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J Am Soc Nephrol 15:380-389, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

Paracetamol-Induced Renal Tubular Injury: A Role for ER Stress

Corina Lorz*, Pilar Justo*, Ana Sanz*, Dolores Subirá{dagger}, Jesús Egido* and Alberto Ortiz*

*Laboratory of Experimental Nephrology and Vascular Pathology, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Madrid, Spain; and {dagger}Unidad de Hematología, Fundación Jiménez Díaz, Madrid, Spain.

Correspondence to: Alberto Ortiz, Laboratorio de Nefrología Experimental y Patología Vascular, Fundación Jiménez Díaz, Av Reyes Católicos 2l, 28040 Madrid, Spain. Phone: 34-915504940; Fax: 34-915494764; E-mail: aortiz{at}fjd.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Paracetamol (also known as acetaminophen) causes acute and chronic renal failure. While the mechanisms leading to hepatic injury have been extensively studied, the molecular mechanisms of paracetamol-induced nephrotoxicity are poorly defined. Paracetamol induced cell death with features of apoptosis in murine proximal tubular epithelial cells. While paracetamol increased the expression of the death receptor Fas on the cell surface, the Fas pathway was not involved in the paracetamol-induced apoptosis of tubular cells. The mitochondrial pathway was not activated during paracetamol-induced apoptosis; there was no dissipation of mitochondrial potential or release of apoptogenic factors such as cytochrome c or Smac/DIABLO. However, paracetamol-induced apoptosis is a caspase-dependent process that involves activation of caspase-9 and caspase-3 in the absence of cytosolic cytochrome c or Smac/DIABLO. The authors also detected induction of endoplasmic reticulum (ER) stress, characterized by GADD153 upregulation and translocation to the nucleus, as well as caspase-12 cleavage. Interestingly, after treatment of murine tubular cells with paracetamol and calpain inhibitors, the caspase-12 cleavage product was still detectable, and calpain inhibitors were unable to protect tubular cells from paracetamol-induced apoptosis. The results suggest that induction of apoptosis may underlie the nephrotoxic potential of paracetamol and identify ER stress as a therapeutic target in nephrotoxicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Paracetamol, also known as acetaminophen, is widely used as an analgesic and antipyretic drug. An acute paracetamol overdose can lead to potentially lethal liver and kidney failure in humans and experimental animals (1–4) and in severe cases to death. Paracetamol is a phenacetin metabolite (5). Phenacetin was considered one of the most nephrotoxic analgesics and has now been withdrawn from the market in most countries (6). A chronic nephrotoxic effect of therapeutic dosing of paracetamol is suggested by case-control studies (7–9). These findings have led to the recommendation that paracetamol be used only in limited amounts and for limited time periods (10). Research into the biologic basis of paracetamol nephrotoxicity has been recently encouraged by a National Kidney Foundation Ad Hoc Committee (10).

Tubular cell loss is a characteristic feature of both acute renal failure and chronic renal disease (11) and is observed when cell death predominates over mitosis. Apoptosis is an active form of cell death that offers the opportunity for therapeutic intervention (11,12). Paracetamol has been shown to promote hepatocyte apoptosis (13–15). However, the mode of renal cell death during paracetamol nephrotoxicity and the mechanisms involved are obscure. Indeed, there is evidence that the molecular basis of nephrotoxicity may differ from those of hepatotoxicity, as N-acetyl-cysteine protects from the latter, but has been shown not to protect from nephrotoxicity (4).

Fas belongs to the tumor necrosis factor receptor family of proteins and plays a critical role in the normal development and homeostasis of T cells (16). However, inappropriate or excessive Fas-mediated apoptosis has been implicated in a number of pathologic conditions. In the context of hepatotoxicity, Fas expression is increased in the liver of animals treated with paracetamol. The severity of liver damage is reduced by oligonucleotide-mediated suppression of Fas expression, demonstrating a role for Fas in paracetamol toxicity in the liver (17). Lethal intracellular proteins include the caspases, a family of 14 proteases widely expressed in a variety of tissues and cell types, that play a central role in promoting apoptosis (18). Studies in human hepatic cells have shown that paracetamol-induced apoptosis is caspase-dependent and that mitochondria are a primary target (15).

