Background: The mechanisms of cyclosporine (CsA)-induced nephrotoxicity are not fully understood. While hemodynamic changes may be involved in vivo, there is also some evidence for tubular involvement. We previously showed direct toxicity of CsA in the LLC-PK1 renal tubular cell line. In the current study we examined mechanisms (apoptosis or necrosis) of cell death induced by CsA in the LLC-PK1 renal proximal tubular cell line. The possible role of the Fas (APO-1/CD95) antigen-Fas ligand system in the mediation of CsA-induced cell death was also investigated.
Methods: Cells were treated with CsA (0.42 nM to 83 microM) for 24 hours and alterations in DNA and protein synthesis and membrane integrity were examined. Flow cytometry was used to investigate: (i) alterations in the DNA content and cell cycle; (ii) the forward (FSC) and side (SSC) light scattering properties (indicators of cell size and granularity, respectively); (iii) the externalization of phosphatidylserine (PS) as a marker of early apoptosis using FITC-annexin V binding; and (iv) expression of the apoptotic Fas protein. DNA fragmentation in apoptotic cells was also determined by the TUNEL assay.
Results: CsA (all doses) caused a block in the G0/G1 phase of the cell cycle as indicated by a decrease in DNA synthesis and supported by an increase in the % of cells in the G0/G1 phase with concurrent decreases of those in the S and G2/M phases. The effect on protein synthesis appeared to be much less. Lower doses of CsA (4.2 nM) caused the appearance of a "sub-G0/G1" peak, indicative of reduced DNA content, on the DNA histogram that was paralleled by a reduction in cell size and an increased cell granularity and an increase in FITC-annexin V binding. DNA fragmentation was evident in these cells as assessed using the TUNEL assay. Higher doses of CsA increased cell size and decreased cell granularity and reduced membrane integrity. Expression of Fas, the cell surface molecule that stimulates apoptosis, was increased following low dose CsA exposure.
Conclusions: These results indicate that CsA is directly toxic to LLC-PK1 cells with reduced DNA synthesis and cell cycle blockade. The mode of cell death, namely apoptosis or necrosis, is dose dependent. Fas may be an important mediator of CsA induced apoptosis in renal proximal tubular cells.