RESUMO
Small-molecule modulators of calcineurin signaling have been proposed as potential therapeutics in Down syndrome and Alzheimer's disease. Models predict that in Down syndrome, suppressed calcineurin-NFAT signaling may be mitigated by proINDY, which activates NFAT, the nuclear factor of activated T-cells. Conversely, elevated calcineurin signaling in Alzheimer's disease may be suppressed with the calcineurin inhibitors cyclosporine and tacrolimus. Such small-molecule treatments may have both beneficial and adverse effects. The current study examines the effects of proINDY, cyclosporine and tacrolimus on behavior, using zebrafish larvae as a model system. To suppress calcineurin signaling, larvae were treated with cyclosporine and tacrolimus. We found that these calcineurin inhibitors induced hyperactivity, suppressed visually-guided behaviors, acoustic hyperexcitability and reduced habituation to acoustic stimuli. To activate calcineurin-NFAT signaling, larvae were treated with proINDY. ProINDY treatment reduced activity and stimulated visually-guided behaviors, opposite to the behavioral changes induced by calcineurin inhibitors. The opposing effects suggest that activity and visually-guided behaviors are regulated by the calcineurin-NFAT signaling pathway. A central role of calcineurin-NFAT signaling is further supported by co-treatments of calcineurin inhibitors and proINDY, which had therapeutic effects on activity and visually-guided behaviors. However, these co-treatments adversely increased excitability, suggesting that some behaviors are regulated by other calcineurin signaling pathways. Overall, the developed methodologies provide an efficient high-throughput platform for the evaluation of modulators of calcineurin signaling that restore neural function, while avoiding adverse side effects, in a complex neural system.
Assuntos
Encéfalo/metabolismo , Inibidores de Calcineurina/farmacologia , Calcineurina/farmacologia , Ciclosporina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismo , Animais , Ciclosporina/antagonistas & inibidores , Hipercinese/induzido quimicamente , Fatores de Transcrição NFATC/metabolismoAssuntos
Humanos , Feminino , Adulto , Transplante de Órgãos/efeitos adversos , Rejeição de Enxerto/tratamento farmacológico , Imunossupressores/análise , Imunossupressores/farmacologia , Azatioprina/uso terapêutico , Tacrolimo/antagonistas & inibidores , Ciclosporina/antagonistas & inibidores , Corticosteroides/administração & dosagem , Sirolimo/antagonistas & inibidores , Inibidores de Calcineurina/uso terapêutico , Everolimo/antagonistas & inibidores , Ácido Micofenólico/uso terapêuticoRESUMO
An albumin-binding CsA analogue 4MCsA was achieved by attachment of a thiol-reactive maleimide group at the side-chain of P4 position of CsA derivative. 4MCsA was semi-synthesized from CsA, and the cell-impermeability of albumin-4MCsA was detected by mass spectrometry and a competitive flow cytometry. 4MCsA exhibits inhibition of chemotaxis activity and inflammation by targeting extracellular CypA without immunosuppressive effect and cellular toxicity. These combined results suggested that 4MCsA can be restricted extracellularly through covalently binding to Cys34 of albumin with its maleimide group, and regulate the functions of cyclophilin A extracellularly.
Assuntos
Albuminas/farmacologia , Ciclofilina A/farmacologia , Ciclosporina/antagonistas & inibidores , Albuminas/química , Sítios de Ligação/efeitos dos fármacos , Ciclofilina A/química , Ciclosporina/metabolismo , Relação Dose-Resposta a Droga , Humanos , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the order Nidovirales In this study, we aimed to identify arterivirus proteins involved in the mode of action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to an â¼2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV's CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely related drug ALV, CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.IMPORTANCE Currently, no approved treatments are available to combat infections with nidoviruses, a group of positive-stranded RNA viruses, including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and they may thus represent a class of pan-nidovirus inhibitors. In this study, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two transmembrane subunits of the viral replication machinery, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of arteriviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and for other RNA viruses inhibited by these compounds.