Caspase-12 is specifically localized on the cytoplasmic side of the endoplasmic reticulum (ER) and connects ER stress to the caspase activation cascade (19). Because caspase-12 is expressed at high levels in the kidney and specifically in renal tubular epithelial cells, the cells affected during paracetamol nephrotoxicity, we examined the role of caspase-12 and ER stress in renal tubular epithelial cell death after paracetamol treatment.

While the mechanisms leading to hepatic injury have been extensively studied (14,15,17), there are virtually no data on the molecular mechanisms of paracetamol-induced nephrotoxicity. We have now investigated the ability of paracetamol to induce apoptosis of cultured mouse renal tubular epithelial cells and the participation of the death receptor Fas, ER stress, and caspases in the process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Cell Culture
MCT cells are a cultured line of proximal tubular epithelial cells harvested originally from the renal cortex of SJL mice (20). The cells were maintained in culture as described previously (20). Primary cultures of murine tubular epithelial cells were obtained as previously published from kidneys of balb/c mice (21). For experiments on the effect of paracetamol, cells were plated and then grown for 24 h in RPMI with 10% fetal calf serum (RPMI-10%). Then the medium was replaced with fresh serum-free RPMI (RPMI-0%), and the cells were grown with the indicated stimuli.

For the experiments with recombinant FasL or blocking FasL antibodies, cells in RPMI-0% medium were treated for 24 h with 100 ng/ml recombinant FasL (Alexis, Switzerland) or 10 µg/ml FasL blocking antibody (MFL3, Pharmingen, San Diego, CA) in the presence or absence of 300 µg/ml paracetamol. MCT cells readily undergo apoptosis when stimulated with this concentration of recombinant FasL following priming with TNF{alpha} + IFN{gamma} + LPS, a mixture that upregulates Fas expression (21). Paracetamol (Sigma, St Louis, MO) was dissolved in 100% ethanol (vehicle). Final concentration of ethanol in culture did not modulate cell death.

The pan-caspase inhibitory peptide benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) was obtained from Bachem (Bubendorf, Switzerland). The caspase-8 inhibitory peptide benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone (zIETD-fmk) and calpain inhibitors I and II were from Sigma. They were dissolved in DMSO. Final concentration of DMSO in culture was 0.1% maximum. This concentration did not modulate cell death (22). N-Acetyl-Cysteine (Sigma) was added to the cell culture 1 h before paracetamol. Staurosporine (STS, from Sigma) was used at a concentration of 100 nM. Tunicamycin (Sigma) was used at a concentration of 1 µg/ml.

Assessment of Apoptosis
Apoptosis was quantified by flow cytometry analysis of DNA content in permeabilized, propidium iodide-stained cells, as described previously (23). The percentage of cells with decreased DNA staining (A0) comprising apoptotic cells with fragmented nuclei was counted.

To assess for the pyknotic nuclear changes seen in apoptosis, cells were plated onto Labtek slides (Nunc Inc, Napersville, IL) in RPMI-10%. After 24 h, the medium was changed to RPMI-0% and then grown for an additional 48 h in the presence of paracetamol or vehicle. The cells were fixed in 10% buffered formalin and stained with propidium iodide as described previously (23).

To identify apoptosis versus necrosis, Annexin V-FITC/7-amino-actinomycin D (7-AAD) staining was performed using the Apotosis Detection Kit I (Pharmingen), and samples were analyzed by flow cytometry within 30 min.

For assessment of internucleosomal genomic DNA fragmentation, low–molecular weight DNA was separated in a 1.5% agarose gel. The DNA fragmentation ladder was demonstrated with ethidium bromide staining of the gel as described previously (23).

