RESUMEN
Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO.
Asunto(s)
Calcio , Oligonucleótidos Antisentido , Canales Catiónicos TRPC , Humanos , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/metabolismo , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/antagonistas & inhibidores , Calcio/metabolismo , Células A549 , Animales , Ratones , Imidazoles/farmacología , Canal Catiónico TRPC6/metabolismo , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/antagonistas & inhibidores , Ácido Egtácico/farmacología , Ácido Egtácico/análogos & derivados , Endosomas/metabolismo , Endosomas/efectos de los fármacos , Línea Celular TumoralRESUMEN
INTRODUCTION: The aim of this study was to explore the renoprotective effects of Klotho on podocyte injury mediated by complement activation and autoantibodies in idiopathic membranous nephropathy (IMN). METHODS: Rat passive Heymann nephritis (PHN) was induced as an IMN model. Urine protein levels, serum biochemistry, kidney histology, and podocyte marker levels were assessed. In vitro, sublytic podocyte injury was induced by C5b-9. The expression of Klotho, transient receptor potential channel 6 (TRPC6), and cathepsin L (CatL); its substrate synaptopodin; and the intracellular Ca2+ concentration were detected via immunofluorescence. RhoA/ROCK pathway activity was measured by an activity quantitative detection kit, and the protein expression of phosphorylated-LIMK1 (p-LIMK1) and p-cofilin in podocytes was detected via Western blotting. Klotho knockdown and overexpression were performed to evaluate its role in regulating the TRPC6/CatL pathway. RESULTS: PHN rats exhibited proteinuria, podocyte foot process effacement, decreased Klotho and Synaptopodin levels, and increased TRPC6 and CatL expression. The RhoA/ROCK pathway was activated by the increased phosphorylation of LIMK1 and cofilin. Similar changes were observed in C5b-9-injured podocytes. Klotho knockdown exacerbated podocyte injury, while Klotho overexpression partially ameliorated podocyte injury. CONCLUSION: Klotho may protect against podocyte injury in IMN patients by inhibiting the TRPC6/CatL pathway. Klotho is a potential target for reducing proteinuria in IMN patients.
Asunto(s)
Citoesqueleto de Actina , Catepsina L , Glomerulonefritis Membranosa , Glucuronidasa , Proteínas Klotho , Podocitos , Transducción de Señal , Canal Catiónico TRPC6 , Podocitos/metabolismo , Podocitos/patología , Glomerulonefritis Membranosa/metabolismo , Glomerulonefritis Membranosa/patología , Animales , Glucuronidasa/metabolismo , Ratas , Canal Catiónico TRPC6/metabolismo , Masculino , Citoesqueleto de Actina/metabolismo , Catepsina L/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Humanos , Modelos Animales de Enfermedad , Proteínas de Microfilamentos/metabolismo , Proteinuria/metabolismo , Ratas Sprague-Dawley , Quinasas Asociadas a rho/metabolismo , Canales Catiónicos TRPC/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismoRESUMEN
Podocyte injury plays a vital role in focal segmental glomerulosclerosis (FSGS), and apoptosis is one of its mechanisms. The transient receptor potential channel 6 (TRPC6) is highly expressed in podocytes and mutations mediate podocyte injury. We found TRPC6 gene mutation (N110S) was a new mutation and pathogenic in the preliminary clinical work. The purpose of this study was to investigate the potential mechanism of mutation in TRPC6 (TRPC6-N110S) in the knock-in gene mouse model and in immortalized mouse podocytes (MPC5). Transmission electron microscopy was used to evaluate renal injury morphology. We measured 24-hour urinary albumin-to-creatinine ratios and major biochemical parameters such as serum albumin, urea nitrogen, and total cholesterol. The results of CCK-8 assay and apoptosis experiments showed that the TRPC6-N110S overexpression group had slower proliferative activity and increased apoptosis than the control group. FluO-3 assay revealed increased calcium influx in the TRPC6-N110S overexpression group. Podocin level was decreased in TRPC6-N110S group, while TRPC6 and desmin levels were increased in TRPC6-N110S group. The 24 h uACR at 6 weeks was significantly higher in the pure-zygotes group than in the WT and heterozygotes groups, and this difference was found at 8 and 10 weeks.TRPC6 levels showed no significant difference between homozygote and WT mice. Compared to homozygote group, expression of podocin and nephrin were increased in WT, but levels of desmin was decreased in WT. Our results suggest that this new mutation causes podocyte injury probably by enhancing calcium influx and podocyte apoptosis, accompanied by increased proteinuria and decreased expression of nephrin and podocin.
