Autophagy

ISSN: 1554-8627 (Print) 1554-8635 (Online) Journal homepage: http://www.tandfonline.com/loi/kaup20

Autophagy protects renal tubular cells against

cyclosporine toxicity

Nicolas Pallet, Nicolas Bouvier, Christophe Legendre, Jerome Gilleron,

Patrice Codogno, Philippe Beaune, Eric Thervet & Dany Anglicheau

To cite this article: Nicolas Pallet, Nicolas Bouvier, Christophe Legendre, Jerome Gilleron,
Patrice Codogno, Philippe Beaune, Eric Thervet & Dany Anglicheau (2008) Autophagy protects
renal tubular cells against cyclosporine toxicity, Autophagy, 4:6, 783-791, DOI: 10.4161/
auto.6477

To link to this article: http://dx.doi.org/10.4161/auto.6477

Published online: 08 Jul 2008.

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 [Autophagy 4:6, 783-791; 16 August 2008]; 2008 Landes Bioscience
Research Paper
Autophagy protects renal tubular cells against cyclosporine toxicity
Nicolas Pallet,1,* Nicolas Bouvier,1 Christophe Legendre,2 Jerome Gilleron,3 Patrice Codogno,4 Philippe Beaune,1 Eric
Thervet1,2 and Dany Anglicheau1,2
1INSERM U775; Centre Universitaire des Saints-Pres; Universit Paris Descartes; Paris, France; 2Service de Transplantation Rnale; Hpital Necker; APHP; Paris, France;
Universit Paris Descartes; Paris, France; 3INSERM U895; Centre Universitaire des Saints-Pres; Universit Paris Descartes; Paris, France; 4INSERM U756; Facult de Pharmacie;
Universit Paris-Sud; Chtenay-Malabry, France
Key words: autophagy, endoplasmic reticulum stress, cyclosporine, nephrotoxicity, tubular cells
A major side effect of the powerful immunosuppressive drug
cyclosporine (CsA) is the development of a chronic nephrotox-
icity whose mechanisms are not fully understood. Recent data
suggest that tubular cells play a central role in the pathogenesis of
chronic nephropathies. We have shown that CsA is responsible for
endoplasmic reticulum (ER) stress in tubular cells. Autophagy has
recently been described to be induced by ER stress and to alleviate
its deleterious effects. In this study, we demonstrate that CsA
induces autophagy in primary cultured human renal tubular cells
through LC3II expression and autophagosomes visualization by
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electron microscopy. Autophagy is dependant on ER stress because
various ER stress inducers activate autophagy, and salubrinal, an
inhibitor of eIF2 dephosphorylation that protects cells against
en
ER stress, inhibited LC3II expression. Furthermore, autophagy
inhibition during CsA treatment with beclin1 siRNA significantly
ci
increases tubular cell death. Finally, immunohistochemical analysis
of rat kidneys demonstrates a positive LC3 staining on injured
s
tubular cells, suggesting that CsA induces autophagy in vivo. Taken
together, these results demonstrate that CsA, through ER stress
io
induction, activates autophagy as a protection against cell death.
B
Introduction
As a highly potent immunosuppressive drug, cyclosporine (CsA)
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remains largely used for the prevention of acute rejection in solid
organ transplantation, and for the treatment of various auto-
nd
immune diseases. However, CsA can lead to a chronic form of renal
damage characterized by a progressive and irreversible deterioration
of renal function associated with interstitial fibrosis, tubular atrophy,
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arteriolar hyalinosis and glomerulosclerosis.1-3 The pathogenesis
of CsA chronic nephrotoxicity is still not fully understood, and
many mechanisms have been proposed. In this issue, the effects
08
of CsA on tubular epithelial cells are of special interest because of
the central role of these cells in the initiation and development of
chronic nephropathies via a variety of mechanisms.4-7 It is of the
20
*Correspondence to: Nicolas Pallet; Unit INSERM U775; Centre Universitaire des
Saints Pres; 45, rue des saints Pres; Paris 75006 France; Tel.: +33142862251;
Fax: +33142862072; Email: nicolas.pallet@univ-paris5.fr
Submitted: 02/12/07; Revised: 06/10/08; Accepted: 06/20/08
Previously published online as an Autophagy E-publication:
http://www.landesbioscience.com/journals/autophagy/article/6477
www.landesbioscience.com
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utmost importance to better understand the role of tubular cells in
the development of interstitial fibrosis in order to develop specific
t
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biomarkers of early graft damage but also to find new therapeutic
targets. A previous study led us to demonstrate that CsA induces
an endoplasmic reticulum stress (ER stress) in tubular cells.8 Cells
that are subject to ER stress activate the unfolded protein response
o
(UPR), a protective mechanism that reduces protein load and
.D
increases nascent protein folding, contributing to cell protection.
However, persistent ER stress may lead to cell death through ill-
defined pathways.9-11
Recent evidence suggests that ER stress drives autophagy.12-14
Autophagy is responsible for the degradation of long-lived proteins
and subcellular organelles and has been shown to be involved in
many physiological or pathological processes. This self-digestion not
only provides nutrients to maintain vital cellular functions during
fasting but also can rid the cell of superfluous or damaged organ-
elles, misfolded proteins and invading microorganisms. Importantly,
autophagy is recognized as a protective mechanism against various
cellular stresses including nutrient deprivation, hypoxia and growth
factor deprivation. Although autophagy is sometimes found to be
associated with non-apoptotic cell death, under most circumstances,
autophagy constitutes a stress adaptation pathway that promotes cell
survival.15-17 Particularly, under ER stress conditions, autophagy has
been shown to alleviate ER stress and to reduce cell death.12
In the current study, we made the assumption that ER stress
induced by CsA could serve as a cellular regulator of autophagy.
We demonstrate that ER stress activates autophagy in response to
CsA exposure. Autophagy inhibition with beclin1 siRNA increases
CsA-induced tubular cell cytotoxicity, suggesting that autophagy
serves as a protective mechanism against CsA toxicity. Finally, we
show in a rat model of CsA nephrotoxicity that injured tubular cells
display intense LC3 staining suggesting that autophagy occurs in
vivo and can be detected.
Results
CsA induces ER stress in human tubular cells. To confirm that
CsA induces ER stress in human renal epithelial cells (HRECs), we
evaluated the expression of glucose-related protein 78 (GRP78 [also
known as BIP]), CHOP (C/EBP Homologous Protein), and HERP
(homo sapiens homocysteine-inducible, endoplasmic reticulum
stress-inducible) transcripts, whose expression characterizes the
Autophagy 783
 Autophagy protects against ER stress induced by CsA