Cell Survival MTT Assay
The methylthiazoletetrazolium (MTT) assay relies on the conversion of MTT to colored formazan by succinate dehydrogenase in metabolically active cells and provides a measurement of cell viability. For viability experiments, 5000 MCT cells/well were placed into 96-well tissue culture plates; after 24 h, the medium was changed to RPMI-0% and cells were treated with paracetamol for defined periods of time. Cells were then washed and allowed to grow for 48 h in RPMI-10%. At the end of the experiment, cell viability was measured by MTT assay as described previously (24). Results are expressed as percent viability.

Western Blot
Western blot analyses were performed as described previously (21). Primary antibodies were: anti-Fas, anti-cytochrome c, and anti-GADD153 (all from Santa Cruz Biotechnology, Santa Cruz, CA); anti-caspase-3, anti-caspase-9, and anti-caspase-12 (all from Cell Signaling, Hertfordshire, UK). Antibodies were diluted in 5% milk PBS/Tween. The appropriate horseradish peroxidase-conjugated secondary antibody (1:2000; Amersham, Aylesbury, UK) was used. Blots were then probed with anti-tubulin antibody to detect differences in loading. Each experiment was performed at least three independent times.

Flow Cytometry Analysis of Fas Expression
For cytofluorography, cells were cultured in the presence of control medium or paracetamol. After washing the culture with PBS, adherent cells were detached with 2.2 mM EDTA, 0.2% BSA in PBS. Single cell (5 x 105) suspensions were incubated in PBS/BSA for 30 min at 4°C with 20 µg/ml of Jo-2 or 20 µg/ml of a control Ig. FITC anti-hamster IgG (dilution, 1:100) was used as a secondary antibody (all from Pharmingen). For analysis, dead cells and debris were excluded by selective gating on the basis of anterior and right angle scatter. At least 10,000 events were collected from each sample, and data were displayed on a logarithmic scale of increasing green fluorescence intensity. Mean cell fluorescence was calculated using LYSIS II software.

Examination of Mitochondrial Transmembrane Potential
Changes in mitochondrial transmembrane potential ({Delta}{Psi}m) were determined by staining the cells with JC-1 (Molecular Probes Europe BV) before flow cytometry analysis, as described previously (25). Data analyses were performed using Cell Quest software by measuring both the green (530 ± 15 nm) and red (585 ± 21 nm) JC-1 fluorescence. The loss in {Delta}{Psi}m is seen as a shift to lower JC-1 red fluorescence accompanied by an increase in JC-1 green fluorescence. At least 10,000 events were collected per sample. The proton translocator carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (CCCP 150 µM) was used as positive controls because it disrupts the mitochondrial electrochemical gradient.

Assay of Cytochrome c and Smac/Diablo Release from Mitochondria
Release of cytochrome c from mitochondria to cytosol was measured by Western blot analysis. Cells (5 x 106) were harvested, washed once with ice-cold PBS, and gently lysed for 6 min in ice with 100 µl of lysis buffer (250 mM sucrose, 80 mM KCl, 500 µg/ml digitonin, 1 mM DTT, 0.1 mM PMSF, protease inhibitors, in PBS). Lysates were centrifuged at 12, 000 x g at 4°C for 5 min to obtain the supernatants (cytosolic extract free of mitochondria) and the pellets (fraction that contains the mitochondria). Supernatants (50 µg) and pellets (50 µg) were electrophoresed on 15% polyacrylamide gels and then analyzed by Western blot as described above. Cytochrome c antibody clone 7H8.2C12 was from Pharmingen, and mouse specific anti-Smac/DIABLO clone 9H10 form Kamiya Biomedical (Seattle, WA). The mitochondrial enzyme cytochrome oxidase subunit IV (Molecular Probes, Leiden, The Netherlands) is not released from mitochondria during apoptosis and was used as control for the technique.

Cytochrome c and GADD153 Immunostaining
For Cytochrome c and GADD153 immunostaining, cells were plated onto Labtek slides in RPMI-10%. After 24 h, the medium was changed to RPMI-0% and cells were incubated from 1 to 24 h with the indicated stimuli. Then cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-10 in PBS for 10 min each. After washing in PBS, cells were incubated overnight at 4°C with anti-cytochrome c (clone 6H2.B4, Pharmingen) or anti-GADD153 followed by 1 h incubation with a FITC-labeled anti-IgG. Cell nuclei were counterstained with DAPI or propidium iodide.