Asunto(s)
Apoptosis , Mutación con Ganancia de Función , Podocitos , Canal Catiónico TRPC6 , Podocitos/metabolismo , Podocitos/patología , Animales , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismo , Apoptosis/genética , Ratones , Mutación con Ganancia de Función/genética , Calcio/metabolismo , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Desmina/genética , Desmina/metabolismo , Proliferación Celular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Masculino , Ratones Endogámicos C57BLRESUMEN
The transient receptor potential canonical 6 (TRPC6) channel, a nonselective cation channel that allows the passage of Ca2+, plays an important role in renal diseases. TRPC6 is activated by Ca2+ influx, oxidative stress, and mechanical stress. Studies have shown that in addition to glomerular diseases, TRPC6 can contribute to renal tubular disorders, such as acute kidney injury, renal interstitial fibrosis, and renal cell carcinoma (RCC). However, the tubule-specific physiological functions of TRPC6 have not yet been elucidated. Its pathophysiological role in ischemia/reperfusion (I/R) injury is debatable. Thus, TRPC6 may have dual roles in I/R injury. TRPC6 induces renal fibrosis and immune cell infiltration in a unilateral ureteral obstruction (UUO) mouse model. Additionally, TRPC6 overexpression may modify G2 phase transition, thus altering the DNA damage checkpoint, which can cause genomic instability and RCC tumorigenesis and can control the proliferation of RCC cells. This review highlights the importance of TRPC6 in various conditions of the renal tubular system. To better understand certain renal disorders and ultimately identify new therapeutic targets to improve patient care, the pathophysiology of TRPC6 must be clarified.
Asunto(s)
Canal Catiónico TRPC6 , Humanos , Canal Catiónico TRPC6/metabolismo , Canal Catiónico TRPC6/genética , Animales , Túbulos Renales/patología , Túbulos Renales/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/etiología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Daño por Reperfusión/metabolismo , Fibrosis , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Renales/genética , Ratones , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPC/genética , Estrés Oxidativo , Enfermedades Renales/metabolismo , Enfermedades Renales/etiologíaRESUMEN
We recently reported that transient receptor potential canonical (TRPC) 6 channel activity contributes to intracellular Zn2+ homeostasis in the heart. Zn2+ has also been implicated in the regulation of intestinal redox and microbial homeostasis. This study aims to investigate the role of TRPC6-mediated Zn2+ influx in the stress resistance of the intestine. The expression profile of TRPC1-C7 mRNAs in the actively inflamed mucosa from inflammatory bowel disease (IBD) patients was analyzed using the GEO database. Systemic TRPC3 knockout (KO) and TRPC6 KO mice were treated with dextran sulfate sodium (DSS) to induce colitis. The Zn2+ concentration and the mRNA expression levels of oxidative/inflammatory markers in colon tissues were quantitatively analyzed, and gut microbiota profiles were compared. TRPC6 mRNA expression level was increased in IBD patients and DSS-treated mouse colon tissues. DSS-treated TRPC6 KO mice, but not TRPC3 KO mice, showed severe weight loss and increased disease activity index compared with DSS-treated WT mice. The mRNA abundances of antioxidant proteins were basically increased in the TRPC6 KO colon, with changes in gut microbiota profiles. Treatment with TRPC6 activator prevented the DSS-induced colitis progression accompanied by increasing Zn2+ concentration. We suggest that TRPC6-mediated Zn2+ influx activity plays a key role in stress resistance against IBD, providing a new strategy for treating colitis.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Canal Catiónico TRPC6 , Animales , Humanos , Ratones , Colon/metabolismo , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/metabolismo , Intestinos , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismoRESUMEN
Transient receptor potential canonical sub-family channel 3 (TRPC3) is considered to play a critical role in calcium homeostasis. However, there are no established findings in this respect with regard to TRPC6. Although the parathyroid gland is a crucial organ in calcium household regulation, little is known about the protein distribution of TRPC channels-especially TRPC3 and TRPC6-in this organ. Our aim was therefore to investigate the protein expression profile of TRPC3 and TRPC6 in healthy and diseased human parathyroid glands. Surgery samples from patients with healthy parathyroid glands and from patients suffering from primary hyperparathyroidism (pHPT) were investigated by immunohistochemistry using knockout-validated antibodies against TRPC3 and TRPC6. A software-based analysis similar to an H-score was performed. For the first time, to our knowledge, TRPC3 and TRPC6 protein expression is described here in the parathyroid glands. It is found in both chief and oxyphilic cells. Furthermore, the TRPC3 staining score in diseased tissue (pHPT) was statistically significantly lower than that in healthy tissue. In conclusion, TRPC3 and TRPC6 proteins are expressed in the human parathyroid gland. Furthermore, there is strong evidence indicating that TRPC3 plays a role in pHPT and subsequently in parathyroid hormone secretion regulation. These findings ultimately require further research in order to not only confirm our results but also to further investigate the relevance of these channels and, in particular, that of TRPC3 in the aforementioned physiological functions and pathophysiological conditions.