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Figure 1. Cyclosporine induces endoplasmic reticulum stress. (A) Real time RT-PCR analysis of the expression of GRP78, HERP and CHOP mRNAs in human
tubular cells after 24 hours of exposure to 6, 8, 10 M cyclosporine or 0.25 M thapsigargin, an ER stress inducer used as positive control. *p < 0.05 versus
20

vehicle-treated cells, n = 3. Fold changes for each tested gene were normalized to the housekeeping gene Ribosomal Protein L13A. (B) Representative west-
ern blot analysis of GRP78 expression in rat kidneys after exposure to 15 mg/kg/day cyclosporine during 28 days (left) and densitometric analysis (right).
*p < 0.05, n = 5. (C) Representative western blot of LC3II expression in human tubular cells after exposure to 0.25 M thapsigargin during 48 hours.

UPR. mRNA isolated from vehicle-treated and CsA-treated HRECs that CsA induces an UPR in cultured HRECs. CsA treatment also
were used to perform RT-PCR. As shown in Figure 1A, transcript increased the transcription of other UPR markers such as GADD34
expression levels were significantly increased by CsA and 0.25 M (Growth Arrest DNA Damage Inducible Protein 34) and GRP94
thapsigargin, a potent ER stress inducer, confirming the hypothesis (Glucose related protein 94) (data not shown). Confirming the

