ß-Arrestin pathway activation by selective ATR1 agonism promotes calcium influx in podocytes, leading to glomerular damage.

Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the ß-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated ß-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine ß-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated ß-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of ß-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the ß-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the ß-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated ß-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, 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 requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage.

Calpain-TRPC6 signaling pathway contributes to propofol-induced developmental neurotoxicity in rats.

Growing animal studies suggest a risk of neuronal damage following early childhood exposure to anesthesia and sedation drugs including propofol. Inhibition of transient receptor potential canonical 6 (TRPC6) degradation has been shown to protect neurons from neuronal damage induced by multiple brain injury models. Our aim was to investigate whether calpain-TRPC6 pathway is a target in propofol-induced neurotoxicity. Postnatal day (PND) 7 rats were exposed to five bolus injections of 25 mg/kg propofol or 10 % intralipid at hourly intervals. Neuronal injury was assessed by the expression pattern of TUNEL staining and cleaved-caspase-3. The Morris water maze test was used to evaluate learning and memory functions in later life. Pretreatments consisting of intracerebroventricular injections of a TRPC6 agonist, TRPC6 inhibitor, or calpain inhibitor were used to confirm the potential role of the calpain-TRPC6 pathway in propofol-induced neurotoxicity. Prolonged exposure to propofol induced neuronal injury, downregulation of TRPC6, and enhancement of calpain activity in the cerebral cortex up to 24 h after anesthesia. It also induced long-term behavioral disorders, manifesting as longer escape latency at PND40 and PND41 and as fewer platform-crossing times and less time spent in the target quadrant at PND42. These propofol-induced effects were attenuated by treatment with the TRPC6 agonist and exaggerated by the TRPC6 inhibitor. Pretreatment with the calpain inhibitor alleviated the propofol-induced TRPC6 downregulation and neuronal injury in the cerebral cortex. In conclusion, our data suggest that a calpain-TRPC6 signaling pathway contributes to propofol-induced acute cortical neuron injury and long-term behavioral disorders in rats.

Qian Yang Yu Yin granule improves hypertensive renal damage: A potential role for TRPC6-CaMKKß-AMPK-mTOR-mediated autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Qian Yang Yu Yin granules (QYYYG) have a long history in the treatment of hypertensive renal damage (HRD) in China. Clinical studies have found that QYYYG stabilizes blood pressure and prevents early renal damage. However, the exact mechanism is not entirely clear. AIM OF THE STUDY: To evaluate the therapeutic effect and further explore the therapeutic mechanism of QYYYG against HRD. MATERIALS AND METHODS: The efficacy of QYYYG in treating HRD was assessed in spontaneous hypertension rats (SHR). Renal autophagy and the TRPC6-CaMKKß-AMPK pathway in rats were evaluated. The regulatory role of QYYYG in angiotensin II (Ang II) induced abnormal autophagy in rat podocytes was determined by detecting autophagy-related proteins, intracellular Ca2+ content, and the TRPC6-CaMKKß-AMPK-mTOR pathway expressions. Finally, we established a stable rat podocyte cell line overexpressing TRPC6 and used the cells to verify the regulatory effects of QYYYG. RESULTS: QYYYG alleviated HRD and reversed the abnormal expression of autophagy-related genes in the SHR. In vitro, QYYYG protected against Ang II-induced podocyte damage. Furthermore, treatment of podocytes with QYYYG reversed Ang II-induced autophagy and inhibited Ang II-stimulated TRPC6 activation, Ca2+ influx and activation CaMKKß-AMPK pathway. Overexpression of TRPC6 resulted in pronounced activation of CaMKKß, AMPK, and autophagy induction in rat podocytes, which were significantly attenuated by QYYYG. CONCLUSIONS: The present study suggested that QYYYG may exert its HRD protective effects in part by regulating the abnormal autophagy of podocytes through the TRPC6-CaMKKß-AMPK-mTOR pathway.

Tetrandrine Attenuates Podocyte Injury by Inhibiting TRPC6-Mediated RhoA/ROCK1 Pathway.

Tetrandrine (Tet), a compound found in a traditional Chinese medicine, presents the protective effect for kidney function. Our study is aimed at clarifying the efficacy and underlying mechanism of Tet on podocyte injury. In this study, podocyte injury was induced in rats with adriamycin (ADR), and MPC5 podocytes were constructed with TRPC6 overexpression. We found that Tet treatment reduced the levels of proteinuria, serum creatinine, and blood urea nitrogen and increased plasma albumin levels in ADR-induced rats. Tet reduced intracellular Ca2+ influx and apoptosis in MPC5 podocytes overexpressing TRPC6. Tet downregulated the expression of renal TRPC6, RhoA, and ROCK1 and upregulated the expression of synaptopodin; meanwhile, it reduced calcineurin activity in vivo and in vitro. In conclusion, Tet protects against podocyte by affecting TRPC6 and its downstream RhoA/ROCK1 signaling pathway.

