Effect of cyclosporin A on nitric oxide production in cultured LLC-PK1 cells

Effect of cyclosporin A on nitric oxide production in cultured LLC-PK1 cells

Author Lima, R. Google Scholar
Serone, A. P. Google Scholar
Schor, N. Google Scholar
Higa, EMS Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract 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-<alpha> + 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-<alpha> +/- 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.
Keywords nitric oxide
nitrite
cyclosporin A
LLC-PK1 cells
tumor necrosis factor-alpha
intracellular calcium
interferon-gamma
Language English
Date 2001-01-01
Published in Renal Failure. New York: Marcel Dekker Inc, v. 23, n. 1, p. 43-52, 2001.
ISSN 0886-022X (Sherpa/Romeo, impact factor)
Publisher Marcel Dekker Inc
Extent 43-52
Origin http://dx.doi.org/10.1081/JDI-100001282
Access rights Closed access
Type Article
Web of Science ID WOS:000167384000005
URI http://repositorio.unifesp.br/handle/11600/26458

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