784 Autophagy 2008; Vol. 4 Issue 6

 Autophagy protects against ER stress induced by CsA
specific involvement of the UPR, other chaperone transcripts unre-
lated to the UPR (Hsp-10, Hsp-27 and Hsp-40) were not altered
by CsA at working concentration (data not shown). We also tested
whether CsA could induce ER stress in vivo and showed that GRP78
expression was significantly increased at the protein level in kidneys
of CsA-treated rats (Fig. 1B). Because ER stress has been linked
to autophagy,12 we evaluated whether thapsigargin could induce
autophagy in vitro using LC3 conversion assay. Two forms of LC3
exist, a cytosolic one, LC3I, and another one, LC3II, which is conju-
gated with phosphatidylethanolamine when autophagy is active, and
which is present only on autophagosomes. LC3 immunoblotting is
the preferred marker of autophagy and is closely correlated to the
number of autophagosomes.18,19 We showed that LC3II expression
was strongly induced by thapsigargin, suggesting that ER stress could
induce autophagy in HRECs in vitro (Fig. 1C).
CsA induces autophagy. To test whether CsA induces autophagy,
LC3 lipidation was evaluated using immunoblot with anti-LC3
antibody. Figure 2A shows that 8 M CsA highly increases the
expression of LC3II expression in HRECs, and this increase is
reversed by the addition of 5 mM 3-methyl adenine (3-MA), an
inhibitor of autophagosome formation, confirming the autopha-
gosome specificity of LC3II in our model (Fig. 2B). Since LC3II
increase can be induced by lysosomal degradation inhibition,19 we
co-incubated CsA-treated cells with 5 M E-64, a lysosomal degra-
dation inhibitor. E64 alone, used as a negative control, increased
LC3II expression, thus determining a basal autophagic flux level.
ce
E-64 in association with CsA induced a higher LC3II expression
than did CsA alone (Fig. 2B), suggesting that CsA and E-64 increase
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LC3II expression through two different ways, autophagy for CsA
and lysosomal degradation for E-64. The hallmark of autophagy is
the presence of autophagosomes, characterized by double membrane
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bound compartments that contain cytoplasmic material and/or
organelles. Ultrastructural analysis of tubular cells exposed to 8
s
M CsA during 48 hours with conventional transmission electron
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microscopy shows that CsA induces typical autophagosomes forma-
tion (Fig. 2C).
B
CsA-induced autophagy is mediated by ER stress. To further
prove that autophagy is mediated by ER stress in HRECs, we
treated tubular cells with various chemical compounds (trans-
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4,5-Dihydroxy-1,2-dithiane [DTTox], tunicamycin, thapsigargin
and brefeldin A) that induce ER stress by different mechanisms
nd
(disulfide bound reduction for DTTox, glycosylation inhibition for
tunicamycin, calcium homeostasis disturbances for thapsigargin, and
inhibition of the anterograde transport to the Golgi apparatus for
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brefeldin A). Thapsigargin and tunicamycin induce a strong LC3II
expression (Figs. 1C and 3A), whereas DTTox increases only weakly
LC3II and brefeldin A seems to have no effect (Fig. 3A). These
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results suggest that ER stress might participate to the autophagic
response of HRECs.
We also tested whether alleviating ER stress with salubrinal (SAL)
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reduces autophagy. SAL inhibits eIF2 dephosphorylation under
ER stress circumstances and inhibits mRNA translation, thereby
reducing protein load.20 In tubular cells treated with CsA, SAL
significantly reduces the expression of the ER stress markers GRP78
and GADD34 (Fig. 3B). SAL also reduces HERP expression, but
the difference did not reach statistical significance (data not shown).
Together, these results suggest that SAL alleviates tubular cells from
www.landesbioscience.com
CsA-induced ER stress. Whereas 8 M CsA increases LC3II forma-
tion at 24, 48 and 72 hours, 50 M SAL completely blocks LC3II
formation in CsA-treated cells, suggesting that SAL inhibits CsA-
induced autophagy (Fig. 3C). These data highlight a functional link
between ER stress and autophagy.
.
Inhibition of autophagy decreases tubular cell viability during
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CsA treatment. As autophagy plays a complex role in promoting
cell life and death that essentially depends on the cell type and
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the kind of stress and its duration, we tested whether autophagy is
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protective or cytotoxic in HRECs. CsA reduces HRECs viability in
a concentration-dependant manner (Fig. 4D). Necrosis, rather than
apoptosis, mediates HRECs cell death induced by CsA,21 and empty
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cytoplasmic spaces visualized on transmission electron microscopy
confirm this.
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Beclin1 siRNA were used to inhibit autophagy in CsA-treated
tubular cells. Beclin1 siRNA dramatically decreased Beclin1 tran-
script and protein expression in HRECs (Fig. 4A and B), and
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inhibited autophagy (Fig. 4C). Cell viability was then quantified in
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CsA-treated tubular cells in presence or absence of beclin1 siRNA
using the MTS assay (Fig. 4D). Autophagy inhibition by beclin1
siRNA significantly increased CsA cytotoxicity (Fig. 4D). Phase
o
contrast microscopy analysis of CsA-treated HRECs also showed
a clear increase of cell death induced by CsA when autophagy was
.D
inhibited (Fig. 4E).
CsA induces autophagy in vivo. To test whether autophagy
occurs in vivo, LC3 expression was evaluated by immunohistochem-
istry in rats treated subcutaneously with 15 mg/kg/day CsA (n = 3)
or placebo (olive oil, n = 3) during 28 days. At 28 days, mean serum
creatinine was 57 3 mol/L in the CsA-treated group vs 37 2
mol/L in the placebo-treated group (p < 0.05), and histological
analysis showed that CsA induced acute nephrotoxicity characterized
by tubular necrosis and cytoplasmic vacuolizations (data not shown).
Whereas LC3 staining was virtually absent in the kidneys of placebo-
treated rats, kidneys of CsA-treated rats displayed a strong tubular
LC3 staining. Interestingly, LC3 positive tubules were injured and
displayed cytoplasmic vacuolizations and tubular necrosis (Fig. 5A).
To confirm that CsA induced autophagy in vivo, we carried out
immunoblot analyses of LC3II expression and showed that LC3II
was significantly increased in the kidneys of CsA-treated rats (Fig.
5B). Together, these results suggest that CsA induces autophagy in
rat kidneys.
Discussion
Our findings identify autophagy as a new cytoprotective mechanism
against ER stress induced by CsA in renal tubular cells. Our study has
several important implications. Deciphering new biological pathways
that contribute to CsA nephrotoxicity is of great importance because
they may lead to the development of early biomarkers of kidney injury
in CsA-treated patients and to new therapeutic options.
Using cDNA microarrays, we previously demonstrated that CsA
induces an ER stress in human tubular cells.8 ER dysfunction may be
induced by calcium disturbances, hypoxia, ATP and glucose depriva-
tion or chemicals.11,17 Of note, these factors are implicated during
various kidney injuries such as ischemia-reperfusion, chronic isch-
emia or calcineurin-inhibitors nephrotoxicity. ER stress has recently
been described as a major process that can activate autophagy
through the PERK-eIF2 and IRE1-JNK pathways. The third
Autophagy 785
 Autophagy protects against ER stress induced by CsA