Cell-Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities.

Pulsed electromagnetic fields (PEMFs) are capable of specifically activating a TRPC1-mitochondrial axis underlying cell expansion and mitohormetic survival adaptations. This study characterizes cell-derived vesicles (CDVs) generated from C2C12 murine myoblasts and shows that they are equipped with the sufficient molecular machinery to confer mitochondrial respiratory capacity and associated proliferative responses upon their fusion with recipient cells. CDVs derived from wild type C2C12 myoblasts include the cation-permeable transient receptor potential (TRP) channels, TRPC1 and TRPA1, and directly respond to PEMF exposure with TRPC1-mediated calcium entry. By contrast, CDVs derived from C2C12 muscle cells in which TRPC1 has been genetically knocked-down using CRISPR/Cas9 genome editing, do not. Wild type C2C12-derived CDVs are also capable of restoring PEMF-induced proliferative and mitochondrial activation in two C2C12-derived TRPC1 knockdown clonal cell lines in accordance to their endogenous degree of TRPC1 suppression. C2C12 wild type CDVs respond to menthol with calcium entry and accumulation, likewise verifying TRPA1 functional gating and further corroborating compartmental integrity. Proteomic and lipidomic analyses confirm the surface membrane origin of the CDVs providing an initial indication of the minimal cellular machinery required to recover mitochondrial function. CDVs hence possess the potential of restoring respiratory and proliferative capacities to senescent cells and tissues.

Design, synthesis and characterization of novel N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors leading to the identification of the selective compound, AC1903.

The transient receptor potential cation channel 5 (TRPC5) has been previously shown to affect podocyte survival in the kidney. As such, inhibitors of TRPC5 are interesting candidates for the treatment of chronic kidney disease (CKD). Herein, we report the synthesis and biological characterization of a series of N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors. Work reported here evaluates the benzimidazole scaffold and substituents resulting in the discovery of AC1903, a TRPC5 inhibitor that is active in multiple animal models of CKD.

A small-molecule inhibitor of TRPC5 ion channels suppresses progressive kidney disease in animal models.

Progressive kidney diseases are often associated with scarring of the kidney's filtration unit, a condition called focal segmental glomerulosclerosis (FSGS). This scarring is due to loss of podocytes, cells critical for glomerular filtration, and leads to proteinuria and kidney failure. Inherited forms of FSGS are caused by Rac1-activating mutations, and Rac1 induces TRPC5 ion channel activity and cytoskeletal remodeling in podocytes. Whether TRPC5 activity mediates FSGS onset and progression is unknown. We identified a small molecule, AC1903, that specifically blocks TRPC5 channel activity in glomeruli of proteinuric rats. Chronic administration of AC1903 suppressed severe proteinuria and prevented podocyte loss in a transgenic rat model of FSGS. AC1903 also provided therapeutic benefit in a rat model of hypertensive proteinuric kidney disease. These data indicate that TRPC5 activity drives disease and that TRPC5 inhibitors may be valuable for the treatment of progressive kidney diseases.

TRPC1 protects dopaminergic SH-SY5Y cells from MPP+, salsolinol, and N-methyl-(R)-salsolinol-induced cytotoxicity.

Neurotoxins and alterations in Ca2+ homeostasis have been associated with Parkinson's disease (PD), but the role of store-operated Ca2+ entry channels is not well understood. Previous studies have shown the neurotoxicity of salsolinol and 1-methyl-4-phenylpyridinium ion on SH-SY5Y cells and cytoprotection induced by transient receptor potential protein 1 (TRPC1). In the present study, N-methyl-(R)-salsolinol was tested for its cellular toxicity and effects on TRPC1 expression. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, DAPI (4',6-diamidino-2-phenylindole), fluorescein isothiocyanate-Annexin-V/propidium iodide, western blot analysis, and JC-1 labeling revealed that the three indicated drugs could induce caspase-dependent, mitochondrial-mediated apoptosis. Exposure of SH-SY5Y cells to the indicated drugs resulted in a significant decrease in thapsigargin-mediated Ca2+ influx and TRPC1 expression. Immunocytochemistry experiments revealed that neurotoxins treatment induced TRPC1 translocation to the cytoplasm. Taken together, our results indicate that treatment with neurotoxins may alter Ca2+ homeostasis and induce mitochondrial-mediated caspase-dependent cytotoxicity, an important characteristic of PD.