Asunto(s)
Regulación hacia Abajo , Hiperparatiroidismo Primario , Glándulas Paratiroides , Canales Catiónicos TRPC , Canal Catiónico TRPC6 , Humanos , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPC/genética , Hiperparatiroidismo Primario/metabolismo , Hiperparatiroidismo Primario/genética , Hiperparatiroidismo Primario/patología , Glándulas Paratiroides/metabolismo , Glándulas Paratiroides/patología , Femenino , Masculino , Canal Catiónico TRPC6/metabolismo , Canal Catiónico TRPC6/genética , Persona de Mediana Edad , Anciano , Adulto , Inmunohistoquímica , Hormona Paratiroidea/metabolismoRESUMEN
TRPV4 channels, which respond to mechanical activation by permeating Ca2+ into the cell, may play a pivotal role in cardiac remodeling during cardiac overload. Our study aimed to investigate TRPV4 involvement in pathological and physiological remodeling through Ca2+-dependent signaling. TRPV4 expression was assessed in heart failure (HF) models, induced by isoproterenol infusion or transverse aortic constriction, and in exercise-induced adaptive remodeling models. The impact of genetic TRPV4 inhibition on HF was studied by echocardiography, histology, gene and protein analysis, arrhythmia inducibility, Ca2+ dynamics, calcineurin (CN) activity, and NFAT nuclear translocation. TRPV4 expression exclusively increased in HF models, strongly correlating with fibrosis. Isoproterenol-administered transgenic TRPV4-/- mice did not exhibit HF features. Cardiac fibroblasts (CFb) from TRPV4+/+ animals, compared to TRPV4-/-, displayed significant TRPV4 overexpression, elevated Ca2+ influx, and enhanced CN/NFATc3 pathway activation. TRPC6 expression paralleled that of TRPV4 in all models, with no increase in TRPV4-/- mice. In cultured CFb, the activation of TRPV4 by GSK1016790A increased TRPC6 expression, which led to enhanced CN/NFATc3 activation through synergistic action of both channels. In conclusion, TRPV4 channels contribute to pathological remodeling by promoting fibrosis and inducing TRPC6 upregulation through the activation of Ca2+-dependent CN/NFATc3 signaling. These results pose TRPV4 as a primary mediator of the pathological response.
Asunto(s)
Calcineurina , Insuficiencia Cardíaca , Canales Catiónicos TRPV , Remodelación Ventricular , Animales , Ratones , Calcineurina/metabolismo , Células Cultivadas , Fibrosis , Insuficiencia Cardíaca/metabolismo , Isoproterenol , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Remodelación Ventricular/genéticaRESUMEN
OBJECTIVE: To summarize the clinical and genetic characteristics, treatment and prognosis of four children with Steroid-resistant nephrotic syndrome (SRNS) due to variants of TRPC6 gene. METHODS: Clinical data of four children with SRNS admitted to Children's Hospital Affiliated to Zhengzhou University between May 2020 and August 2022 were collected. Peripheral blood samples were collected from the children and their parents, and whole exome sequencing was carried out. Sanger sequencing was used to verify the pathogenicity of the candidate variants among the children and their parents. RESULTS: All of the four children were found to harbor heterozygous variants of the TRPC6 gene, including c.523C>T (p.R175W), c.1327T>A (p.F443I), c.430G>C (p.E144Q) (unreported previously), and c.523C>T (p.R175W), which were all missense variants. Two of the children have shown a simple type, whilst two have shown a nephritis type, none had extrarenal phenotype. Comprehensive renal pathology of three children revealed focal segmental glomerulosclerosis (FSGS). Two children were treated with steroids combined with calcineurin inhibitors (CNIs), among whom one showed significant improvement in symptoms. CONCLUSION: Discoveries of the novel c.430G>C variant and the new SRNS phenotype of the c.1327T>A variant have expanded the mutational and phenotypic spectrum of the TRPC6 gene, which has provided a reference for clinical diagnosis and genetic counseling for the families.