Assuntos
Equartevirus/genética , RNA Polimerase Dependente de RNA/genética , Proteínas não Estruturais Virais/metabolismo , Arterivirus/genética , Linhagem Celular , Ciclofilinas/metabolismo , Ciclosporina/antagonistas & inibidores , Equartevirus/metabolismo , Células HEK293 , Humanos , Mutação , Nidovirales/genética , Nidovirales/metabolismo , Inibidores da Síntese de Ácido Nucleico/metabolismo , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
An increasing number of experiments and clinical trials have demonstrated the safety, feasibility, and efficacy of mesenchymal stem cells (MSCs)-based therapies for the treatment of various diseases. The main drawbacks of MSC therapy are the lack of specific homing after systemic infusion and early death of injected cells because of the injury micro-environment. We pretreated bone mesenchymal stem cells (BMSCs) with erythropoietin (EPO) to investigate their positive effect on cyclosporine A (CsA)-induced nephrotoxicity. BMSCs were incubated with different concentrations of EPO (10, 100, 500, and 1000 IU/mL) for 24 and 48 h, and their proliferation rate, cytoskeletal morphology, migration ability, and the expression of CXCR4 were evaluated to determine the optimal pretreatment conditions. To investigate the therapeutic effects of BMSCs pretreated with EPO in CsA-induced nephrotoxicity, we established CsA-induced in vitro and in vivo toxicity models. In our in vitro study, preconditioning of BMSCs with 500 IU/mL EPO for 48 h induced a marked increase in their proliferation rate, cytoskeletal rearrangement, migration in the scrape-healing assay, and migration toward injured HK2 cells. In vivo, EPO-BMSCs showed higher ability to improve renal function than BMSCs, and in CsA-induced rats treated with EPO-BMSCs, interstitial lymphocyte infiltration, tubular swelling, necrosis, and interstitial fibrosis decreased. We demonstrated that pretreatment with 500 IU/mL EPO before infusion markedly increased the homing ability of BMSCs, and obviously ameliorate CsA-induced nephrotoxicity in rats.
Assuntos
Ciclosporina/antagonistas & inibidores , Eritropoetina/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Nefrite/terapia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Ciclosporina/toxicidade , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Expressão Gênica/efeitos dos fármacos , Sobrevivência de Enxerto , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Testes de Função Renal , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Nefrite/induzido quimicamente , Nefrite/metabolismo , Nefrite/patologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Receptores CXCR4/genética , Receptores CXCR4/metabolismoRESUMO
OBJECTIVE: To investigate the role of cortistatin (CST) on cyclosporine A (CsA)-induced myocardial apoptosis in rats and determine its effect on the expressions of myocardial apoptosis genes. MATERIALS AND METHODS: H9C2 cells were treated with different concentrations of CsA solution (0.04, 0.2, 1 and 5 µM) for 24, 48 and 72 h, respectively. The cell viability was detected via methyl thiazolyl tetrazolium (MTT) assay, and the appropriate dose and time were compared and determined. At the same time, CST in different concentrations (0.08, 0.04, 0.2, 1, 5 and 25 µM) was added into cell culture, and the appropriate dose was identified using MTT assay. The cellular morphology in each group was observed, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed for the detection of cell apoptosis. Moreover, in molecular mechanism research, the apoptosis-associated factors, factor associated suicide (Fas), Fas ligand (FasL) and B-cell lymphoma-2-associated X protein (Bax), were detected via quantitive Real-time polymerase chain reaction (qPCR). Finally, the levels of a protein related to myocardial apoptosis in rats were investigated via Western blotting. RESULTS: The treatment with 1 µM CsA for 48 h caused significant apoptosis. The results of TUNEL staining showed the inhibitory role of CST on the myocardial apoptosis in rats induced by CsA. The detection of apoptosis factors via Real-time PCR revealed that after the induction of CsA, the expressions of Fas, FasL and Bax mRNA in cells were significantly higher than those in control group, but were significantly decreased after administration of CST. Western blotting showed that the protein expressions of Caspase 3 and Caspase 9 were remarkably elevated in cells after the use of CsA, but were significantly reduced after administration of CST (p < 0.01). CONCLUSIONS: CST contributes to antagonistic function against the CsA-induced apoptosis of rat myocardial cells, and its effect is related to the down-regulation of expressions of apoptotic factors, Fas, FasL, Bax, Caspase 3, and Caspase 9.