Estudo dos mensageiros intracelulares cálcio e óxido nítrico em células LLC-PK1 em cultura, tratadas com ciclosporina A

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Lima, Roberta [UNIFESP]
Higa, Elisa Mieko Suemitsu [UNIFESP]
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Cyclosporin A (CsA) is an immunossupressive agent largely used in the treatment of organ transplant rejection, but nephrotoxicity is the major side effect which limits its use. The pathophysioiogy of this side effect is stili controversial, but it seems to be mediated by renal vasoconstriction provoked by this drug. The proximal tubules are likely its main target segments in the kidney. The aim of the present study is to investigate the effect of CsA in the synthesis of nitric oxide (NO), a potent vasodilator, in cultured LLC-PKL1 cells. The intracellular calcium([Ca+2]i), a mediator involved in the mechanisms of vasoconstriction, was also studied in CsA treated LLC-PK1 cells. The cells were incubated during 72hr with vehicle (control group, CTL), CsA (lOmg/ml), TNF-a (l5O U/ml) + IFN-g (interferon-g 5OOU/ml), used as a positive control, or CsA + TNF-a + IFN-g. After these treatments the culture medium was collected and nitrite (NO), a stable metabolite of NO was determined by Griess method. The results were corrected by the protein harvested from these cells and measure by the method of Lowry. Viability was determined in all groups by acridine orange method. The [Ca +2] was determined in CTL and CsA treated groups using the fluorescence indicator fura=2 AM, before and after vasopressin (AVP, 10-5 M) stimuli. ln CsA treated cells, the NO (pmoles/mg of protein) was decreased when compared to the CTL (l8.8 n O.6 vs 12.8 n O.5; p < O.05, both n =8). TNF- a + IFN- g tread cells presented a significant increase in NO2 (52.0 + O.2; n= 6; p < O.05) compared to CTL. This effect was partially reversed by the simultaneous treatment with CsA (CsA + TNF-a + IFN- g = 38.8 n O.3; p < O.05; n=6). Cell viability in TNF- a + IFN- g treated group was not different from CTL (93.00 n O.30 vs 93.60 n O.15; both n=1O). However, CsA treated cells presented a significantly reduced viability as compared to CTL : CsA, 84.70 n O.23 ; n = 10 and TNF- a+ IFN-g, 85.00 n O.21 ; n=1O. The basal levels of [Ca + 2]i, in CsA treated cells were not different from CTL cells ( 1.92 n O.08 vs 1.76 n O.12; n= 6, n=8, respectively). Conversely, after AVP stimuli, CsA treated group presented a significant increase of [Ca +2]i when compared to the CTL group (2.72 n O.18; n= 6 vs 2.22 O.14; n=8, p < O.05). Our data suggest a reduced no production in CsA treated LLC- PK1 cells, which could be a consequence of as cell damage provoked by this drug, as seen by its reduced cell...(au)
São Paulo: [s.n.], 1999. 85 p. ilustabgraf.