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.
Highlights
Podocytes are terminally differentiated multipolar cells that cover the outer surface of the glomerular capillary and form the final barriers for glomerular filtration
We report that 20-hydroxyeicosatetraenoic acid (20-HETE) exposure increases the steady-state surface expression of TRPC6 subunits and increases cationic currents through TRPC6 channels in podocytes
In the present study we show that 20-HETE increases current flowing through podocyte TRPC6 channels, as has been observed
Summary
Podocytes are terminally differentiated multipolar cells that cover the outer surface of the glomerular capillary and form the final barriers for glomerular filtration. Changes in podocyte ultrastructure often occur in response to glomerular injuries and in a variety of disease processes. If sufficient numbers of podocytes detach or die, the entire nephron will be lost (Kim et al, 2001; Kriz and LeHir, 2005). Adult-onset familial forms of focal and segmental glomerulosclerosis (FSGS) occur with an autosomal dominant mode of inheritance in patients with mutations in the gene encoding canonical transient receptor potential-6 (TRPC6) channels. Most of these mutations result in a
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