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclosporina/farmacologia , Miocárdio/citologia , Neuropeptídeos/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciclosporina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Regulação para Baixo , Expressão Gênica/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Masculino , Miocárdio/metabolismo , RatosRESUMO
Honokiol, a bioactive compound isolated from the cone and bark of Magnolia officinalis, has been shown to have various activities including inhibition of the growth of Candida albicans. We investigated the roles of the Hsp90-calcineurin pathway in the antifungal activity of honokiol. The pharmacologic tool was employed to evaluate the effects of Hsp90 and calcineurin in the antifungal activity of honokiol. We also evaluated the protective effects of the calcineurin inhibitor cyclosporin A (CsA) on honokiol-induced mitochondrial dysfunction by the fluorescence staining method. The Hsp90 inhibitor potentiated the antifungal activity of honokiol. A C. albicans strain with the calcineurin gene deleted displayed enhanced sensitivity to honokiol. However, co-treatment with calcineurin inhibitor CsA attenuated the cytotoxic activity of honokiol due to the protective effect on mitochondria. Our results provide insight into the action mechanism of honokiol.
Assuntos
Antifúngicos/farmacologia , Compostos de Bifenilo/farmacologia , Calcineurina/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lignanas/farmacologia , Apoptose/efeitos dos fármacos , Calcineurina/genética , Inibidores de Calcineurina/metabolismo , Candida albicans/efeitos dos fármacos , Ciclosporina/antagonistas & inibidores , Magnolia/química , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacosRESUMO
AIMS: Cyclosporine (CSA) elevates blood pressure (BP) and alters arterial baroreflex sensitivity (BRS) and vasoreactivity. In this study we determined whether the renin-angiotensin system (RAS) interplays with other vasopressor pathways in mediating the CSA actions. MATERIALS AND METHODS: Whole animal and isolated vascular preparations were employed to determine the effects of pharmacologic interruption of angiotensin II (Ang II), endothelin (ET), or thromboxane (TXA2) signaling on the adverse cardiovascular effects of CSA. KEY FINDINGS: CSA (25mg/kg/day i.p. for 7days) caused significant increases in BP that were paralleled with (i) reduced BRS measured by phenylephrine (BRSPE) or sodium nitroprusside (BRSSNP), (ii) enhanced aortic contractile responses to Ang II and U-46619 (thromboxane analogue), and (iii) reduced aortic eNOS expression and acetylcholine, but not SNP, vasorelaxations. Except for the reduced BRSSNP, the CSA effects disappeared upon concurrent administration of losartan (angiotensin AT1 receptor antagonist), captopril (angiotensin converting enzyme inhibitor), or their combination. Moreover, CSA augmentation of Ang II contractions was abolished after cyclooxygenase inhibition (indomethacin) or endothelin ETA/ETB receptor blockade (atrasentan/BQ788). By contrast, the blockade of thromboxane receptors (terutroban) failed to alter the CSA-evoked facilitation of Ang II responsiveness. SIGNIFICANCE: The facilitation of baroreflex control and inhibition of vascular responsiveness to Ang II and thromboxane contribute to the BP lowering effect of RAS inhibitors in CSA-treated rats. Further, endothelin receptors and vasoconstrictor prostanoids contribute to the CSA-evoked exaggeration of Ang II vascular responsiveness and hypertension.