Caspase-3 Activity Assay
Caspase-3 activity was measured following the manufacturer’s instructions (Biomol, Plymouth, PA). In brief, cell extracts (30 µg of protein) were incubated in half-area 96-well plates for 3 h at 37°C with 200 µM Asp-Glu-Val-Asp-pNA (DEVD-pNA) peptide in a total volume of 50 µl. The color of free pNA, generated as a result of cleavage of the aspartate-pNA bond, was monitored continuously over 3 h by measuring absorbance at 405 nm in a spectrophotometer plate reader. The emission from each well was plotted against time, and linear regression analysis of the initial velocity (Vmax) for each curve yielded the activity. As a control, caspase-3 activity in the samples was inhibited with Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO).

Statistical Analyses
Results are expressed as mean ± SD. Significance at the 95% level was established using one-way ANOVA and t test. The presence of significant differences between groups was examined by a post hoc test (Bonferroni method) by means of the SigmaStat statistical software (Jandel, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Paracetamol Induces Apoptosis in Primary Cultures of Renal Tubular Epithelial Cells and in the Tubular Epithelial Cell Line MCT
Cell death of tubular epithelium caused by paracetamol has features of apoptosis. These include characteristic nuclear morphology (Figure 1A) and internucleosomal DNA degradation (Figure 1B). Significantly, MCT cells treated with paracetamol were positive for annexin-V but did not show uptake of the vital dye 7-AAD (Figure 1C). This indicates that, at the concentration used in our experiments, paracetamol induced apoptosis and not necrosis of tubular epithelial cells. In addition, decreased DNA content was present in paracetamol-treated murine tubular epithelial MCT cells and in primary cultures of murine tubular epithelial cells (Figure 1D).



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  		Figure 1. Paracetamol-induced tubular cell death has features of apoptosis. (A) Characteristic shrunk, pyknotic-fragmented nuclei (arrows) are present among fixed, propidium iodide-stained, paracetamol-treated tubular epithelial MCT cells, but not among control cells. (B) Internucleosomal DNA degradation in MCT cells treated with increasing concentrations of paracetamol for 24 h. (C) Nonpermeabilized MCT cells treated with paracetamol are positive for annexin-V, but they do not show 7-AAD staining. (D) Presence of apoptotic, hypodiploid (A0) cells among MCT cells and primary cultures of murine tubular epithelial cells as shown by flow cytometry. Except otherwise stated, cells were treated under serum-free conditions with 300 µg/ml paracetamol for 24 h.

 
Paracetamol-induced apoptosis increased with time (Figure 2A) and dose (Figure 2B). Paracetamol plasma concentrations of 10 to 20 µg/ml are associated with antipyretic activity (26). Although the therapeutic analgesic plasma concentration for paracetamol is not well defined, new protocols are using higher concentrations, which result in mean paracetamol plasma levels of 30 µg/ml (27). An increased rate of apoptosis was observed with as little as 30 µg/ml paracetamol (P < 0.05 versus control), a concentration that can be reached during therapeutic dosing (27). For further experiments we chose a concentration of 300 µg/ml of paracetamol, which induces about a 50% of the cells to undergo apoptosis in 24 h (Figure 2A). This concentration of paracetamol can be reached during clinical paracetamol toxicity (28).



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  		Figure 2. Paracetamol-induced apoptosis is time-dependent and concentration-dependent. Cell death was assessed by flow cytometry after culture in the presence of 300 µg/ml paracetamol for different time periods (A) and in cells cultured in the presence of different concentrations of paracetamol for 72 h (B) under serum-free conditions. *P < 0.05 versus control. (C) After 8 h of paracetamol treatment, 50% of the cells are committed to die. MCT cells were cultured for the indicated times with 300 µg/ml of paracetamol; they were then allowed to recover for 48 h in 10% FCS medium. Cell viability was measured by MTT assay. Results are expressed as percent viability.

