APOPTOSIS AND NECROSIS - MECHANISMS OF CELL-DEATH INDUCED BY CYCLOSPORINE-A IN A RENAL PROXIMAL TUBULAR CELL-LINE

Background. The mechanisms of cyclosporine (CsA)-induced nephrotoxicit y are not fully understood. While hemodynamic changes may be involved in vivo, there is also some evidence for tubular involvement. We previ ously showed direct toxicity of CsA in the LLC-PK1 renal tubular cell line. In the current study we examined mechanisms (apoptosis or necros is) 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 liga nd system in the mediation of CsA-induced cell death was also investig ated. Methods. Cells were treated with CsA (0.42 nM to 83 mu M) for 24 hours and alterations in DNA and protein synthesis and membrane integ rity were examined. Flow cytometry was used to investigate: (i) altera tions in the DNA content and cell cycle; (ii) the forward (FSC) and si de (SSC) light scattering properties (indicators of cell size and gran ularity, respectively); (iii) the externalization of phosphatidylserin e (PS) as a marker of early apoptosis using FITC-annexin V binding; an d (iv) expression of the apoptotic Fas protein. DNA fragmentation in a poptotic cells was also determined by the TUNEL assay. Results. CsA (a ll doses) caused a block in the G(0)/G(1) phase of the cell cycle as i ndicated by a decrease in DNA synthesis and supported by an increase i n the % of cells in the G(0)/G(1) phase with concurrent decreases of t hose in the S and G(2)/M phases. The effect on protein synthesis appea red to be much less. Lower doses of CsA (4.2 nM) caused the appearance of a ''sub-G(0)/G(1)'' peak, indicative of reduced DNA content, on th e 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 TU NEL assay. Higher doses of CsA increased cell size and decreased cell granularity and reduced membrane integrity. Expression of Fas, the cel l 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 c ycle blockade. The mode of cell death, namely apoptosis or necrosis, i s dose dependent. Fas may be an important mediator of CsA induced apop tosis in renal proximal tubular cells.