Assuntos
Barorreflexo/efeitos dos fármacos , Ciclosporina/farmacologia , Hipertensão/fisiopatologia , Sistema Renina-Angiotensina/fisiologia , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Acetilcolina/farmacologia , Angiotensina II/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Atrasentana , Pressão Sanguínea/efeitos dos fármacos , Captopril/farmacologia , Ciclosporina/antagonistas & inibidores , Hipertensão/induzido quimicamente , Indometacina/farmacologia , Losartan/farmacologia , Masculino , Naftalenos/farmacologia , Óxido Nítrico Sintase Tipo III/biossíntese , Nitroprussiato/farmacologia , Oligopeptídeos/farmacologia , Fenilefrina/farmacologia , Piperidinas/farmacologia , Propionatos/farmacologia , Pirrolidinas/farmacologia , Ratos , Sistema Renina-Angiotensina/efeitos dos fármacosRESUMO
We investigated the renal protective effects of phophodiesterase type 5 (PDE5) inhibitors in mice with cyclosporine A (CyA; a calcineurin phosphatase inhibitor) induced nephrotoxicity. Fifty male mice were divided into five groups of 10. Group 1 received no treatment, group 2 received only saline orally, group 3 received 30 mg/kg/day CyA by subcutaneous injection, group 4 received only 30 mg/kg/day vardenafil orally, and group 5 received 30 mg/kg/day CyA by subcutaneous injection and 30 mg/kg/day vardenafil orally. At 28 days, platelet-derived growth factor A (PDGF-A) and C (PDGF-C), transforming growth factor-beta 1 (TGF-ß1), cyclo-oxygenase 1 and 2 (COX-1 and COX-2), and P glycoprotein (Pgp) expression levels were measured in the renal tissues. In addition, expressions of COX-1 and COX-2 genes were determined using real-time PCR. PDE5 inhibitor administration ameliorated decreased PDGF-A and C, TGF-ß1, COX-1 and -2, and Pgp expression levels by modulation of cyclic guanosine monophosphate (cGMP) activity in kidneys. The relative expressions of COX-1 and COX-2 genes to GAPDH revealed that the maximum increase was obtained in the group treated with CyA and vardenafil for both COX-1 and COX-2 genes. Our study revealed that long term oral treatment with vardenafil protects kidneys from CyA induced nephrotoxicity. We showed that long term oral treatment with PDE5 prevents pathological kidney changes caused by CyA induced nephrotoxicity.
Assuntos
Ciclosporina/antagonistas & inibidores , Ciclosporina/toxicidade , Imunossupressores/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/prevenção & controle , Inibidores da Fosfodiesterase 5/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Imuno-Histoquímica , Nefropatias/patologia , Masculino , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Dicloridrato de Vardenafila/farmacologiaRESUMO
BACKGROUND: Previously, we have reported that flow cytometry analysis of fine-needle aspirates can accurately predict rejection in kidney transplants treated with cyclosporine-azathioprine-prednisolone. In this study, we examined this technique's accuracy using current immunosuppression. METHODS: Kidney transplant recipients were treated with calcineurin inhibitors, mycophenolate mofetil, and prednisolone: 92 remained rejection-free - Group I - and 37 developed acute rejection - Group II. An allograft aspiration specimen and peripheral blood were collected from Group I on post-transplant day 7 and from Group II on the day of clinical rejection. RESULTS: Significant changes were seen in both aspiration and peripheral blood samples in several T cell subsets when comparing Groups I and II. A sensitivity of 94.6%, specificity of 85%, and AUC = 0.966 were observed through combining CD8DR with CD3CD69 values from aspiration specimen; the corresponding AUC in peripheral blood was 0.847. Irreversible rejections displayed a significantly higher activation score (p = 0.024). CONCLUSIONS: Flow cytometry analysis of aspiration specimen achieved high diagnostic performance in renal transplants through studying CD8DR and CD3CD69 under current immunosuppressive therapy. Peripheral blood analysis, although not significant, showed the same trend. The activation score anticipated the irreversibility of rejection. The data suggest this test, through an easily tolerated technique, merits further diagnostic use.