 
Cell survival studies showed that at 8 h of paracetamol incubation 50% of the cells were committed to die (Figure 2C). Even though, at this time point the rate of apoptosis was NS in the cell cultures compared with control cells (Figure 2A).

In vivo treatment of paracetamol toxicity with N-acetylcysteine can prevent hepatic damage in most cases (29,30). Nevertheless, relatively high doses of N-acetylcysteine (10 mM) failed to prevent apoptosis of MCT cells induced by 300 µg/ml paracetamol at 24 h (Paracetamol 56 ± 2%, N-acetylcysteine + paracetamol 57 ± 2% apoptotic cells, NS).

The Fas Pathway Is Not Involved in Paracetamol-Induced Apoptosis in Tubular Cells
A direct role for Fas in paracetamol toxicity has been previously shown in the liver (17). Figure 3A shows that paracetamol increased Fas protein expression in MCT cells and that the receptor was expressed on the cell surface (Figure 3B). These results raised the possibility that, as in the liver, in the tubular epithelium Fas is involved in the apoptotic process induced by paracetamol. To test this possibility, we cultured MCT cells with recombinant FasL or a FasL blocking antibody, in the presence of paracetamol (Figure 3C). The addition of recombinant FasL did not increase the amount of paracetamol-induced cell death. Likewise, blocking FasL with an anti-FasL blocking antibody did not protect against apoptosis caused by paracetamol. Furthermore, the caspase-8 inhibitor, zIETD-fmk, was unable to protect tubular cells from paracetamol-induced apoptosis (Figure 3D), and no caspase-8 activity was detected on activity assays (data not shown). Together, these results indicate that, contrary to the findings in liver, paracetamol does not induce apoptosis of renal tubular epithelial cells through the Fas pathway.



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  		Figure 3. Paracetamol-induced apoptosis of tubular cells does not involve the Fas pathway. (A) Western blot studies revealed that Fas expression increases in MCT cells treated with increasing doses of paracetamol for 24 h. (B) Fas is expressed in the surface of cells treated with paracetamol (24 h, 300 µg/ml) compared with untreated cells. Flow cytometry of nonpermeabilized cells. Control IgG–stained cells display a peak that overlies that of the untreated cells. (C) Incubation of paracetamol-treated cells with recombinant FasL did not increase drug-induced cell death. Also paracetamol-induced apoptosis of MCT cells was not prevented in the presence of a FasL blocking antibody. (D) MCT cells were treated for 2 h with the caspase-8 inhibitor zIETD-fmk (200 µM) before paracetamol incubation. Cell death was assessed by flow cytometry after 24 h.

 
Paracetamol Does Not Induce Cytochrome c Release or Changes in the Mitochondrial Transmembrane Potential of Tubular Epithelial Cells
Mitochondrial changes that lead to apoptosis include release of caspase-activating proteins and/or loss of mitochondrial transmembrane potential ({Delta}{Psi}m). We investigated whether paracetamol treatment of renal tubular epithelial cells induced changes in the mitochondria. While the proton translocator CCCP caused a significant membrane depolarization, no loss of {Delta}{Psi}m was detected in the tubular epithelial cells treated with paracetamol (Figure 4). Cytochrome c release from the mitochondria was studied by Western blot and by immunofluorescence (Figures 5 and 6Go). We did not detect any cytochrome c release from the mitochondria of MCT cells treated with paracetamol, even at time points when around 50% of the cells were undergoing apoptosis and caspases had already been activated (Figure 7). Cytochrome c release per se, however, is functional in MCT cells, because treatment with staurosporine (an inducer of the mitochondrial pathway) induced apoptosis at a similar level of lethality than paracetamol with a significant release of cytochrome c. Similar findings were observed with the cellular distribution of Smac/DIABLO. Taken together, these results suggest that in MCT cells treated with paracetamol mitochondria do not suffer evident alterations.



