Abstract: PUB198
Effects of a Direct TRPC6 Activator on Albumin Permeability, Junctional Biochemistry, and Cell Death in Podocytes
Session Information
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Author
- Dryer, Stuart E., University of Houston, Houston, Texas, United States
Background
TRPC6 channels have been implicated in the pathogenesis of glomerular diseases. Inhibition, inactivation or deletion of TRPC6 channels results in reduced severity in several animal models, including models of nephrosis, autoimmune disease, and interstitial fibrosis. TRPC6 channels in podocytes are rapidly activated during various G protein-coupled signal cascades (e.g., in response to Ang II or ATP) and during mechanical stimuli. However, the physiological roles of TRPC6 channels remain unclear. Here we examined the role of TRPC6 channels in modulating albumin permeability across podocyte monolayers, alteration of junctional biochemistry, and in driving apoptotic cell death .
Methods
FITC-albumin movement across confluent layers of podocytes (PAlb) was assessed in transwell assays using mouse MPC-5 podocytes. Occludin abundance was determined by immunoblot. Shedding of nephrin ectodomains was measured by ELISA. Cell death was assessed by cleaved caspase-3 and annexin V. TRPC6 was activated using the directly acting compound PPZ2 (2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide). To assess specificity, some preparations were simultaneously treated with the TRPC6 inhibitor SAR-7334.
Results
Exposing podocyte monolayers to PPZ2 for 1 h or 24 h had no effect on PAlb, in marked contrast to suPAR (which increases PAlb after 24 h) or the TLR4 agonist CRX-527 (which can increase PAlb in as little as 1 h). PPZ2 treatment for 24 h caused significant shedding of nephrin ectodomains, accompanied by an increase in total expression of the tight junctional protein occludin. This effect was blocked by SAR-7334. Exposing podocytes to PPZ2 for an even longer period of time (72 h) evoked increases in apoptotic cell death.
Conclusion
TRPC6 does not regulate albumin flux across a podocyte monolayer in this in vitro model. It is possible that its rapid activation of TRPC6 plays a role in regulation of glomerular filtration rate as opposed to regulation of glomerular macromolecular permeabilty. The effects of TRPC6 on junctional protein dynamics, especially nephrin shedding, raise the possibility that it contributes to foot process effacement over longer timeframes. More sustained continuous activation of TRPC6 can induce podocyte cell death, which may be relevant to the pathogenesis of glomerular disease.
Funding
- NIDDK Support