Assuntos
Citometria de Fluxo/métodos , Rejeição de Enxerto/diagnóstico , Imunossupressores/farmacologia , Falência Renal Crônica/cirurgia , Transplante de Rim , Linfócitos/patologia , Adulto , Idoso , Biópsia por Agulha , Ciclosporina/antagonistas & inibidores , Feminino , Seguimentos , Taxa de Filtração Glomerular , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/etiologia , Humanos , Imunossupressores/uso terapêutico , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Risco , Subpopulações de Linfócitos T/imunologia , Adulto JovemRESUMO
BACKGROUND: L-carnitine has protective effects against various types of injury. This study was designed to evaluate the beneficial effects of L-carnitine on pancreatic and renal injuries caused by cyclosporine (CsA). METHODS: Rats maintained on a low sodium diet were given vehicle (olive oil, 1 mL/kg/d), CsA (15 mg/kg/d), L-carnitine (50 or 200 mg/kg/d), or a combination of CsA and L-carnitine for 4 weeks. The impact of L-carnitine on pancreatic injury was assessed by blood glucose levels, plasma insulin concentrations, and hemoglobulin A1c (HbA1c). In addition, the protective effects of L-carnitine against CsA-induced kidney injury were evaluated in terms of renal function, histopathology (inflammatory cell influx and tubulointerstitial fibrosis), oxidative stress (8-hydroxy 2'-deoxyguanosine, 8-OHdG), transforming growth factor-betal (TGF-ß1), apoptosis (caspase-3), and autophagy (LC3-II). RESULTS: CsA treatment caused diabetes, renal dysfunction, tubulointerstitial inflammation (ED-1-positive cells), and fibrosis, which were accompanied by an increase in 8-OHdG production and upregulation of TGF-ß1, caspase-3, and LC3-II. Concomitant administration of L-carnitine increased plasma insulin concentrations, decreasing plasma glucose and HbA1c levels. In the kidney, L-carnitine induced dose-dependent improvement of renal function, inflammation, and fibrosis in parallel with suppression of the expression of TGF-ß1 and 8-OHdG. Furthermore, the administration of L-carnitine at a high dose inhibited the expression of caspase-3 and LC3-II. CONCLUSION: These findings suggest that L-carnitine has a protective effect against CsA-induced pancreatic and renal injuries.
Assuntos
Carnitina/farmacologia , Ciclosporina/antagonistas & inibidores , Rim/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Animais , Western Blotting , Masculino , Ratos , Ratos Sprague-DawleyAssuntos
Apoptose/efeitos dos fármacos , Ciclosporina/antagonistas & inibidores , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Imunossupressores/antagonistas & inibidores , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Lovastatina/farmacologia , Junções Íntimas/efeitos dos fármacos , AnimaisRESUMO
Although calcineurin inhibitors (CNIs) are very useful in preventing allograft rejection, they can mediate a rapid progression of post-transplantation malignancies. The CNI cyclosporine A (CsA) can promote renal tumor growth through activation of the proto-oncogene ras and over-expression of the angiogenic cytokine VEGF; the ras activation also induces over-expression of the cytoprotective enzyme HO-1, which promotes survival of renal cancer cells. Here, we show that the natural product honokiol significantly inhibited CsA-induced and Ras-mediated survival of renal cancer cells through the down-regulations of VEGF and HO-1. Thus, honokiol treatment may help to prevent tumor-promoting effects of CsA in transplant patients.