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  		Figure 4. Changes in mitochondrial transmembrane potential ({Delta}{Psi}m) were analyzed by JC-1 staining. The loss in {Delta}{Psi}m is seen as a shift to lower JC-1 red fluorescence. Results show the percentage of cells with reduced {Delta}{Psi}m. MCT cells were treated with 300 µg/ml paracetamol for 24 h. As a positive control, cells were treated for 4 h with 150 µM CCCP.

 


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  		Figure 5. Mitochondria do not release cytochrome c during paracetamol treatment. Western blot analyses of cytochrome c and Smac/DIABLO in cytosolic and mitochondrial extracts of MCT cells treated for different time with 300 µg/ml paracetamol or 100 nM staurosporine (STS). Cytochrome oxidase subunit IV and {alpha} Tubulin are controls for fraction separation and loading.

 


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  		Figure 6. Cytochrome c immunostaining and the corresponding DAPI staining of MCT cells treated with: (A) vehicle 6 h; (B) paracetamol 6 h; (C) STS 6 h; (D) paracetamol 24 h. Cytochrome c labeling appears punctuate in control cells, where it is localized at the mitochondria. When cytochrome c is released from the mitochondria, the labeling becomes diffuse (arrow in C). We could not detect cytochrome c release, even in late paracetamol treated apoptotic cells (arrowheads in D).

 


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  		Figure 7. Paracetamol induces caspase-3 and caspase-9 activation in tubular cells. (A) Western blot analyses of the processing of caspase-3 and caspase-9 during paracetamol-induced apoptosis. The migration position of the caspase-9 cleavage products is indicated. (B) Caspase-3–like activity of extracts of tubular cells treated with paracetamol (300 µg/ml) or STS (100 nM) for the indicated times. Ac-DEVD-CHO was used to inhibit caspase-3 like activity.

 
Paracetamol-Induced Cell Death Is Caspase-Dependent
Paracetamol induced processing of caspase-3 starting at 6 h, as shown by the appearance of caspase-3 cleavage product in Western blots (Figure 7A) and by the presence of caspase-3–like activity in cell extracts treated with paracetamol (Figure 7B), with maximal activity detected at 6 to 8 h. Caspase-3 is mainly activated by caspase-8 or caspase-9. Caspase-8 does not play a role in paracetamol-induced cell death; we therefore studied caspase-9 cleavage by Western blot. Paracetamol induced caspase-9 processing starting at 6 h (Figure 7A). Two bands of 39 kD and 37 kD, corresponding to the cleaved fragments of caspase-9, were observed on Western blots. Tunicamycin, an inducer of ER stress, also induced cleavage of caspase-3 and caspase-9 in MCT cells. zVAD-fmk is a irreversible, broad-spectrum inhibitor of caspases (31). zVAD-fmk afforded complete protection against nuclear features of apoptosis induced by paracetamol (Figure 8) and inhibited paracetamol-induced caspase-3 activity at the concentrations used.



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  		Figure 8. Effect of the pan-caspase inhibitor zVAD-fmk on apoptosis induced by 300 µg/ml paracetamol for 24 h. (A) Apoptosis quantified as hypodiploid cells in permeabilized, propidium iodide–stained cells. For the expression of specific protection, apoptosis in the presence of paracetamol alone was considered to be 100%, and apoptosis in the presence of the caspase inhibitor and paracetamol was expressed as a percentage of this. (B) Internucleosomal DNA degradation in MCT cells treated with 300 µg/ml paracetamol in the presence or absence of 200 µM zVAD-fmk for 24 h.

 
Paracetamol Treatment Induces ER Stress and Caspase-12 Cleavage in Tubular Epithelial Cells
To determine whether paracetamol induced ER stress in murine tubular cells, we studied the expression and localization of GADD153, a transcription factor that is induced during cell death triggered by ER stress (32). GADD153 expression was upregulated in tubular cells treated with paracetamol similarly to cells treated with the ER stress-inducer tunicamycin (Figure 9A). Consistent with its role as a transcription factor, we detected translocation of GADD153 from the cytosol to the nucleus in cells treated with paracetamol (Figure 9B).



