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ß-arrestin/TRPC6 mediated calcium influx in human podocytes

Mariia Stefanenko1, Marharyta Semenikhina1, Mykhailo Fedoriuk1, Joshua H. Lipschutz1,2, and Oleg Palygin1,3

1Department of Medicine, Division of Nephrology, Medical University of South Carolina, Charleston, SC
2Department of Medicine, Ralph H. Johnson VAMC, Charleston, SC
3Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC

AT1 receptor antagonism is one of the primary strategies for treating hypertension and
chronic kidney diseases. TRV120027 (TRV) is a novel AT1R-biased agonist which
acts selectively through the β-arrestin pathway. However, despite the evidence of the
beneficial effect of β-arrestins signaling in hypertension, there is a knowledge gap
regarding its action on the glomerular and podocyte cells.

We used a conditionally immortalized human podocyte cell line to determine βarrestin’s involvement in podocyte calcium signaling and cytoskeletal reorganization.
Intracellular Ca2+ influx (Fluo-4 AM) and NO response (DAF-FM) to Ang II or TRV
applications were detected by confocal microscopy. The role of TRPC involvement
was tested by electrophysiology. Western blotting assay for apoptosis-associated
proteins, TUNEL staining to detect DNA cleavage, and actin cytoskeleton
rearrangements were performed in podocytes exposed to TRV or Ang II treatment.

Results: Our experiments determined that TRV-mediated β-arrestin pathway
activation promotes the rapid elevation of intracellular Ca2+ concentration in
podocytes. Interestingly, the amplitude of this response was four times higher than in
response to Ang II. The pharmacological blockade of TRPC6, but not TRPC5,
significantly reduces Ca2+ influx in response to β-arrestin pathway activation.
Moreover, TRV does not modulate AT2R-mediated NO oxide production in cells,
supporting the antagonism only of the AT1R receptor signaling pathway. Single
channel analyses show rapid activation of TRPC activity in response to acute TRV
application in podocytes. Overall prolonged activation of the β-arrestin pathway in
podocytes results in enhanced actin bundle thickness, abnormal actin cytoskeleton
distribution, and an increase in cell apoptotic markers.

Conclusions: TRV-mediated β-arrestin signaling in podocyte promote high ionotropic
TRPC6 channel-mediated Ca2+ influx, cytoskeleton rearrangement, and apoptosis,
possibly leading to severe defects in glomerular filtration barrier integrity and kidney
health. Under these circumstances, the potential therapeutic application of TRV for
hypertension treatment is controversial and requires detailed investigation.