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

The effect of Cyclosporin A on nitric oxide production was studied in cultu red 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 necro sis factor (TNF-alpha, 150 U/ml) + interferon (IFN-gamma, 500 U/ml) to upre gulate NOS synthesis, and therefore, NO production (used as a positive cont rol), or CsA + TNF-alpha + IFN-gamma. After 72 hours the culture medium was collected and nitrite was determined by the Griess method. The results wer e normalized to the protein harvested from these cells as measured by the L owry method. Viability was determined by the exclusion of the fluorescent d yes (acridine orange and ethidium bromide). Intracellular calcium was measu red spectrophotometrically using the fluorescent calcium indicator fura-2 A M. In CsA treated cells, the nitrite (pmoles/mg of protein) was decreased w hen 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 th e simultaneous treatment with CsA (38.8 +/- 0.3; p < 0.05; n = 6). Cell via bility 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 wit h 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 wi th 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 intracellula r calcium levels, however CsA treatment did reduce cellular viability. We s uggest that, in part, the decreased levels of NO production are a secondary consequence of direct cell damage. However, CsA may also be exerting direc t effects on NO synthesis through its interactions with both iNOS and cNOS. These results also provide a dual mechanism of action for CsA induced neph rotoxicity, that is, direct cell damage and interference with the NO system within the nephron.