J Am Soc Nephrol 15: 380389, 2004

Paracetamol-Induced Renal Tubular Injury: A Role for ER

Stress
,
CORINA LORZ,* PILAR JUSTO,* ANA SANZ,* DOLORES SUBIRA
S EGIDO,* and ALBERTO ORTIZ*
JESU
*Laboratory of Experimental Nephrology and Vascular Pathology, Fundacin Jimnez Daz, Universidad
Autnoma de Madrid, Madrid, Spain; and Unidad de Hematologa, Fundacin Jimnez Daz, Madrid, Spain.

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.

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now been withdrawn from the market in most countries (6).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 (79). 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 (1315). 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 paracetamolinduced 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

Corina Lorzs present affiliation: Weston Laboratory, IRDB, Department of

PO&G, Imperial College of Sciences, Technology and Medicine, Hammersmith Campus, London, UK.
Received July 25, 2003. Accepted November 16, 2003.
Correspondence to: Alberto Ortiz, Laboratorio de Nefrologa Experimental y
Patologa Vascular, Fundacin Jimnez Daz, Av Reyes Catlicos 2l, 28040
Madrid, Spain. Phone: 34-915504940; Fax: 34-915494764; E-mail:
aortiz@fjd.es
1046-6673/1502-0380
Journal of the American Society of Nephrology
Copyright 2004 by the American Society of Nephrology
DOI: 10.1097/01.ASN.0000111289.91206.B0

J Am Soc Nephrol 15: 380389, 2004

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

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 g/ml FasL
blocking antibody (MFL3, Pharmingen, San Diego, CA) in the presence or absence of 300 g/ml paracetamol. MCT cells readily undergo apoptosis when stimulated with this concentration of recombinant FasL following priming with TNF IFN 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-AlaDL-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 g/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 RPMI10%. 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-aminoactinomycin 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,
lowmolecular weight DNA was separated in a 1.5% agarose gel. The

Paracetamol: A Role for ER Stress

381

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 antiGADD153 (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 (5 105) suspensions were incubated in PBS/BSA for 30 min at
4C with 20 g/ml of Jo-2 or 20 g/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 (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 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 M) 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 (5 106) were harvested,
washed once with ice-cold PBS, and gently lysed for 6 min in ice with
100 l of lysis buffer (250 mM sucrose, 80 mM KCl, 500 g/ml

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digitonin, 1 mM DTT, 0.1 mM PMSF, protease inhibitors, in PBS).

Lysates were centrifuged at 12, 000 g at 4C for 5 min to obtain the
supernatants (cytosolic extract free of mitochondria) and the pellets
(fraction that contains the mitochondria). Supernatants (50 g) and
pellets (50 g) 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 4C with anticytochrome 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 manufacturers
instructions (Biomol, Plymouth, PA). In brief, cell extracts (30 g of
protein) were incubated in half-area 96-well plates for 3 h at 37C
with 200 M Asp-Glu-Val-Asp-pNA (DEVD-pNA) peptide in a total
volume of 50 l. 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
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).

J Am Soc Nephrol 15: 380389, 2004

Paracetamol-induced apoptosis increased with time (Figure

2A) and dose (Figure 2B). Paracetamol plasma concentrations
of 10 to 20 g/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 g/ml (27). An increased rate of apoptosis was
observed with as little as 30 g/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 g/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).
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 g/ml
paracetamol at 24 h (Paracetamol 56 2%, N-acetylcysteine
paracetamol 57 2% apoptotic cells, NS).

The Fas Pathway Is Not Involved in ParacetamolInduced 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 antiFasL 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.

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 (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 m was detected in the tubular epithelial cells treated with

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383

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 g/ml paracetamol for 24 h.

paracetamol (Figure 4). Cytochrome c release from the mitochondria was studied by Western blot and by immunofluorescence (Figures 5 and 6). 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.

Paracetamol-Induced Cell Death Is CaspaseDependent

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

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J Am Soc Nephrol 15: 380389, 2004

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).
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

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 g/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 g/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.

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.

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

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 paracetamolinduced 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 (79). 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

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385

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 g/ml) compared with untreated cells. Flow cytometry of nonpermeabilized cells. Control IgGstained 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 M) before paracetamol incubation. Cell death was assessed by
flow cytometry after 24 h.

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

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

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J Am Soc Nephrol 15: 380389, 2004

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 g/ml paracetamol or 100 nM staurosporine (STS). Cytochrome oxidase subunit IV and Tubulin are controls
for fraction separation and loading.

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) Caspase3like activity of extracts of tubular cells treated with paracetamol
(300 g/ml) or STS (100 nM) for the indicated times. Ac-DEVDCHO was used to inhibit caspase-3 like activity.

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).

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 stressmediated 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

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Figure 8. Effect of the pan-caspase inhibitor zVAD-fmk on apoptosis

induced by 300 g/ml paracetamol for 24 h. (A) Apoptosis quantified
as hypodiploid cells in permeabilized, propidium iodidestained 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 g/ml paracetamol in the presence or absence
of 200 M zVAD-fmk for 24 h.

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 caspasemediated mechanism that involves caspase-9 and caspase-3 in

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 g/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.

388

Journal of the American Society of Nephrology

a cytochrome c and Smac/DIABLOindependent 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 Espaola de Nefrologa, Instituto
Reina Sofia de Investigaciones Nefrolgicas, and EU project QLG1CT-2002 01215. PJ was supported by Fondo de Investigaciones
Sanitarias. AS was supported by Conchita Rbago de Fundacin
Jimnez Daz. CL was supported by Ministerio de Educacio n, Ciencia
y Deporte.

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