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Figure 2. Cyclosporine induces autophagy. (A) Representative western blot analysis of LC3I and LC3II expression in human tubular cells after exposure to
8 M cyclosporine during 24, 48 and 72 hours (left), and densitometric analysis (right), n = 3. (B) Representative western blot analysis of LC3I and LC3II
expression in human tubular cells after exposure to 8 M cyclosporine during 48 hours with or without 5 M of the cathepsin inhibitor E-64, or 5 mM of
the autophagosome formation inhibitor 3-methyl adenine (3MA) (left), and densitometric analysis (right), n = 3. (C) Representative transmission electronic
08

microscopy image analysis of tubular cells exposed to 8 M cyclosporine during 48 hours. Autophagosomes are intracellular formations characterized by
a double membrane (arrow) that contains cytoplasmic organelles (arrow head). Aut symbolizes autophagosomes, Mit labeled organelles are mitochondria,
Lys are lysosomes and Ne symbolizes empty formations sometimes referred as necrosis vacuoles. Magnification x14000.
20

mediator of the UPR, ATF6, does not seem to induce autophagy.12- infection and amino acid starvation induce autophagy by eIF2
14,16 Here, we demonstrate that ER stress and autophagy are linked

phosphorylation.22 Here, we demonstrate that SAL, a selective

in HRECs exposed to CsA. inhibitor of the eIF2 dephosphorylation, reduces LC3 conver-
Our results point out the role of eIF2 in the regulation of sion during CsA treatment. These results are conflicting with the
autophagy. During ER stress, eIF2 phosphorylation by PERK known role of eIF2 phosphorylation in inducing autophagy.
reduces protein synthesis and alleviates cell from death.14 Viral One can speculate that both the magnitude and duration of eIF2