Assuntos
Compostos de Bifenilo/farmacologia , Inibidores de Calcineurina , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Lignanas/farmacologia , Proteínas ras/metabolismo , Apoptose/efeitos dos fármacos , Calcineurina/genética , Calcineurina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclosporina/antagonistas & inibidores , Ciclosporina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes ras , Heme Oxigenase-1/biossíntese , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Fosforilação/efeitos dos fármacos , Proto-Oncogene Mas , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Transfecção , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genéticaRESUMO
We used mouse cortical collecting duct principal cells (mpkCCDc14 cell line) as a model to determine whether statins reduce the harmful effects of cyclosporine A (CsA) on the distal nephron. The data showed that treatment of cells with CsA increased transepithelial resistance and that the effect of CsA was abolished by lovastatin. Scanning ion conductance microscopy showed that CsA significantly increased the height of cellular protrusions near tight junctions. In contrast, lovastatin eliminated the protrusions and even caused a modest depression between cells. Western blot analysis and confocal microscopy showed that lovastatin also abolished CsA-induced elevation of both zonula occludens-1 and cholesterol in tight junctions. In contrast, a high concentration of CsA induced apoptosis, which was also attenuated by lovastatin, elevated intracellular ROS via activation of NADPH oxidase, and increased the expression of p47phox. Sustained treatment of cells with lovastatin also induced significant apoptosis, which was attenuated by CsA, but did not elevate intracellular ROS. These results indicate that both CsA and lovastatin are harmful to principal cells of the distal tubule, but via ROS-dependent and ROS-independent apoptotic pathways, respectively, and that they counteract probably via mobilization of cellular cholesterol levels.
Assuntos
Apoptose/efeitos dos fármacos , Ciclosporina/antagonistas & inibidores , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Imunossupressores/antagonistas & inibidores , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Lovastatina/farmacologia , Junções Íntimas/efeitos dos fármacos , Análise de Variância , Animais , Western Blotting , Células Cultivadas , Colesterol/biossíntese , Corantes , Ciclosporina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Imunossupressores/farmacologia , Túbulos Renais Coletores/ultraestrutura , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Varredura , NADPH Oxidases/metabolismo , Permeabilidade , Espécies Reativas de Oxigênio/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1/biossínteseRESUMO
Cyclosporine A (CsA) nephrotoxicity has been linked to reactive oxygen species (ROS) production in renal cells. We have demonstrated that the antioxidant Vitamin E (Vit E) abolished renal toxicity in vivo and in vitro models. As one of the main sources of intracellular ROS are mitochondria, we studied the effects of CsA on several mitochondrial functions in LLC-PK1 cells. CsA induced ROS synthesis and decreased reduced glutathione (GSH). The drug decreased mitochondrial membrane potential (ΔΨm) and induced physiological modifications in both the inner (IMM) and the outer mitochondrial membranes (OMM). In the IMM, CsA provoked mitochondrial permeability transition pores (MPTP) and cytochrome c was liberated into the intermembrane space. CsA also induced pore formation in the OMM, allowing that intermembrane space contents can reach cytosol. Furthermore, CsA altered the mitochondrial dynamics, inducing an increase in mitochondrial fission; CsA increased the expression of dynamin related protein 1 (Drp1) that contributes to mitochondrial fission, and decreased the expression of mitofusin 2 (Mfn2) and optic atrophy protein 1 (Opa1), proteins involved in the fusion process. All these phenomena were related to apoptosis. These effects were inhibited when cells were treated with the antioxidant Vit E suggesting that they were mediated by the synthesis of ROS.