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Glomeruloesclerosis Focal y Segmentaria , Síndrome Nefrótico , Niño , Humanos , Síndrome Nefrótico/tratamiento farmacológico , Síndrome Nefrótico/genética , Síndrome Nefrótico/diagnóstico , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/uso terapéutico , Fenotipo , Riñón , Genotipo , Mutación , Glomeruloesclerosis Focal y Segmentaria/genéticaRESUMEN
Cardiovascular disease is the leading cause of death, medical complications, and healthcare costs. Although recent advances have been in treating cardiovascular disorders linked with a reduced ejection fraction, acutely decompensate cardiac failure remains a significant medical problem. The transient receptor potential cation channel (TRPC6) family responds to neurohormonal and mechanical stress, playing critical roles in cardiovascular diseases. Therefore, TRP C6 channels have great promise as therapeutic targets. Numerous studies have investigated the roles of TRP C6 channels in pain neurons, highlighting their significance in cardiovascular research. The TRPC6 protein exhibits a broad distribution in various organs and tissues, including the brain, nerves, heart, blood vessels, lungs, kidneys, gastrointestinal tract, and other bodily structures. Its activation can be triggered by alterations in osmotic pressure, mechanical stimulation, and diacylglycerol. Consequently, TRPC6 plays a significant role in the pathophysiological mechanisms underlying diverse diseases within living organisms. A recent study has indicated a strong correlation between the disorder known as TRPC6 and the development of cardiovascular diseases. Consequently, investigations into the association between TRPC6 and cardiovascular diseases have gained significant attention in the scientific community. This review explores the most recent developments in the recognition and characterization of TRPC6. Additionally, it considers the field's prospects while examining how TRPC6 might be altered and its clinical applications.
Asunto(s)
Enfermedades Cardiovasculares , Canal Catiónico TRPC6 , Humanos , Pulmón/metabolismo , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6/metabolismoRESUMEN
OBJECTIVE: To assess the effect of platycodin D (PD) for alleviating pulmonary fibrosis in mice and explore the underlying mechanism. METHODS: C57BL/6J mouse models of pulmonary fibrosis induced by bleomycin injection into the airway were treated with daily intragastric administration of 10 mg/kg PD for 28 days. The changes of pulmonary fibrosis and the expression and distribution of transient receptor potential cation channel subfamily C member 6 (TRPC6) were evaluated with immunohistochemistry, HE staining and Sirius Red staining. Western blotting was used to detect α-SMA expression in the lung tissues of the mice. Primary cultures of mouse lung fibroblasts were pretreated with PD (2.5, 5.0, and 10 µmol/L) or larixyl acetate (LA; 10 µmol/L) before exposure to 10 ng/mL transforming growth factor-ß1 (TGF-ß1), and the changes in cell survival rate, expressions of collagen â , α-SMA and TRPC6, reactive oxygen species (ROS) production, mitochondrial membrane potential, and cell proliferation capacity were assessed. Network pharmacology analysis was performed to explore the mechanism by which PD alleviated pulmonary fibrosis. RESULTS: PD treatment significantly alleviated pulmonary fibrosis and reduced α-SMA expression in BLM-induced mouse models (P<0.05). In TGF-ß1-induced primary mouse lung fibroblasts, PD effectively inhibited the cell proliferation, reduced ROS production (P<0.0001), rescued the reduction of mitochondrial membrane potential (P<0.001), and inhibited the expressions of α-SMA and collagen â (P<0.05). Network pharmacology analysis suggested that TRPC6 mediated the effect of PD for alleviating pulmonary fibrosis. Immunohistochemistry showed that PD significantly reduced TRPC6 expression in the lung tissues of BLM-induced mice. In primary mouse lung fibroblasts, PD significantly inhibited TGF-ß1-induced TRPC6 expression (P<0.05), and LA treatment obviously lowered the expression levels of TRPC6, α-SMA and collagenâ (P<0.05). CONCLUSION: PD alleviated pulmonary fibrosis in mice possibly by down-regulating TRPC6 and reducing ROS production.
Asunto(s)
Fibrosis Pulmonar , Saponinas , Triterpenos , Ratones , Animales , Fibrosis Pulmonar/inducido químicamente , Especies Reactivas de Oxígeno/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Canal Catiónico TRPC6/metabolismo , Canal Catiónico TRPC6/uso terapéutico , Ratones Endogámicos C57BL , Pulmón/patología , Fibroblastos , Bleomicina/efectos adversos , Colágeno Tipo IRESUMEN
BACKGROUND: Expression and function of TRPC3 and TRPC6 in the pancreas is a controversial topic. Investigation in human tissue is seldom. We aimed to provide here a detailed description of the distribution of TRPC3 and TRPC6 in the human exocrine and endocrine pancreas. METHODS: We collected healthy samples from cadavers (n = 4) and visceral surgery (n = 4) to investigate the respective expression profiles using immunohistochemical tracing with knockout-validated antibodies. RESULTS: TRPC3- and TRPC6-proteins were detected in different pancreatic structures including acinar cells, as well as epithelial ductal cells from intercalate, intralobular, and interlobular ducts. Respective connective tissue layers appeared unstained. Endocrine islets of Langerhans were clearly and homogenously immunolabeled by the anti-TRPC3 and anti-TRPC6 antibodies. Insular α, ß, γ, and δ cells were conclusively stained, although no secure differentiation of cell types was performed. CONCLUSIONS: Due to aforementioned antibody specificity verification, protein expression in the immunolabeled localizations can be accepted. Our study in human tissue supports previous investigations especially with respect to acinar and insular α and ß cells, while other localizations are here reported for the first time to express TRPC3 and TRPC6, ultimately warranting further research.