786 Autophagy 2008; Vol. 4 Issue 6

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Figure 3. Autophagy is mediated by ER stress during cyclosporine treatment. (A) Representative western blot analysis of LC3I and LC3II expression in human
tubular cells after exposure to 10 mM trans-4,5-Dihydroxy-1,2-dithiane (DTTox), 10 M brefeldin A and 0.5 M tunicamycin during 48 hours (left), and den-
sitometric analysis (right). (B) Real time RT-PCR analysis of the expression of GRP78 and GADD34 mRNAs in human tubular cells after 24 hours of exposure
08

to 8 M cyclosporine, 50 M salubrinal or their association. *p < 0.05 versus cyclosporine-treated cells, n = 3. Fold changes for each tested gene were
normalized to the housekeeping gene Ribosomal Protein L13A. (C) Representative western blot analysis of LC3I and LC3II expression in human tubular cells
after exposure to 8 M cyclosporine with or without 50 M salubrinal during 24, 48 and 72 hours (left), and densitometric analysis (right), n = 3.
20

hosphorylation might be important factors in adjusting the

p during ER stress in CsA-treated HRECs remain imprecise and need
autophagic response to ER stress. Moreover, due to the beneficial to be elucidated.

effects of SAL in reducing protein load and the magnitude of the
unfolded protein response, other pathways that activate autophagy
during ER stress, such as IRE1/TRAF2/JNK might be downregu-
lated, thus reducing autophagic process.14 The precise mechanisms
and signaling pathways involved in the activation of autophagy
In nonrenal cells, CsA has been shown to inhibit autophagy, in
particular via its inhibitory effect on the mitochondrial transition
pore.23,24 In these studies, cells were exposed to CsA for 30 to 60
minutes, whereas in our study we analyzed autophagy after 24 to 48
hours of treatment. These discrepancies suggest that the effects of

www.landesbioscience.com Autophagy 787

 Autophagy protects against ER stress induced by CsA

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Figure 4. Autophagy inhibition increases tubular cell toxicity induced by cyclosporine. (A) Real time RT-PCR analysis of the expression of Beclin1 mRNA in
human tubular cells 48 hours after Beclin1 siRNA transfection. (B) Western blot analysis of Beclin1 expression in human tubular cells 48 hours after Beclin1
siRNA transfection. (C) Western blot analysis of LC3 expression in human tubular cells transfected with Beclin1 or control siRNA after 48 hours of CsA treat-
ment. (D) Cell viability was determined using the MTS assay 48 hours after incubation with 8 or 10 M cyclosporine with or without transfection with control
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or Beclin1 siRNA. Values are percentage of cell viability reported to vehicle-treated cells. *p < 0.05 vs control siRNA, n = 3. (E) Phase contrast microscopy
of tubular cells transfected with control siRNA (left) or beclin1 siRNA (right) after 48 hours of treatment with 8 M cyclosporine.
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CsA on autophagy might depend on the incubation times and cell known about the role of autophagy in mediating programmed cell
types. A similar paradox is described for the pro- and anti-apoptotic death, also called as type II cell death. The functional relationship
20

roles of CsA. Indeed, CsA inhibits mitochondrial mediated apoptosis between cell death or survival and autophagy is complex: in several
but also induces mitochondrial apoptotic cell death in the kidney.21 scenarios, autophagy constitutes an adaptation to stress that avoids
The increase of tubular cell cytotoxicity during autophagy inhibi- cell death, whereas in other cellular settings, autophagy constitutes an

tion suggests that autophagy protects HRECs against CsA toxicity.
Many organisms use autophagy as a protective mechanism during
stress such as nutrient deprivation, oxidative stress or viral infec-
tions. Because autophagosomes are often visualized in dying cells,
autophagy has been implicated in cell death activation, but little is
alternative pathway to cellular demise.25 However, growing evidence
suggests that autophagy is a protective mechanism rather than a
death inducer.
New insights into the molecular mechanisms of autophagy may
lead to the discovery of exciting new potential drug targets. Indeed,

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Figure 5. Cyclosporine activates autophagy in vivo. (A) LC3 expression was analyzed by immunohistochemistry and western blotting in kidneys of
cyclosporine-treated rats using rabbit anti-rat LC3 polyclonal antibody. Staining for LC3 was found neither in kidneys of cyclosporine-treated rats without
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primary antibody (upper), nor in kidneys of vehicle-treated rats (middle). Kidneys of cyclosporine-treated rats displayed intense cytoplasmic staining (lower),
particularly in injured (vacuolized and necrotic) tubules (magnification x20 and x40). (B) Representative western blot analysis of LC3 expression in rat kidneys
after 28 days of treatment with 15 mg/kg/day cyclosporine or vehicle (left), and densitometric analysis (right). *p < 0.05, n = 5.
08