Assuntos
Apoptose/efeitos dos fármacos , Ciclosporina/toxicidade , Túbulos Renais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Ciclosporina/antagonistas & inibidores , Antagonismo de Drogas , Transferência Ressonante de Energia de Fluorescência , Glutationa/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Células LLC-PK1 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/metabolismo , Permeabilidade/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Suínos , Vitamina E/farmacologiaRESUMO
BACKGROUND AND OBJECTIVE: Ciclosporin A (CsA)-induced gingival overgrowth is attributed to an exaggerated accumulation of extracellular matrix, which is mainly due to an increased expression of transforming growth factor-ß1 (TGF-ß1). Herein, the in vitro investigation of effects of overexpression of Smad7, a TGF-ß1 signaling inhibitor, in the events associated with CsA-induced extracellular matrix accumulation was performed. MATERIAL AND METHODS: The effects of Smad7 were assessed by stable overexpression of Smad7 in fibroblasts from normal gingiva. Smad7-overexpressing cells and control cells were incubated with CsA, and synthesis of type I collagen, production and activity of MMP-2 and cellular proliferation were evaluated by ELISA, zymography, growth curve, bromodeoxyuridine incorporation assay and cell cycle analysis. The effects of CsA on cell viability and apoptosis of fibroblasts from normal gingiva were also evaluated. Western blot and immunofluorescence for phospho-Smad2 were performed to measure the activation of TGF-ß1 signaling. RESULTS: Although the treatment with CsA stimulated TGF-ß1 production in both control and Smad7-overexpressing fibroblasts, its signaling was markedly inhibited in Smad7-overexpressing cells, as revealed by low levels of phospho-Smad2. In Smad7-overexpressing cells, the effects of CsA on proliferation, synthesis of type I collagen and the production and activity of MMP-2 were significantly blocked. Smad7 overexpression blocked CsA-induced fibroblast proliferation via p27 regulation. Neither CsA nor Smad7 overexpression induced cell death. CONCLUSION: The data presented here confirm that TGF-ß1 expression is related to the molecular events associated with CsA-induced gingival overgrowth and suggest that Smad7 overexpression is effective in blocking these events, including proliferation, type I collagen synthesis and MMP-2 activity.
Assuntos
Ciclosporina/efeitos adversos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Crescimento Excessivo da Gengiva/induzido quimicamente , Proteína Smad7/farmacologia , Antimetabólitos , Apoptose/efeitos dos fármacos , Bromodesoxiuridina , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ciclosporina/antagonistas & inibidores , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Gengiva/citologia , Gengiva/metabolismo , Humanos , Masculino , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Fosforilação , Inibidores de Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/efeitos dos fármacos , Proteína Smad7/genética , Transfecção , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Adulto JovemRESUMO
AIMS: To bring further insight into the mechanism of cyclosporine A (CsA)-induced hepatotoxicity, the acute effect of CsA on local hepatic blood flow (LHBF) and its association with systemic hemodynamics, histopathological and biochemical indicators of liver toxicity were studied in guinea pigs in vivo. The association of endothelin (ET) and/or Cremophor-EL (C-EL, vehicle in parenteral CsA preparation) with CsA effects was also investigated. MAIN METHODS: Animals were assigned into five groups; control, CsA, C-EL, Bosentan (non-selective ET receptor antagonist)+CsA, and BQ-123 (ET(A) receptor antagonist)+CsA. CsA was infused intravenously (i.v.) at 20 and 10mg/kg doses by 15 min interval. Antagonists were administered 15 min before CsA infusion. LHBF and mean arterial blood pressure (MAP) changes were simultaneously recorded. Blood and liver samples were collected for biochemical and histopathological examinations. KEY FINDINGS: CsA, but not C-EL, decreased LHBF by 53.3% at the end of 30 min. Although being non-significant, CsA slightly increased MAP suggesting that, CsA-induced acute decrease in LHBF was likely independent of MAP changes. Bosentan (5mg/kg, i.v.) and BQ-123 (1mg/kg, i.v.) pre-treatments prevented the CsA-induced decrease in LHBF suggesting that CsA decreases LHBF through an ET-related mechanism. Additionally, CsA, but not its vehicle C-EL, caused marked acute pathological changes in the liver morphology. SIGNIFICANCE: CsA-induced findings of acute hepatotoxicity were prevented by bosentan and BQ-123 pre-treatments. Thus, CsA seems to exert acute hepatotoxic effect through ET-related mechanisms.
