Background

The pathogenesis of cyclosporine A (CsA) nephrotoxicity has not been completely elucidated.

Methods

The ability of CsA to induce apoptosis in cultured murine tubular epithelial cells and its regulation by the cell microenvironment and inhibitors of caspases were studied.

Results

This study found that CsA induces apoptotic death in murine proximal tubular epithelial MCT cells in a dose- (0.1 to 15 g/ml) and time-dependent (24 to 72 hr) manner. Death caused by CsA is additive to apoptosis induced by deprivation of the survival factors present in serum. Primary cultures of murine tubular epithelial cells are also sensitive to CsA-induced apoptosis. Peptide inhibitors of caspases such as zVAD-fmk (which inhibits caspases 8 and 9) and DEVD-CHO (which inhibits caspase 3 and related caspases) prevented CsA-induced apoptosis in MCT cells, although zVAD-fmk was effective at lower concentrations.

Conclusion

These data suggest that tubular cell apoptosis mediated by caspases may play a role in CsA nephrotoxicity and that the microenvironment modulates resistance to CsA lethality as low local levels of survival factors may potentiate nephrotoxicity. Caspases my be new therapeutic targets in the management of nephrotoxic injury.

METHODS

Cells

Murine proximal tubular epithelial (MCT) cells^8,9 were cultured in RPMI 1640 (GIBCO, Grand Island, NY, USA), 10% decomplemented fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin and 100 g/ml streptomycin, in 5% CO₂ at 37°C. CsA (Sandoz Pharma, Barcelona, Spain) was dissolved in ethanol. Primary cultures of murine proximal tubular epithelial cells (a kind gift from Carlos Peñaranda, Fundación Jiménez Díaz) were also tested for their susceptibility to CsA-induced apoptosis.

Studies of cell death and apoptosis

For quantification of cell death and apoptosis 20000 cells were seeded in 24-well plates (Costar, Cambridge, MA, USA) in 10% FCS RPMI. CsA (0.01 to 15 g/ml), or vehicle (ethanol, final concentration 0.1%) were added to cells grown in 10% FCS RPMI or to cells that had been deprived of serum for 24 hours. The caspase inhibitory peptides Acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO) and benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) were obtained from Bachem (Bubendorf, Switzerland) and used at concentrations previously shown to protect from apoptosis in other cell systems [reviewed in⁶. DEVD-CHO was dissolved in water and zVAD-fmk in DMSO. Final concentration of DMSO was 0.05% and it did not influence MCT cell apoptosis. Both peptides or vehicle were added to the cell cultures three hours prior to addition of CsA. Cells were cultured in the presence of stimuli for an additional 24 to 72 hours. Cells attached to the plate were detached with trypsin/EDTA and mixed with detached cells present in the supernatant. Cell survival was determined in pooled cells by trypan blue exclusion. At least 200 cells were counted in quadruplicate wells.

Apoptosis was characterized by morphologic and functional criteria. Nuclei of formalin-fixed cells were stained with propidium iodide in the presence of RNAse A to observe the typical morphological changes, as previously described^10,11. Cells exposed to CsA were also fixed in glutaraldehyde/paraformaldehyde and processed for electron microscopy¹². For assessment of apoptosis by flow cytometry adherent cells were pooled with spontaneously detached cells, and stained in 100 g/ml propidium iodide, 0.05% NP-40, 10 g/ml RNAse A in PBS and incubated at 4°C for more than one hour. The percentage of apoptotic cells with decreased DNA staining (A₀) was counted as previously described¹³. The protection from apoptosis offered by peptide inhibitors of caspases was expressed as a percentage of specific apoptosis induced by CsA in the presence of the peptide vehicle, which was considered to be 100%. Specific apoptosis induced by CsA was calculated by subtracting apoptosis encountered in cells cultured in the presence of the CsA vehicle, 0.1% ethanol. Internucleosomal DNA fragmentation was investigated in genomic DNA obtained from attached or detached cells that were lysed in hypotonic lysis buffer¹⁰. DNA was separated in a 1.5% agarose gel, and stained with ethidium bromide.

Statistics

Results are expressed as mean SD. Significance at the 95% level was established using one-way ANOVA and Bonferroni's method by means of the SigmaStat statistical software (Jandel, San Rafael, CA, USA).

