Abstract
Atypical N-methyl-d-aspartate (NMDA) receptors are expressed in podocytes. Sustained (≥24 h) application of 50 to 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 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 l-homocysteic 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.
Footnotes
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01-DK8259]; and by an American Society for Nephrology James M. Scherbenske Award.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- TRPC
- transient receptor potential canonical
- NMDA
- N-methyl-d-aspartic acid
- ROS
- reactive oxygen species
- MK-801
- dizocilpine maleate
- l-HCA
- l-homocysteic acid
- NOX
- NADPH oxidase
- CsA
- cyclosporine
- NFAT
- nuclear factor of activated T cells
- SKF-96365
- 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole
- MnTBAP
- manganese(III) tetrakis(4-benzoic acid)porphyrin chloride
- DAPI
- 4′-6-diamidino-2-phenylindole
- OAG
- 1-oleoyl-2-acetyl-sn-glycerol
- TUNEL
- terminal deoxynucleotidyl transferase dUTP nick-end labeling.
- Received April 17, 2012.
- Accepted July 23, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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