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Páncreas Exocrino , Canales Catiónicos TRPC , Canal Catiónico TRPC6 , Humanos , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6/metabolismo , Páncreas Exocrino/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Anciano , Adulto , InmunohistoquímicaRESUMEN
BACKGROUND: Skeletal muscle ischaemia-reperfusion injury (IRI) is a prevalent condition encountered in clinical practice, characterised by muscular dystrophy. Owing to limited treatment options and poor prognosis, it can lead to movement impairments, tissue damage, and disability. This study aimed to determine and verify the influence of transient receptor potential canonical 6 (TRPC6) on skeletal muscle IRI, and to explore the role of TRPC6 in the occurrence of skeletal muscle IRI and the signal transduction pathways activated by TRPC6 to provide novel insights for the treatment and intervention of skeletal muscle IRI. METHODS: In vivo ischaemia/reperfusion (I/R) and in vitro hypoxia/reoxygenation (H/R) models were established, and data were comprehensively analysed at histopathological, cellular, and molecular levels, along with the evaluation of the exercise capacity in mice. RESULTS: By comparing TRPC6 knockout mice with wild-type mice, we found that TRPC6 knockout of TRPC6 could reduced skeletal muscle injury after I/R or H/R, of skeletal muscle, so as therebyto restoringe some exercise capacity inof mice. TRPC6 knockdown can reduced Ca2+ overload in cells, therebyo reducinge apoptosis. In additionAdditionally, we also found that TRPC6 functionsis not only a key ion channel involved in skeletal muscle I/R injury, but also can affects Ca2+ levels and then phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signalling pathway. by knocking downTherefore, knockdown of TRPC6, so as to alleviated the injury inducedcaused by skeletal muscle I/R or and H/R. CONCLUSIONS: These findingsdata indicate that the presence of TRPC6 exacerbatescan aggravate the injury of skeletal muscle injury after I/Rischemia/reperfusion, leading towhich not only causes Ca2+ overload and apoptosis., Additionally, it impairsbut also reduces the self- repair ability of cells by inhibiting the expression of the PI3K/Akt/mTOR signalling pathway. ETo exploringe the function and role of TRPC6 in skeletal muscle maycan presentprovide a novelew approachidea for the treatment of skeletal muscle ischemia/reperfusion injury.
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Apoptosis , Ratones Noqueados , Músculo Esquelético , Daño por Reperfusión , Transducción de Señal , Canal Catiónico TRPC6 , Animales , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Canal Catiónico TRPC6/metabolismo , Canal Catiónico TRPC6/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Calcio/metabolismoRESUMEN
beta3-adrenergic activation causes Ca2+ release from the mitochondria and subsequent Ca2+ release from the endoplasmic reticulum (ER), evoking store-operated Ca2+ entry (SOCE) due to Ca2+ depletion from the ER in mouse brown adipocytes. In this study, we investigated how Ca2+ depletion from the ER elicits SOCE in mouse brown adipocytes using fluorometry of intracellular Ca2+ concentration ([Ca2+]i). The administration of cyclopiazonic acid (CPA), a reversible sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump blocker in the ER, caused an increase in [Ca2+]i. Moreover, CPA induced SOCE was suppressed by the administration of a Ca2+ free Krebs solution and the transient receptor potential canonical 6 (TRPC6) selective blockers 2-APB, ML-9 and GsMTx-4 but not Pico145, which blocks TRPC1/4/5. Administration of TRPC6 channel agonist 1-oleoyl-2-acetyl-sn-glycerol (OAG) and flufenamic acid elicited Ca2+ entry. Moreover, our RT-PCR analyses detected mRNAs for TRPC6 in brown adipose tissues. In addition, western blot analyses showed the expression of the TRPC6 protein. Thus, TRPC6 is one of the Ca2+ pathways involved in SOCE. These modes of Ca2+ entry provide the basis for heat production via activation of Ca2+-dependent dehydrogenase and the expression of uncoupling protein 1 (UCP1). Enhancing thermogenic metabolism in brown adipocytes may serve as broad therapeutic utility to reduce obesity and metabolic syndrome.