RESULTS

CsA induces tubular cell death

CsA was toxic to cultured renal tubular epithelial cells. In the presence of survival factors (10% FCS), trypan blue exclusion detected a significant lethality in cells treated with CsA for 24 hours (0.1% ethanol, 10.9 0.9%, 10 g/ml CsA, 36.9 3.4% dead cells, P < 0.0001). However, most cells were only lightly stained by trypan blue and had decreased cell size. Only occasional dark blue cells with increased size, consistent with primary necrosis, were observed. A dose-response (0.1 to 15 g/ml CsA) relationship was observed (not shown). CsA-induced cell death increased from 24 to 72 hours. Serum deprivation induced, per se, tubular cell death. CsA toxicity was additive to that induced by serum deprivation.

Death induced by CsA has features of apoptosis

The mechanism of CsA-induced cell death was assessed by morphological and functional studies. Propidium iodide staining revealed the characteristic bright, shrunk, fragmented nuclei of apoptosis among cells treated with CsA Figure 1a. The percentage of apoptotic bodies was increased among cells treated with CsA that remained attached to the slide (at 24 hr, in 10% FCS, 0.1% ethanol 0.3 0.1%, 10 g/ml CsA, 3.8 0.5% apoptotic cells, P < 0.001). A higher percentage of apoptotic cells was found in the pool of attached and detached cells, with values similar to those obtained when hypodiploid cells were quantified in pooled cells by flow cytometry. This indicates that, as expected, most apoptotic cells detach from the culture substrate. Electron microscopy confirmed that cells treated with CsA display the apoptotic morphology (not shown)^10,13. Functionally, the internucleosomal DNA degradation typical of apoptosis was present among detached, CsA-treated cells Figure 1b. Flow cytometry analysis of cell DNA content demonstrated a distinct peak of hypodiploid (A₀), apoptotic cells among CsA-treated cells Figure 2a. The percentage of apoptotic cells was higher in the absence Figure 3 than in the presence of serum, that was used as a source of survival factors (in 10% FCS A₀ cells at 24 hr, 0.1% ethanol 2.9%, 10 g/ml CsA 25.7%). Apoptosis induced by CsA was time-dependent, that is, it was already noted in cells treated with 10 g/ml CsA for 24 hours and increased at later time points Figure 3a. At 24 hours there was no evident increase in apoptosis with the lower concentrations of CsA (0.1 to 1 g/ml), but at later time points an increased rate of apoptosis was induced by 0.1 g/ml CsA and a dose-response relationship was observed Figure 3b. CsA (10 g/ml) also induced apoptosis in primary cultures of murine tubular epithelial cells (not shown).

Figure 1.

Cyclosporine A (CsA) induces apoptosis of tubular epithelial cells. (A) Cells grown in serum-free conditions were fixed in 10% formalin and stained with propidium iodide. Tubular cells grown in 0.1% ethanol vehicle displayed pale, oval nuclei. Bright, condensed and fragmented nuclei (apoptotic cells) were observed after treatment with 10 g/ml CsA for 48 hours (CsA). The caspase inhibitory peptides 200 M zVAD-fmk and 400 M DEVD-CHO protected from CsA-induced apoptosis. (B) Gel electrophoresis of DNA obtained from cells grown in the presence of 10% FCS demonstrated the presence of internucleosomal DNA degradation in detached cells (detach) treated with 10 g/ml CsA for 24 hours, which was absent in cells treated with the ethanol vehicle (Et). The DNA of cells that remained attached (attach) to the plate was mostly intact, as assessed by its high molecular weight. This is consistent with the low percentage (<5%) of cells with apoptotic morphology that remained attached to the plate, even after treatment with CsA in the presence of 10% FCS. Abbreviation pGEM is molecular weight markers.

Full figure and legend (179K)

Figure 2.

Flow cytometry assessment of CsA-induced apoptosis: Prevention by caspase inhibitors. (A) Flow cytometry showed that cells treated with 15 g/ml CsA for 24 hours in the absence of serum displayed a distinct peak of apoptotic cells with hypodiploid DNA content. This peak was negligible in ethanol vehicle-treated cells or cells pretreated with the caspase inhibitory peptides 200 M zVAD-fmk and 400 M DEVD-CHO. (B) Caspase inhibitors protect from CsA-induced apoptosis. Protection from apoptosis is expressed as a percentage of apoptosis induced by CsA, which was considered to be 100%. Cells were incubated for 24 hours under serum-free conditions in the presence of inhibitors and 15 g/ml CsA and apoptosis was quantified by flow cytometry. Mean SD of three independent experiments. * p ≷ 0.005 versus CsA alone.

Full figure and legend (68K)

Figure 3.