therapeutic strategies that induce autophagy might have potential of accumulated proteins. Autophagy can also be induced by an
protective effect against CsA nephrotoxicity. Interestingly, among the mTOR independent route by lowering inositol-1,4,5-triphosphate
two evolutionary sensors involved in autophagy regulation, mamma- (IP3) levels. This can be achieved with drugs such as lithium,
20

lian target of rapamycin (mTOR) is a major inhibitory signal that carbamazepine or sodium valproate, which decrease IP3.27
shuts off autophagy during nutrient abundance. Its inhibition with The expression of LC3 in the kidneys of CsA-treated rats suggests

the immunosuppressive drug rapamycin is also known to activate
autophagy. Induction of autophagy by rapamycin treatment in intra-
cytosolic proteinopathies that cause neurodegenerative disorders is
experimentally effective in mice.26 In this setting, autophagy induc-
tion by mTOR inhibition is an effective way to increase the clearance
that autophagy also occurs in vivo and might be easily detected. The
detection of autophagy in kidney from CsA-treated patients might
be useful to detect early tissue injury and identify ongoing insult.
Indeed, autophagy activation means that a cellular stress occurs that
could lead to cell death and, ultimately to organ dysfunction. Its early

www.landesbioscience.com Autophagy 789

 Autophagy protects against ER stress induced by CsA
detection could then lead to therapeutic modifications such as CsA
dosage reduction. Evaluating autophagy in clinical samples has been
difficult mainly due to the lack of appropriate autophagy-specific
markers. Recently, LC3 immunohistochemical staining has been
shown to be a useful surrogate marker for autophagy in surgically
resected cancer specimens.28 It was demonstrated that accumulated
LC3 protein detected by immunohistochemistry in colonic neoplasia
samples was involved in the autophagosome formation. Our immu-
nohistochemical results clearly show an intense tubular staining
of LC3, especially in vacuolized and atrophic tubes whereas LC3
remained virtually absent in the control group. Thus, in addition to
our in vitro results, these data strongly suggest that LC3 is induced
by CsA, and demonstrate that autophagy may be detected in vivo.
In conclusion, we describe autophagy as a new mechanism by
which human renal tubular cells under ER stress conditions protect
against CsA toxicity. Our data also suggest that LC3 detection could
serve as a biomarker of CsA-induced nephrotoxicity.
Materials and Methods
Materials and reagents. All chemicals, otherwise specified, were
obtained from Sigma-Aldrich (Saint Quentin Fallavier, France).
Salubrinal was obtained from Calbiochem (VWR, Fontenay sous
Bois, France). The cell culture medium and the other cell culture
products were supplied by Life Technologies (Cergy Pontoise,
France).
Cell culture. Normal human renal epithelial cells were recovered
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from human nephrectomy specimens removed for renal cell carci-
noma and cultured according to previously published methods.29,30
en
Cells were cultured in Dulbeccos Modified Eagle Medium (DMEM)
containing 5 g/mL insulin, 10 g/mL human apotransferrin, 500
ng/mL hydrocortisone, 10 ng/mL EGF, 6.5 ng/mL triiodothyronin,
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5 ng/mL sodium selenite, 1% FCS, 25 IU/mL penicillin, 25 g/
mL streptomycin and 10 mM HEPES buffer. Cells were incubated
s
at 37C in 5% CO2 and 95% air. Due to tubular cell isolation
io
method, the cell culture could contain different cell types. However,
the characterization of our cellular model confirmed the proximal
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descent of the vast majority of the cultured tubular epithelial cells.30
Tubular cells (1.105/mL) were seeded in complete medium in 6, 12
or 96 wells plates 24 hours before experimentation. Experiments
es
were not performed with cells beyond the third passage. CsA
working concentration for autophagy detection was 8 M because
nd
at this concentration, CsA significantly reduced tubular cell viability
and cells began to display cytoplasmic vacuoles, suggestive of CsA
toxicity.
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RNA extraction and RT-PCR. Total RNA was extracted using
the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) following the
manufacturers protocol. The yield and purity of RNA were measured
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using a NanoDrop ND-1000 spectrophotometer (Nanodrop
Technologies, Inc., Rockland, Maine). Transcripts expression levels
were quantified by SYBR green Real-Time PCR (RT-PCR) using an
20
ABI PRISM 7900 sequence detector system (Applied Biosystems,
Foster City, California). Vehicle-treated samples were used as
controls, and fold changes for each tested gene were normalized