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Canales de Potencial de Receptor Transitorio , Ratones , Animales , Canal Catiónico TRPC6/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Canales Catiónicos TRPC/metabolismo , Calcio/metabolismo , Adipocitos Marrones/metabolismo , Retículo Endoplásmico/metabolismo , Señalización del CalcioRESUMEN
BACKGROUND: Canonical transient receptor potential channels play a crucial role in cancer cell proliferation. While TRPC6 subtype detection in submandibular glands and the relevance of some TRPC channels in this gland have been shown in animal models, its histological detection in human lacrimal and submandibular glands, as well as related tumors, lacks systematic study. Studying TRPC6 in humans could lead to new therapeutic options. This research aimed to immunohistochemically detect TRPC6 in human samples of physiological lacrimal and submandibular glands and of adenoid cystic carcinoma and mucoepidermoid carcinoma. METHODS: Seven fixed body donors and samples of six cancer patients were examined. The ten tissue samples collected from the submandibular and lacrimal glands were then processed into histological slides and stained with hematoxylin-eosin. Tumor samples were provided as sections. TRPC6 presence was determined by immunohistochemistry, which was performed by indirect detection with a primary TRPC6 antibody, a secondary HRP-conjugated antibody and the chromogen diaminobenzidine. RESULTS: Results confirm TRPC6 expression in all ten physiological gland samples: all samples showed a immunohistochemical signal with varying intensity. No significant gender-specific differences could be observed. TRPC6 was detected in four of six submandibular adenoid cystic carcinoma and the mucoepidermoid carcinoma samples, especially in tumor cells' cytoplasma and nuclei. Excretory ducts consistently showed TRPC6. Mucous tubules, their nuclei and the nuclei of adipocytes generally showed no signal while serous acini and their nuclei showed a weak TRPC6 signal. CONCLUSION: The discovery of TRPC6 in glandular tissue indicates a role in salivary gland function and calcium homeostasis is a basis for further research into its significance for tumor development in adenoid cystic carcinoma and mucoepidermoid carcinoma of salivary glands. TRPC6 could be used as a target for treatment of these tumors. However, the correlation between TRPC6 and submandibular and lacrimal gland diseases requires further exploration.
Asunto(s)
Carcinoma Adenoide Quístico , Inmunohistoquímica , Aparato Lagrimal , Neoplasias de las Glándulas Salivales , Glándula Submandibular , Canal Catiónico TRPC6 , Humanos , Femenino , Masculino , Neoplasias de las Glándulas Salivales/patología , Neoplasias de las Glándulas Salivales/metabolismo , Persona de Mediana Edad , Aparato Lagrimal/patología , Aparato Lagrimal/metabolismo , Carcinoma Adenoide Quístico/patología , Carcinoma Adenoide Quístico/metabolismo , Canal Catiónico TRPC6/metabolismo , Glándula Submandibular/patología , Glándula Submandibular/metabolismo , Anciano , Adulto , Carcinoma Mucoepidermoide/patología , Carcinoma Mucoepidermoide/metabolismo , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/análisisRESUMEN
OBJECTIVE: Renal fibrosis is the ultimate pathway of various forms of acute and chronic kidney damage. Notably, the knockout of transient receptor potential channel 6 (TRPC6) has shown promise in alleviating renal fibrosis. However, the regulatory impact of TRPC6 on renal fibrosis remains unclear. METHODS: In vivo, TRPC6 knockout (TRPC6-/-) mice and age-matched 129 SvEv (WT) mice underwent unilateral renal ischemia-reperfusion (uIR) injury surgery on the left renal pedicle or sham operation. Kidneys and serum were collected on days 7, 14, 21, and 28 after euthanasia. In vitro, primary tubular epithelial cells (PTECs) were isolated from TRPC6-/- and WT mice, followed by treatment with transforming growth factor ß1 (TGFß1) for 72 h. The anti-fibrotic effect of TRPC6-/- and the underlying mechanisms were assessed through hematoxylin-eosin staining, Masson staining, immunostaining, qRT-PCR, and Western blotting. RESULTS: Increased TRPC6 expression was observed in uIR mice and PTECs treated with TGFß1. TRPC6-/- alleviated renal fibrosis by reducing the expression of fibrotic markers (Col-1, α-SMA, and vimentin), as well as decreasing the apoptosis and inflammation of PTECs during fibrotic progression both in vivo and in vitro. Additionally, we found that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase 3 beta (GSK3ß) signaling pathway, a pivotal player in renal fibrosis, was down-regulated following TRPC6 deletion. CONCLUSION: These results suggest that the ablation of TRPC6 may mitigate renal fibrosis by inhibiting the apoptosis and inflammation of PTECs through down-regulation of the PI3K/AKT/GSK3ß pathway. Targeting TRPC6 could be a novel therapeutic strategy for preventing chronic kidney disease.
