Navegando por Palavras-chave "LLC-PK1 cells"
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- ItemSomente MetadadadosEffect of cyclosporin A on nitric oxide production in cultured LLC-PK1 cells(Marcel Dekker Inc, 2001-01-01) Lima, R.; Serone, A. P.; Schor, N.; Higa, EMS; Universidade Federal de São Paulo (UNIFESP)The effect of Cyclosporin A on nitric oxide production was studied in cultured LLC- PK1 cells. for this purpose the cells were incubated with vehicle (olive oil, 10 mug/ml in DMSO), Cyclosporin A (CsA, 10 mug/ml), tumor necrosis factor (TNF-alpha, 150 U/ml) + interferon (IFN-gamma, 500 U/ml) to upregulate NOS synthesis, and therefore, NO production (used as a positive control), or CsA + TNF-alpha + IFN-gamma. After 72 hours the culture medium was collected and nitrite was determined by the Griess method. the results were normalized to the protein harvested from these cells as measured by the Lowry method. Viability was determined by the exclusion of the fluorescent dyes (acridine orange and ethidium bromide). Intracellular calcium was measured spectrophotometrically using the fluorescent calcium indicator fura-2 AM. in CsA treated cells, the nitrite (pmoles/mg of protein) was decreased when compared to control (12.8 +/- 0.5 vs. 18.3 +/- 0.6; p < 0.05; both n = 8). TNF- + IFN-gamma increased the nitrite synthesis (52.0 +/- 0.2; p < 0.05 vs. control; n = 6). This effect was decreased significantly by the simultaneous treatment with CsA (38.8 +/- 0.3; p < 0.05; n = 6). Cell viability in CsA group was decreased when compared to the cdntrol (84.7 +/- 0.2% vs. 93.6 +/- 0,1%; p < 0.05: both n = 10). TNF- +/- IFN-gamma had no effect on viability (93.0 +/- 0.3%; n = 10). However, when combined with CsA, viability was decreased relative to the control (85.0 +/- 0.2%; p < 0.05; n = 10). Acute (1 h) or chronic (72 h) treatment of LLC- PK1 cells with CsA had no effect on basal calcium levels.Our results demonstrate a reduced level of nitric oxide production in LLC- PK1 cells treated with CsA. There was no effect of the drug on intracellular calcium levels, however CsA treatment did reduce cellular viability. We suggest that, in part, the decreased levels of NO production are a secondary consequence of direct cell damage. However, CsA may also be exerting direct effects on NO synthesis through its interactions with both iNOS and cNOS. These results also provide a dual mechanism of action for CsA induced nephrotoxicity, that is, direct cell damage and interference with the NO system within the nephron.
- ItemAcesso aberto (Open Access)Gentamicin-induced preconditioning of proximal tubular LLC-PK1 cells stimulates nitric oxide production but not the synthesis of heat shock protein(Associação Brasileira de Divulgação Científica, 2009-07-01) Pessoa, Edson de Andrade [UNIFESP]; Convento, Marcia.Bastos [UNIFESP]; Silva, Ronaldo Goncalves da [UNIFESP]; Oliveira, Accary Souza Bulle [UNIFESP]; Borges, Fernanda Teixeira [UNIFESP]; Schor, Nestor [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Nephrotoxicity is the main side effect of antibiotics such as gentamicin. Preconditioning has been reported to protect against injuries as ischemia/reperfusion. The objective of the present study was to determine the effect of preconditioning with gentamicin on LLC-PK1 cells. Preconditioning was induced in LLC-PK1 cells by 24-h exposure to 2.0 mM gentamicin (G/IU). After 4 or 15 days of preconditioning, cells were again exposed to gentamicin (2.0 mM) and compared to untreated control or G/IU cells. Necrosis and apoptosis were assessed by acridine orange and HOESCHT 33346. Nitric oxide (NO) and endothelin-1 were assessed by the Griess method and available kit. Heat shock proteins were analyzed by Western blotting. After 15 days of preconditioning, LLC-PK1 cells exhibited a significant decrease in necrosis (23.5 ± 4.3 to 6.5 ± 0.3%) and apoptosis (23.5 ± 4.3 to 6.5 ± 2.1%) and an increase in cell proliferation compared to G/IU. NO (0.177 ± 0.05 to 0.368 ± 0.073 µg/mg protein) and endothelin-1 (1.88 ± 0.47 to 2.75 ± 0.53 pg/mL) production significantly increased after 15 days of preconditioning compared to G/IU. No difference in inducible HSP 70, constitutive HSC 70 or HSP 90 synthesis in tubular cells was observed after preconditioning with gentamicin. The present data suggest that preconditioning with gentamicin has protective effects on proximal tubular cells, that involved NO synthesis but not reduction of endothelin-1 or production of HSP 70, HSC 70, or HSP 90. We conclude that preconditioning could be a useful tool to prevent the nephrotoxicity induced by gentamicin.
- ItemSomente MetadadadosHSP70 induced by hyperosmotic stress partially protects LLC-PK1 cells against nephrotoxic drugs(Karger, 2003-07-01) Leitao, A. A.; Leite, C. A.; Santos, B. C.; Boim, M. A.; Universidade Federal de São Paulo (UNIFESP)Accumulation of HSP70 is related to the cytoprotection. It was evaluated whether hyperosmotic stress induces HSP70 accumulation in LLC-PK1 cells, and protects cells against toxicity provoked by cisplatin (Cis) and cyclosporine A (CyA). Cells were maintained in isosmotic (Iso) or hyperosmotic (H) culture medium for 24 h and then exposed to Cis or CyA for an additional period of 12 or 24 h (groups H+Cis and H+CyA). the H medium did not induce cell death and increased both HSP70 mRNA and protein levels, suggesting a role in cell adaptation to H condition. H medium produced partial cytoprotection against Cis and CyA compared with control cells. Despite the cytoprotection, there was a reduction in HSP70 mRNA and protein levels in H+Cis group. in contrast, the H+CyA group presented high levels of HSP70 mRNA and protein. the induction of HSP70 by H medium was associated with tolerance of LLC-PK1 cells against Cis and CyA cytotoxicity but this protection was induced by different mechanisms and depended on the characteristics of the drug used. Copyright (C) 2003 S. Karger AG, Basel.
- ItemAcesso aberto (Open Access)Nephrotoxicity Of Polymyxin B: Experimental Study In Cells And Implications For Nursing Practice(Universidade de São Paulo, Escola de Enfermagem, 2014-04-01) Neiva, Luciana Barros de Moura; Borges, Fernanda Teixeira; Watanabe, Mirian; Pessoa, Edson de Andrade; Barbosa, Dulce Aparecida [UNIFESP]; Vattimo, Maria de Fatima Fernandes; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)The aim of the study was to characterize the cell damage mechanisms involved in the pathophysiology of cytotoxicity of polymyxin B in proximal tubular cells (LLC - PK1) and discuss about the nurses interventions to identify at risk patients and consider prevention or treatment of nephrotoxicity acute kidney injury. This is a quantitative experimental in vitro study, in which the cells were exposed to 375μM polymyxin B sulfate concentration. Cell viability was determined by exclusion of fluorescent dyes and morphological method with visualization of apoptotic bodies for fluorescence microscopy. Cells exposed to polymyxin B showed reduced viability, increased number of apoptotic cells and a higher concentration of the enzyme lactate dehydrogenase. The administration of polymyxin B in vitro showed the need for actions to minimize adverse effects such as nephrotoxicity.