Sustained Activation of N-Methyl-d-Aspartate Receptors in Podoctyes Leads to Oxidative Stress, Mobilization of Transient Receptor Potential Canonical 6 Channels, Nuclear Factor of Activated T Cells Activation, and Apoptotic Cell Death

Abstract

Atypical N-methyl-D-aspartate (NMDA) receptors are expressed in podocytes. Sustained (Ն24 h) application of 50 to 100 M NMDA to immortalized mouse podocytes evoked a marked increase in the production of reactive oxygen species (ROS) such as H 2 O 2 . This effect of NMDA was associated with increased cell-surface expression of p47 (phox) , a cytosolic regulatory subunit of the NADPH oxidase NOX2. NMDA-evoked generation of ROS drove an increase in steady-state surface expression of transient receptor potential canonical (TRPC) 6 channels, which was blocked by the NMDA antagonist dizocilpine (MK-801) and by a membrane-permeable scavenger of ROS. The effect of NMDA on TRPC6 was observed using cell surface biotinylation assays and also with whole-cell recordings made under conditions designed to facilitate detection of current through TRPC6. NMDA mobilization of TRPC6 channels was blocked by concurrent treatment with the NMDA antagonist MK-801 and by a membrane-permeable scavenger of ROS. Mobilization of TRPC6 was also evoked by Lhomocysteic acid. NMDA treatment also increased nuclear localization of endogenous nuclear factor of activated T cells, which could be blocked by MK-801, by scavenging ROS, by the calcineurin inhibitor cyclosporine, and by the TRPC channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole (SKF-96365). NMDA treatment also evoked robust activation of Rho but not Rac, consistent with previous studies of downstream effectors of TRPC6 activation. Exposing cells to NMDA for 24 h reduced total and cell surface expression of the podocyte markers nephrin and podocin, but there was no loss of cells. With longer NMDA exposure (72 h), we observed loss of cells associated with nuclear fragmentation and increased expression of caspase-3, caspase-6, and Bax, suggesting an apoptotic process.

Opposing effects of podocin on the gating of podocyte TRPC6 channels evoked by membrane stretch or diacylglycerol

“…In our initial studies, we examined whether mechanical stimuli could evoke current through activation of endogenously expressed TRPC6 channels of differentiated immortalized podocyte cell lines. We described the whole cell recording procedures and some of the general properties of macroscopic currents through podo- cyte TRPC6 channels in previous studies (31,32). The recording conditions are designed to eliminate any current contributions from K ϩ channels known to be expressed in podocytes.…”

“…Whole cell recordings were made as described in detail elsewhere (31,32). The control bath solution (100%) contained 150 mM NaCl, 5.4 mM CsCl, 0.8 mM MgCl 2, 5.4 mM CaCl2, and 10 mM HEPES (pH 7.4).…”

“…Cell culture protocols are described elsewhere (27)(28)(29)(30)(31)(32). Briefly, mouse podocyte cell lines (originally obtained from Dr. Peter Mundel) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin, with recombinant mouse ␥-interferon, in humidified 5% CO 2 incubators and propagated at 33°C.…”

“…This was done in normal and 70% hyposmotic solutions, which in some experiments also contained drugs or inhibitors. Whole cell currents were quantified at ϩ80 mV, as described previously (31,32). To evoke inward deformations of the membrane, glass micropipettes with tip diameters of ϳ1 m were filled with normal external saline containing 60% sucrose and positioned 80 -100 m from a podocyte cell body under whole cell clamp.…”

Prevention of PKG1α oxidation augments cardioprotection in the stressed heart

“…Reduced channel suppression by PKG1α oxidation fits this observation. Redox sensitivity of PKG1α suppression of TRPC6 may also have therapeutic implications beyond the heart, including in dystrophinopathies (24) and glomerulosclerosis (26,27), where ROS and TRPC6 play a pathophysiological role. Future studies are needed to identify the protein partners that control PKG1α translocation and develop strategies to selectively sustain its reduced state to maximize the efficacy of its activation against myocardial disease.…”