM 20-HETE in cell treated with non-targeting siRNA. (D) Typical response to 1 μM 20-HETE in cell treated with siRNA targeting podocin. (E) Summary of fold increase in current at +80 mV evoked by 1 μM 20-HETE compared to baseline in cells treated with non-targeting siRNA (control) and siRNA targeting podocin. Asterisk indicates P < 0.05 (unpaired t-test, n = 6 cells per group).
Figure 6
Figure 6
Schematic diagrams suggesting mechanisms surrounding 20-HETE modulation of TRPC6 in podocytes. (A) In many cell types, 20-HETE can be produced during G protein coupled signaling cascades, for example by G protein coupled receptor (GPCR) pathways activated by angiotensin II. In foot processes and in immortalized podocyte cell lines, a population of TRPC6 channels is part of a larger complex organized by podocin, a cholesterol-binding hairpin loop protein. In the presence of podocin, NOX enzymes are able to assemble in close proximity to TRPC6 channels, including for example formation of a ternary complex between gp91phox and TRPC6 (Kim et al., 2013). This can lead to highly localized and rapid generation of ROS within the immediate vicinity of the channels. The effect of 20-HETE on podocyte TRPC6 channels requires G proteins, which may reflect a requirement of Rac activation for NOX2 or NOX4 activation (Bedard and Krause, 2007), or in other aspects of TRPC6 trafficking to the cell surface. (B) Data from many laboratories indicate that TRPC6 channels become active in presence of ROS, most likely though a combination of increased open probability of surface channels and increased steady-state localization at the cell surface.
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