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Fibrosis , Glucógeno Sintasa Quinasa 3 beta , Ratones Noqueados , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Canal Catiónico TRPC6 , Animales , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Masculino , Riñón/patología , Riñón/metabolismo , Enfermedades Renales/metabolismo , Enfermedades Renales/genética , Enfermedades Renales/patología , Enfermedades Renales/etiología , Células Epiteliales/metabolismo , Células Epiteliales/patología , ApoptosisRESUMEN
Diabetic wounds exhibit delayed and incomplete healing, usually due to vascular and nerve damage. Dysregulation of cellular Ca2+ homeostasis has recently been shown to be closely related to insulin resistance and type 2 diabetes mellitus. However, the involvement of this dysregulation in diabetic wound complications remains unknown. In this study, we found calcium dysregulation in patients with diabetic ulcers via tissue protein profiling. High glucose and glucometabolic toxicant stimulation considerably impaired the function of TRPC6, a pore subunit of transient receptor potential channels mediating Ca2+ influx, and mitochondria, which regulate calcium cycling and metabolism. Furthermore, we found that mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEVs) could play a dual role in restoring the function of TRPC6 and mitochondria by delivering transcription factor SP2 and deubiquitinating enzyme USP9, respectively. MSC-sEVs could transfer SP2 that activated TRPC6 expression by binding to its specific promoter regions (-1519 to -1725 bp), thus recovering Ca2+ influx and downstream pathways. MSC-sEVs also promoted mitophagy to restore mitochondrial function by transporting USP9 that stabilized the expression of Parkin, a major player in mitophagy, thereby guaranteeing Ca2+ efflux and avoidance of Ca2+ overload. Targeting the regulation of calcium homeostasis provides a perspective for understanding diabetic wound healing, and the corresponding design of MSC-sEVs could be a potential therapeutic strategy.
Asunto(s)
Diabetes Mellitus Tipo 2 , Vesículas Extracelulares , Células Madre Mesenquimatosas , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Canal Catiónico TRPC6/metabolismo , Calcio/metabolismo , Cicatrización de Heridas/fisiología , Células Madre Mesenquimatosas/metabolismo , Vesículas Extracelulares/metabolismo , Mitocondrias/metabolismoRESUMEN
Diabetic kidney disease (DKD), one of the common complications of type2 diabetes mellitus (T2DM), has become the principal cause of endstage kidney disease. Transient receptor potential channel 6 (TRPC6), one of nonselective cation channels with significant calciumpermeability, is associated with renal fibrosis. However, the mechanism of TRPC6 in T2DMinduced renal fibrosis is still not entirely understood. The present study explored the potential mechanism of Trpc6 knockout in T2DMinduced renal fibrosis in Trpc6/ mice. The results showed that Trpc6 knockout inhibited the loss of body weight and the increase of fasting blood glucose (FBG) and significantly improved renal dysfunction and glomerular fibrosis in T2DM mice. The present study also indicated that Trpc6 knockout significantly lowered the expression of phosphorylated (p)SMAD2/3, TGFß, calcineurin (CN), nuclear factor of activated Tcell (NFAT)2 and Nodlike receptor (NLR) 3 inflammasomeassociated proteins. Calcium imaging results revealed that Trpc6 knockdown could decrease the levels of [Ca2+]i and inhibited calcium homeostasis imbalance. Moreover, it was found that knockout of Trpc6 had no significant influence on lipid disposition and reactive oxygen species generation in the kidney cortex. The present study suggested that knockout of Trpc6 may alleviate glomerular fibrosis and delay DKD progression by reducing [Ca2+]i overload and inhibiting the CNNFAT2 pathway in T2DM mice.