CsA induces apoptosis of murine proximal tubular epithelial cells (MCT) in a time- and dose-dependent manner. (A) Time-response curve of apoptosis induced by 10 g/ml CsA in serum-deprived MCT cells. (B) Dose response curve of apoptosis induced by exposure of serum-deprived cells to CsA for 72 hours. Mean SD of three independent experiments. * p ≷ 0.005 versus CsA alone.

Full figure and legend (17K)

Peptide inhibitors of caspases protect MCT cells from CsA-induced apoptosis

Cells treated with 200 M zVAD-fmk were protected from CsA-induced apoptosis as assessed by morphology or DNA content Figures 1a and 2. DEVD-CHO was also protective, although it required higher concentrations Figures 1a and 2. Neither peptide significantly inhibited apoptosis induced by serum deprivation under the conditions of the study.

DISCUSSION

This paper reports that CsA induces apoptosis in a murine proximal tubular epithelial cell line and in primary cultures of murine tubular epithelial cells. CsA-induced apoptosis was already noted at concentrations similar to those found in the blood of patients treated with CsA (0.1 to 0.5 g/ml), and it increased with dose and time. The capacity of CsA to induce apoptosis of tubular cells may be related to its nephrotoxicity. Even small increases in the rate of apoptosis can result in a significant loss of cells in vivo. The half life of a cell displaying the apoptotic morphology has been calculated to be one to two hours. In the developing central nervous system the presence of <1% of apoptotic cells in tissue sections results in the loss of more than 50% of the cell mass after 72 hours¹⁴. Thus, small rates of apoptotic death of tubular cells may lead, after weeks or months, to the chronic tubular atrophy observed in animals and patients receiving long-term CsA therapy^1,2.

The pathways that lead to CsA-induced apoptosis are unclear. The present study provides evidence that caspases play a prominent role in CsA-induced apoptosis. zVAD-fmk protected from CsA apoptosis. This effect may point to a new target in the prevention and therapy of nephrotoxicity, as zVAD-fmk has been successfully used to prevent apoptosis in vivo¹⁵. ZVAD-fmk inhibits caspase-8 (MACH/FLICE/Mch5), an upstream effector of death receptor mediated apoptosis¹⁶. This may suggest a role for the autocrine activation of a death receptor system, such as the Fas ligand-Fas system, in CsA-induced apoptosis. We have indeed observed that CsA increases the expression of the Fas receptor in MCT cells (unpublished observation), and these cells do express the Fas ligand¹¹. Other drugs, such as chemotherapeutic agents, are known to induce apoptosis through the activation of the Fas ligand-Fas system¹⁷. The possibility that CsA-induced apoptosis in tubular cells is mediated through this pathway deserves further studies. There are other zVAD-fmk targets, such as caspase 9. Caspase 9 (Mch6/ICE-LAP6) is an upstream caspase that binds to Apaf-1 and Apaf-2 (cytochrome c), forming a complex that activates caspase 3 and other caspases¹⁸. The main target of the inhibitory peptide DEVD-CHO are caspase 3 (yama/apopain/CPP32) and caspase 3-like enzymes¹⁹. Caspase-3 is implicated in the final common pathway in several modes of apoptosis⁶ and is not inhibited by zVAD-fmk.

Another interesting finding of our study is that the rate of apoptosis induced by CsA varies with the microenvironment. CsA-induced cell death is decreased in the presence of a cytokine microenvironment that favors cell survival, that is, the presence of serum. The dependence of CsA-induced lethality on the microenvironment may have direct clinical consequences. Adverse conditions for cell survival may be encountered during rejection or ischemic insult to the kidney, in which the production of survival factors may be decreased²⁰. Our in vitro observations may be relevant to the observed impaired recovery from post-transplant acute renal failure in kidney graft recipients treated with CsA.

In conclusion, our data suggest that apoptosis may play a role in the loss of tubular cells in CsA nephrotoxicity, and that the cell microenvironment may be important in determining the extent of CsA-induced tubular cell damage. Several caspases, most probably caspases 8, 9 and 3, may be involved in CsA-induced tubular cell apoptosis and are potential targets for therapeutic intervention.

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Acknowledgments

This work was supported, in part, by grants from Sandoz Pharma (Barcelona, Spain), FISSS 94/0370, 98/0637, Ministerio de Educación y Ciencia (PB 94/0211 and PM 95/0093), Comision Interministerial de Ciencia y Tecnología (SAF 97/0071), and Fundación Renal Iñigo Alvarez de Toledo. C.L. was supported by Universidad Autónoma de Madrid and Ministerio de Educación y Ciencia, and M.P.C. was supported by Fundación Conchita Rábago de Jiménez Díaz.