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  		Figure 9. Paracetamol induces ER stress and caspase-12 cleavage in tubular cells. (A) Western blot analyses of the expression of GADD153 and caspase-12 cleavage during paracetamol-induced apoptosis. (B) Confocal images of GADD153 (green) immunostaining of MCT cells treated with 300 µg/ml paracetamol for 24 h. In red nuclei stained with propidium iodide. (C) Western blot analyses of caspase-12 cleavage during paracetamol-induced apoptosis in the presence of caspase inhibitor zVAD-fmk or calpain inhibitor I. Similar results were obtained with calpain inhibitor II. (D) Densitometric analyses of at least three independent experiments for the Western blots shown on panels A and C. *P < 0.005 versus control.

 
We detected caspase-12 cleavage in MCT cells treated with paracetamol starting at 6 h (Figure 9A). Caspase-12 cleavage product was not present in cells treated with the caspase inhibitor zVAD-fmk before paracetamol incubation (Figure 9C). Calpains are a family of cysteine proteases that are activated by elevated intracellular calcium. They have been involved in the activation of caspase-12 upon disturbance of intracellular calcium homeostasis (33). Nevertheless, caspase-12 cleavage product was still detectable in tubular cells that had been treated with paracetamol and calpain inhibitors (Figure 9C). Calpain inhibitors I and II did not protect tubular cells from paracetamol-induced apoptosis (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Paracetamol overdose causes acute renal failure, and chronic exposure to paracetamol has been linked to chronic renal failure (9). While the mechanisms of paracetamol-induced hepatotoxicity have been extensively studied (14,15,17,34–37), information about the specific molecular pathways that lead to apoptosis of tubular cells during nephrotoxic injury is incomplete (11). The mechanisms involved in paracetamol-induced apoptosis in nephrotoxicity may differ to those during hepatotoxicity, as suggested by the fact that N-acetylcysteine can prevent in vivo paracetamol hepatic damage (29,30) but did not prevent apoptosis of tubular cells. Because tubular cell death is a feature of both acute and chronic renal failure, we examined the ability of paracetamol to induce cell death in murine proximal tubular cells and the intracellular mechanisms involved.

Paracetamol induced a mild degree of tubular cell apoptosis, even at concentrations found during therapeutic dosing. These findings are consistent with the chronic long-term toxicity of the drug (7–9). To explore the molecular mechanisms of paracetamol-induced apoptosis, we examined the effect of a concentration of paracetamol that is reached in humans during acute paracetamol toxicity (28). Upon treatment with paracetamol, primary cultures of murine tubular epithelial cells and the murine proximal tubular cell line MCT showed morphologic changes associated with apoptosis, such as chromatin condensation and internucleosomal DNA fragmentation. Moreover, the loss of membrane asymmetry observed after paracetamol treatment as detected by annexin-V staining without loss of membrane integrity suggests that apoptosis is the primary mode of cell death in tubular cells treated with paracetamol.

A variety of cytototoxins such as chemotherapeutic agents (38), toxic bile salts (39), and paracetamol (17) may induce apoptosis by upregulating the death receptor Fas expression. This prompted the study of the Fas pathway during paracetamol nephrotoxicity. We found that Fas expression was increased in tubular cells upon paracetamol treatment. The Fas receptor could theoretically be activated by autocrine FasL, as tubular epithelium constitutively expresses FasL (21). Nevertheless, neither Fas receptor activation by recombinant FasL nor FasL neutralization significantly modified the rate of cell death induced by paracetamol treatment alone. Together with the fact that we did not detect caspase-8 activation in treated cells, and caspase-8 inhibitors were unable to protect from paracetamol-induced apoptosis, we can conclude that, contrary to paracetamol hepatotoxicity, the Fas receptor pathway is not involved in paracetamol-induced cell death in murine proximal tubular cells.