Assuntos
Ciclosporina/toxicidade , Endotelinas/fisiologia , Circulação Hepática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Animais , Bosentana , Ciclosporina/antagonistas & inibidores , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Glicerol/análogos & derivados , Glicerol/farmacologia , Cobaias , Fígado/irrigação sanguínea , Fígado/enzimologia , Fígado/patologia , Circulação Hepática/fisiologia , Masculino , Peptídeos Cíclicos/farmacologia , Sulfonamidas/farmacologiaAssuntos
Anti-Hipertensivos/efeitos adversos , Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Hipertensão/terapia , Nefropatias/induzido quimicamente , Transplante de Rim , Animais , Ciclosporina/efeitos adversos , Ciclosporina/antagonistas & inibidores , Rejeição de Enxerto/prevenção & controle , Humanos , Nefropatias/cirurgiaRESUMO
Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the induction of chronic nephrotoxicity including endoplasmic reticulum (ER) stress in tubular cells. Recently, it was reported that autophagy is induced by ER stress and serves to alleviate the associated deleterious effects. In the current study, CsA treatment (0-100 microm) decreased cell survival of rat pituitary GH3 cells in a dose-dependent manner. At concentrations ranging from 1.0 to 10 microm, CsA induced a dose-dependent increase in the expression of microtubule-associated protein 1 light chain 3 (LC3)-I and LC3-II. Cells treated with 2.5 microm CsA exhibited cytoplasmic vacuolation, indicating that CsA induces autophagy in rat pituitary GH3 cells. In the presence of 1.0-10 microm CsA, the expression of catalase decreased while that of the ER stress markers, ER luminal binding protein (BiP) and inositol-requiring enzyme 1 alpha (IRE1alpha), increased as compared those levels in untreated cells. These results suggested that CsA-induced autophagy is dependent on ER stress. To determine whether melatonin would protect cells against CsA-induced autophagy, we treated rat pituitary GH3 cells with melatonin in the presence of CsA. Melatonin treatment (100 and 200 microm) suppressed autophagy induced by 2.5 and 5 microm CsA. Furthermore, co-treatment with 100 microm melatonin inhibited LC3-II expression, and increased catalase and phosphorylated p-ERK levels in the presence of 2.5 and 5 microm CsA. BiP and IRE1alpha expression in melatonin-co-treated cells was superior to that in cells treated with 2.5 and 5 microm CsA alone. Thus, melatonin suppresses CsA-mediated autophagy in rat pituitary GH3 cells.
Assuntos
Autofagia/efeitos dos fármacos , Ciclosporina/antagonistas & inibidores , Melatonina/farmacologia , Hipófise/efeitos dos fármacos , Análise de Variância , Animais , Western Blotting , Catalase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Interações Medicamentosas , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Endorribonucleases/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Hipófise/citologia , Hipófise/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Estresse Fisiológico , Proteína X Associada a bcl-2/metabolismoRESUMO
This study was conducted to investigate the possible protective effect of ellagic acid (EA) on cyclosporine A (CsA)-induced testicular and spermatozoal damages associated with oxidative stress in male rats. Forty adult male Sprague-Dawley rats were divided into 4 groups of 10 animals each. Control group was used as placebo. Cyclosporine group received CsA at the dose of 15 mg/kg/day. Ellagic acid group was treated with EA (10 mg/kg/day). Cyclosporine plus ellagic acid group received CsA+EA. Reproductive organs were weighed and epididymal sperm characteristics and histopathological structure of testes were examined along with malondialdehyde (MDA) and glutathione (GSH) levels, glutathione-peroxidase (GSH-Px) and catalase (CAT) activities in testicular tissue. CsA significantly decreased the weights of testes and ventral prostate, epididymal sperm concentration, motility, testicular tissue glutathione (GSH), glutathione-peroxidase (GSH-Px) and catalase (CAT), diameters of seminiferous tubules and germinal cell layer thickness, and it significantly increased malondialdehyde (MDA) level and abnormal sperm rates along with degeneration, necrosis, immature germ cells, congestion and atrophy in testicular tissue. However, the CsA plus EA treatment attenuated all the CsA-induced negative changes observed in the testicular tissue, sperm and oxidant/antioxidant parameters. In conclusion, CsA-induced oxidative stress leads to the structural and functional damages in the testicular tissue and sperm quality of rats, and also EA has a protective effect on these damages.