Purpose: One of the major limiting factors in the use of FK506 (Tacrolimus) is nephrotoxicity, but the mechanisms of nephrotoxicity are not fully understood. In order to elucidate the pathogenesis of FK506 tubulotoxicity, we examined mechanisms of cellular injury induced by FK506 in cultured LLC-PK1 renal tubular cell line.
Methods: The 3H-thymidine uptake and flow cytometric analysis of apoptosis following FK506 treatment were evlauated. The changes of the Fas and Bcl-2 protein in FK506-induced renal tubular cell injury were also investigated by Western blotting.
Results: FK506 treatment significantly decreased 3H-thymidine uptake (M+/-S.D., 3591.6+/-274.3 cpm/well vs 2217.6+/-79.7 at 1 microgram/mL, p<0.05), in a dose dependent manner upto 50microgram/mL, indicating that DNA damage is a sensitive indicator of FK506- induced nephrotoxicity. The addition of FK506 (50 microgram/mL) for 96 hours also induced a significant increase in the percentage of cells displaying annexin-V binding from 3.9+/-2.2% in control cells to 34.9+/-8.5% in treated cells (P<0.05), indicating early apoptotic cellwith an increase of Bcl-2 protein levels up to 6.2 fold (mean) on Western blot analysis, and the dose-dependent further increase of Bcl-2 protein was observed at a dose above 5microgram/mL. death. This finding was supported by electrophoretic analysis of the DNA extracted from FK506-treatedcells, where a series of bands correspondingto integer multiples of 180 to 200 base pairs was visualized. The treatment with FK506 at a concentration higher than 1 microgram/mL for 96 hours was seen to cause a significant 3.7 (mean) fold elevation in the expression of the 45 kD Fas protein by Western blot analysis. The exposure to FK506 at dose of 5microgram/mL for 96 hours was also associated with an increase of Bcl-2 protein levels up to 6.2 fold (mean) on Western blot analysis, and the dose-dependent further increase of Bcl-2 protein was observed at a dose above 5 microgram/mL.
Conclusion: FK506 is directly toxic to renal tubular cells with inhibiting DNA synthesis and inducing cell death in the form of apoptosis. The changes of Fas antigen system and Bcl-2 protein may play roles in mediating FK506-induced apoptosis of renal tubular cells.
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