Asunto(s)
Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Ratones , Animales , Canal Catiónico TRPC6/genética , Calcineurina/metabolismo , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Calcio/metabolismo , Nefropatías Diabéticas/metabolismo , Transducción de Señal , Diabetes Mellitus Tipo 2/complicaciones , Fibrosis , Ratones NoqueadosRESUMEN
Heart failure is a primary cause of global mortality. In the present study, whether larixyl acetate, an inhibitor of transient receptor potential cation channel subfamily C member 6, attenuates pressure overloadinduced heart failure in mice was investigated. To test this hypothesis, a transverse aortic constriction (TAC) animal model and an angiotensin II (Ang II)treated H9c2 cell model were used. Cardiac and cellular structure, function and the expression levels of hypertrophy, endoplasmic reticulum (ER) stress, apoptosis, autophagy and pmTOR/mTOR related mRNAs or proteins were assessed to explore the underlying molecular mechanisms. The results indicated that treatment with TAC or Ang II leads to significant hypertrophy and dysfunction of the heart or H9c2 cells, accompanied by an increase in ER stress, apoptosis and activation of the mTOR signaling pathway, and a decrease in autophagy. The administration of larixyl acetate attenuated these impairments, which can be reversed by inhibiting autophagy through the activation of the mTOR signaling pathway. These findings suggested that larixyl acetate can effectively protect against pressure overloadinduced heart failure by enhancing autophagy and limiting ER stress and apoptosis through inhibition of the mTOR pathway.
Asunto(s)
Acetatos , Estenosis de la Válvula Aórtica , Insuficiencia Cardíaca , Naftalenos , Ratones , Animales , Canal Catiónico TRPC6 , Miocitos Cardíacos/metabolismo , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Autofagia , Estenosis de la Válvula Aórtica/metabolismo , Hipertrofia/metabolismo , Cardiomegalia/tratamiento farmacológico , Cardiomegalia/metabolismo , ApoptosisRESUMEN
BACKGROUND: Post-traumatic stress disorder (PTSD) refers to a chronic impairing psychiatric disorder occurring after exposure to the severe traumatic event. Studies have demonstrated that medicinal cannabis oil plays an important role in neuroprotection, but the mechanism by which it exerts anti-PTSD effects remains unclear. METHODS: The chronic complex stress (CCS) simulating the conditions of long voyage stress for 4 weeks was used to establish the PTSD mice model. After that, behavioral tests were used to evaluate PTSD-like behaviors in mice. Mouse brain tissue index was detected and hematoxylin-eosin staining was used to assess pathological changes in the hippocampus. The indicators of cell apoptosis and the BDNF/TRPC6 signaling activation in the mice hippocampus were detected by western blotting or real-time quantitative reverse transcription PCR experiments. RESULTS: We established the PTSD mice model induced by CCS, which exhibited significant PTSD-like phenotypes, including increased anxiety-like and depression-like behaviors. Medicinal cannabis oil treatment significantly ameliorated PTSD-like behaviors and improved brain histomorphological abnormalities in CCS mice. Mechanistically, medicinal cannabis oil reduced CCS-induced cell apoptosis and enhanced the activation of BDNF/TRPC6 signaling pathway. CONCLUSIONS: We constructed a PTSD model with CCS and medicinal cannabis oil that significantly improved anxiety-like and depressive-like behaviors in CCS mice, which may play an anti-PTSD role by stimulating the BDNF/TRPC6 signaling pathway.
Asunto(s)
Ansiedad , Factor Neurotrófico Derivado del Encéfalo , Depresión , Modelos Animales de Enfermedad , Hipocampo , Transducción de Señal , Trastornos por Estrés Postraumático , Canal Catiónico TRPC6 , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/efectos de los fármacos , Ratones , Transducción de Señal/efectos de los fármacos , Ansiedad/tratamiento farmacológico , Ansiedad/metabolismo , Masculino , Depresión/tratamiento farmacológico , Depresión/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Trastornos por Estrés Postraumático/tratamiento farmacológico , Trastornos por Estrés Postraumático/metabolismo , Canal Catiónico TRPC6/metabolismo , Conducta Animal/efectos de los fármacos , Marihuana Medicinal/farmacología , Ratones Endogámicos C57BL , Apoptosis/efectos de los fármacos , Aceites de Plantas/farmacología , Aceites de Plantas/administración & dosificación , Estrés Psicológico/tratamiento farmacológico , Estrés Psicológico/metabolismoRESUMEN
Collapsing glomerulopathy (CG) is a proliferative disease characterized by segmental or global wrinkling of the glomerular basement membrane and the formation of pseudocrescents, whereas focal segmental glomerulosclerosis (FSGS) is characterized by podocytopenia, and focal and segmental sclerosis of the glomeruli. Mutations in NPHS1, NPHS2, WT1, PLCE1, CD2AP, ACTN4, and TRPC6 have been reported in steroid-resistant FSGS patients. The mutations p.R895C and p.R895L in Exon 13 are the only ones in TRPC6 causing CG reported to date. Here, we present the case of a 17-year-old male patient with a collapsing variant of familial FSGS caused by a mutation in TRPC6 (p.R895C) who presented with rapidly progressive (crescentic) and proliferative glomerulonephritis.