to the housekeeping gene Ribosomal Protein L13A. The relative
expression levels between samples were calculated using the compara-
tive delta Ct (threshold cycle number) method with vehicle-treated
samples as the reference point.31 All mRNA levels were quantified
in triplicate.
790
Protein extraction and western blot analysis. Total protein
lysate from HRECs was separated by sodium-dodecyl-sulfate poly-
acrylamide gel electrophoresis under denaturing conditions and
transferred to PVDF membrane (GE Healthcare, Aulnay sous bois,
France). Membranes were blocked with phosphate-buffered saline
.
and 0.1% Tween 20 containing 5% non-fat dry milk. Rabbit anti-
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bodies were used to detect LC3B (N2775; dilution 1:1000; Cell
Signaling Technologies, Saint Quentin en Yvelines, France) and actin
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(N2668, 1:5000, Sigma Aldrich, Saint Quentin Fallavier, France),
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and were revealed with a horseradish peroxidase-conjugated poly-
clonal secondary antibody (Dako, Trappes, France), and detected
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with ECL reagent (GE Healthcare).
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SiRNA transfections. Beclin1 small interfering RNA (siRNA)
(Hs_BECN1_4, cat No: si00055594) and control siRNA (Allstars
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Negative Control siRNA, Cat No: 1027280) were designed and
obtained from Qiagen (Courtabeouf, France). Tubular cells (1.105/
mL) were seeded in complete medium in 6, 12 or 96 wells plates
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before transfection. The confluency of the cells was 50% the day of
no
transfection. 20 nM of siRNA and the adequate Hiperfect volume
transfection reagent (Qiagen) were diluted in serum free medium
and mixed for 10 minutes at room temperature. siRNA-Hiperfect
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were then added dropwise to the cells. siRNA-treated cells were
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incubated for 48 hours.
Viability studies. HRECs were seeded in 96-well plates (104 cells/
ml). Twenty-four hours later, cells were treated. Three days afterwards,
the relative number of living cells per well was determined on the basis
of mitochondrial integrity by assay with 3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-fophenyl)-2H-tetrazolium
(MTS) (Promega, Charbonnieres, France), according to the manu-
facturers instructions.
Electronic microscopy. Cell pellets were fixed with 1.6% glutar-
aldehyde at 4C, followed by treatment with osmium tetroxide, then
dehydrated and embedded in epon resin. Ultrathin sections were
cut on an LKB-III ultra-microtome, stained for contrast with uranyl
acetate and lead citrate, and examined with a Zeiss EM 902 transmis-
sion electron microscope.
In vivo study. Adult male Sprague-Dawley rats (Charles River
laboratories, LArbresle, France) weighing 170180 g were housed
in a temperature and light-controlled environment. The experi-
mental protocol was approved by the animal care committee of
the University Paris Descartes. CsA was diluted in olive oil and
administered subcutaneously at a dose of 15 mg/kg/day. The vehicle-
treated group received olive oil at 1 mL/kg/day subcutaneously. Rats
were anesthetized with intraperitoneal ketamine, the abdomen was
opened through a midline incision, and the aorta was retrogradely
cannulated below the renal arteries with an 18-gauge needle. With
the aorta occluded by ligation above the renal arteries, and the
renal vein opened by a small incision for outflow, the kidneys were
perfused with 20 mL of cold heparinized saline. After the experi-
ment, the animals were euthanized by deep anesthesia with ketamine
followed by exsanguination. Kidney specimens were fixed in 10%
formalin neutral buffered. After fixation, tissues were dehydrated
with a graded series of ethanol and xylene, embedded in paraffin,
and cut into 3-m sections. All samples were then deparaffinized,
rehydrated, and heated for 20 min at 97C in citrate buffer (pH 6).
Endogenous peroxidase was inactivated by incubation for 10 min at
room temperature in 0.03% H2O2. Next, the sections were incubated
Autophagy 2008; Vol. 4 Issue 6
 Autophagy protects against ER stress induced by CsA

overnight at 4C with phosphate-buffered saline containing 1/10
anti-LC3B rabbit polyclonal antibody (Cell Signaling Technologies,
Saint Quentin en Yvelines, France). The sections were then incu-
bated with anti-rabbit antibody conjugated with peroxidase-labeled
polymer (Dako, Trappes, France). Immunoreactive proteins were
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values of less than 0.05 were considered significant.

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