Beta-arrestin-biased agonism promotes TRPC6-mediated calcium influx in human podocytes

Abstract

Background: Angiotensin Receptor blockers (ARBs) are the first-line treatment for hypertension and other chronic kidney diseases and act by inhibiting signaling through the angiotensin 1 receptor (AT1R). AT1R activation initiates signaling through G protein-coupled receptor (GPCR) effector signaling and β-arrestins. Recently, a novel AT1R agonist TRV120027 (TRV) that selectively activates the β-arrestin cascade without impacting the GCPR pathway has become available. Therefore, we hypothesized that β-arrestin and G-protein signaling pathways via AT1R have distinct downstream effects in podocytes. Furthermore, we propose that β-arrestin signaling initiates TRPC6-mediated Ca 2+ entry. Methods: We used a conditionally immortalized human podocyte cell line to determine β-arrestin’s involvement in podocyte calcium signaling and cytoskeletal reorganization. Intracellular Ca 2+ influx (Fluo-4 AM) and NO response (DAF-FM) to Ang II or TRV applications were analyzed with confocal microscopy. The role of TRPC channels was tested by single-channel electrophysiology and confocal imaging using a variety of commercially available inhibitors (AC1903 for TRPC5 and GSK283350A, SAR7334 for TRPC6). Western blotting 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 in podocytes promotes rapid elevation of intracellular Ca 2+ in a dose-dependent manner (IC 50 =15μM, ligand-receptor binding fit modeled with Levenberg–Marquardt algorithm, adj. R 2 =0.93). Interestingly, the amplitude of β-arrestin-mediated Ca 2+ influx was four times higher than the response to similar Ang II concentrations (t-test, n≥30, p<0.05). Moreover, the TRV response was significantly increased under hyperglycemic stress conditions, and promoted rapid apoptosis in podocytes (one-way ANOVA, n≥21, p=0.003). The pharmacological blockade of TRPC6, but not TRPC5, significantly attenuated Ca 2+ influx in response to β-arrestin. In contrast to Ang II, TRV does not induce AT1R-mediated NO production, suggesting that β-arrestin activation only targets AT1R, and not AT2R. Single-channel analyses show rapid activation of TRPC activity in response to acute TRV application in podocytes (one-way ANOVA, n=8, p=0.004). Overall prolonged activation of the β-arrestin pathway in podocytes results in enhanced actin bundle thickness, abnormal actin cytoskeleton distribution, and increased apoptotic cell markers. Conclusions: TRV-mediated β-arrestin signaling in podocytes promotes high ionotropic TRPC6 channel-mediated Ca 2+ 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 further investigation. DK126720 (to OP), DK129227 (to AS and OP), and NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (to OP), endowed funds from the SC SmartState Centers of Excellence (to OP), Veterans Affairs Support Veterans Affairs (Merit Award I01 BX000820 to JL), research grant from Dialysis Clinic, Inc (to JL). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.