@article {Kimmol.112.079376, author = {Eun Young Kim and Marc Anderson and Stuart E Dryer}, title = {Sustained Activation of NMDA Receptors in Podoctyes Leads to Oxidative Stress, Mobilization of TRPC6 Channels, NFAT Activation, and Apoptotic Cell Death}, elocation-id = {mol.112.079376}, year = {2012}, doi = {10.1124/mol.112.079376}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Atypical NMDA receptors are expressed in podocytes. Sustained (>= 24 hr) application of 50-100 μM NMDA to immortalized mouse podocytes evoked a marked increase in the production of reactive oxygen species (ROS) such as H2O2. 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 TRPC6 channels, which was blocked by the NMDA antagonist MK-801 and by a membrane-permeable scavenger of ROS. Mobilization of TRPC6 was also evoked by L-homocysteic acid (L-HCA). 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. NMDA treatment also increased nuclear localization of endogenous NFAT, which could be blocked by MK-801, by scavenging ROS, by the calcineurin inhibitor cyclosporine, and by the TRPC channel inhibitor 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 hr reduced total and cell surface expression of podocyte markers nephrin and podocin but there was no loss of cells. With longer NMDA exposure (72 hr) we observed loss of cells associated with increased expression of caspase-3, caspase-6 and Bax, as well as nuclear fragmentation, suggesting an apoptotic process.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/early/2012/07/24/mol.112.079376}, eprint = {https://molpharm.aspetjournals.org/content/early/2012/07/24/mol.112.079376.full.pdf}, journal = {Molecular Pharmacology} }