Numerous pro-apoptotic signal transduction and damage pathways converge on the mitochondria to induce dissipation of mitochondrial membrane potential and release of proteins that are normally strictly confined to the mitochondrial intermembrane space, such as cytochrome c and Smac/DIABLO. Cytochrome c stimulates the cytosolic assembly of the apoptosome by binding to Apaf-1. This leads to caspase-9 oligomerization and activation and to caspase-3 cleavage. Cytochrome c release from mitochondria followed by caspase-9 and caspase-3 cleavage has been reported during paracetamol toxicity in human hepatic cells (15). Paracetamol treatment of renal tubular epithelial cells, at the concentrations used in our studies, did not induce loss of mitochondrial transmembrane potential or release of the proapoptotic factors cytochrome c and Smac/DIABLO into the cytosol. Nevertheless, paracetamol-induced apoptosis is a caspase-dependent process, as shown by the fact that zVAD-fmk protects against features of apoptosis induced by paracetamol. Indeed, paracetamol treatment leads to activation of caspase-9 and caspase-3 in renal tubular epithelial cells.

Paracetamol hepatotoxicity is a process characterized by calcium deregulation (13,34–37). Recently, the endoplasmic reticulum has been shown to participate in the initiation of apoptosis in response to calcium signaling (19). There is increasing evidence to suggest that the ER stress apoptotic pathway is important in the kidney, specifically in tubular epithelial cells. Tunicamycin has been reported to induce ER stress-mediated apoptosis in renal proximal tubules in mice, followed by the development of a histologic picture similar to the human condition known as acute tubular necrosis (32). We found that the expression of GADD153, a marker of ER stress, is increased in tubular epithelial cells during paracetamol-induced apoptosis. GADD153 is a transcription factor that promotes apoptosis (40). Consistent with its role as a transcription factor, we detected GADD153 translocation to the nucleus in tubular cells treated with paracetamol, both in cell culture and in the whole animal (unpublished observation).

Caspase-12 is ubiquitously expressed in mouse tissues, and it is expressed at high levels in kidney, specifically in renal proximal tubular epithelial cells, but not in the glomerulus. Caspase-12 is activated by ER stress, but apparently not by death receptor-mediated or mitochondria-targeted apoptotic signals (19). We detected caspase-12 cleavage in tubular cells treated with paracetamol; this cleavage was prevented by zVAD-fmk. Caspase-12 has been reported to be cleaved by calpains (33), a family of cysteine proteases that are activated by elevated intracellular calcium. Nevertheless, after treatment of murine tubular cells with paracetamol and calpain inhibitors, the caspase-12 cleavage product was still detectable, and calpain inhibitors were unable to protect tubular cells from paracetamol-induced apoptosis.

The present study shows that paracetamol induces apoptosis of cultured murine tubular epithelial cells through a caspase-mediated mechanism that involves caspase-9 and caspase-3 in a cytochrome c and Smac/DIABLO–independent manner. Caspase-12 has been reported to cleave caspase-9 in vitro in the absence of cytochrome c (41); this raises the possibility that caspase-12 is the apical caspase in paracetamol-induced apoptosis in tubular epithelial cells. Nevertheless, we cannot exclude the possibility that other factors (released or not from the mitochondria) are responsible for paracetamol-induced caspase-9 activation. Paracetamol causes ER stress in tubular cells, leading to GADD153 upregulation and translocation to the nucleus, as well as caspase-12 cleavage. Our results suggest that induction of apoptosis may underlie the nephrotoxic potential of paracetamol and identify ER stress as a therapeutic target in nephrotoxicity.


   Acknowledgments
 
This work was supported by grants FISSS 01/0199, Comunidad de Madrid (08.2/0030/2000), Sociedad Española de Nefrología, Instituto Reina Sofia de Investigaciones Nefrológicas, and EU project QLG1-CT-2002–01215. PJ was supported by Fondo de Investigaciones Sanitarias. AS was supported by Conchita Rábago de Fundación Jiménez Díaz. CL was supported by Ministerio de Educación, Ciencia y Deporte.


   Footnotes
 
Corina Lorz’s present affiliation: Weston Laboratory, IRDB, Department of PO&G, Imperial College of Sciences, Technology and Medicine, Hammersmith Campus, London, UK.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication July 25, 2003. Accepted for publication November 16, 2003.




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