In vivo and in vitro assessment of pathways involved in contrast media-induced renal cells apoptosis.

Abstract

Contrast-induced nephropathy accounts for >10% of all causes of hospital-acquired renal failure, causes a prolonged in-hospital stay and represents a powerful predictor of poor early and late outcome. Mechanisms of contrast-induced nephropathy are not completely understood. In vitro data suggests that contrast media (CM) induces a direct toxic effect on renal tubular cells through the activation of the intrinsic apoptotic pathway. It is unclear whether this effect has a role in the clinical setting. In this work, we evaluated the effects of CM both in vivo and in vitro. By analyzing urine samples obtained from patients who experienced contrast-induced acute kidney injury (CI-AKI), we verified, by western blot and immunohistochemistry, that CM induces tubular renal cells apoptosis. Furthermore, in cultured cells, CM caused a dose-response increase in reactive oxygen species (ROS) production, which triggered Jun N-terminal kinases (JNK1/2) and p38 stress kinases marked activation and thus apoptosis. Inhibition of JNK1/2 and p38 by different approaches (i.e. pharmacological antagonists and transfection of kinase-death mutants of the upstream p38 and JNK kinases) prevented CM-induced apoptosis. Interestingly, N-acetylcysteine inhibited ROS production, and thus stress kinases and apoptosis activation. Therefore, we conclude that CM-induced tubular renal cells apoptosis represents a key mechanism of CI-AKI.

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Invivoandinvitroassessmentofpathwaysinvolvedin
contrast media-induced renal cells apoptosis
C Quintavalle1,2, M Brenca1,2, F De Micco3, D Fiore1,2, S Romano4, MF Romano4, F Apone5, A Bianco6, MA Zabatta6, G Troncone6,
C Briguori*,3and G Condorelli*,1,2
Contrast-induced nephropathy accounts for 410% of all causes of hospital-acquired renal failure, causes a prolonged in-
hospital stay and represents a powerful predictor of poor early and late outcome. Mechanisms of contrast-induced nephropathy
arenot completelyunderstood. Invitro data suggeststhat contrastmedia(CM) induces adirecttoxic effecton renal tubular cells
through the activation of the intrinsic apoptotic pathway. It is unclear whether this effect has a role in the clinical setting. In this
work, we evaluated the effects of CM both in vivo and in vitro. By analyzing urine samples obtained from patients who
experienced contrast-induced acute kidney injury (CI-AKI), we verified, by western blot and immunohistochemistry, that CM
induces tubular renal cells apoptosis. Furthermore, in cultured cells, CM caused a dose–response increase in reactive oxygen
species (ROS) production, which triggered Jun N-terminal kinases (JNK1/2) and p38 stress kinases marked activation and thus
apoptosis. Inhibition of JNK1/2 and p38 by different approaches (i.e. pharmacological antagonists and transfection of kinase-
death mutants of the upstream p38 and JNK kinases) prevented CM-induced apoptosis. Interestingly, N-acetylcysteine inhibited
ROS production, and thus stress kinases and apoptosis activation. Therefore, we conclude that CM-induced tubular renal cells
apoptosis represents a key mechanism of CI-AKI.
Cell Death and Disease (2011) 2, e155; doi:10.1038/cddis.2011.38; published online 12 May 2011
Subject Category: Experimental Medicine
Acute kidney injury (AKI) represents a frequent and devastat-
ing problem in hospitalized adults with persistently high rates
of mortality and morbidity. Studies of large adult cohorts have
revealed that contrast-induced AKI (CI-AKI) is the third most
common cause of hospital-acquired AKI, accounting for 410%
of cases. Approximately half of these cases are of patients
undergoing contrast media (CM) exposure because of the
cardiac catheterization and angiography, and about one-third
follow computed tomography. A clear comprehension of the
mechanisms of CI-AKI may, therefore, have important clinical
advantages. A toxic effect of CM on renal tubules has been
shown in both clinical trials and animal experiments.1–3In an
experimental study, we previously observed that CM induces a
dose- and time-dependent renal cell apoptosis through the
activation of the intrinsic pathway.4However, it is unclear
whetherthiseffecthasaroleintheclinicalsetting.Furthermore,
little is known about the molecular mechanisms underlying this
contrast-induced renal cell apoptosis.
Apoptosis is an evolutionarily conserved mechanism of
elimination of the unwanted cells.5The extrinsic pathway is
activated by the engagement of death receptors on the cell
surface. The intrinsic pathway is triggered by various
intracellular and extracellular stresses whose signals con-
verge mainly to the mitochondria.6,7Studies in humans
indicate that reactive oxygen species (ROS) contribute to
contrast-induced acute kidney injury (CI-AKI).8–10Indeed,
several animal experiments showed that CI-AKI is accom-
paniedbytheincreasedproductionofROS.11,12ROSactivate
stress kinases,13,14such as the mitogen-activated protein
kinases (MAPKs).15MAPKs include at least three main
subgroups: theextracellular
(ERK1/2 or p42/44MAPK), the c-Jun N-terminal kinases
(JNK 1/2), andp38MAPK. Although structurally related MAPK
families undergo activation in response to extracellular stimuli
through distinct upstream dual specificity kinases, thereby
functioning in separate MAPK cascades.16The Raf/ERK
kinase1/2/ERK1/2 cascade is stimulated by mitogenic and
survival stimuli, largely through the Ras-Raf-1-dependent
pathway.17At variance, JNK1/2 and p38MAPK are primarily
activated by cellular stresses, including oxidative agents, UV
signal-regulated kinases
Received 15.2.11; revised 09.3.11; accepted 23.3.11; Edited by V De Laurenzi
1Department of Cellular and Molecular Biology and Pathology, ‘Federico II’ University of Naples, Naples, Italy;2IEOS, CNR Naples, Naples, Italy;3Laboratory of
Interventional Cardiology and Department of Cardiology, Clinica Mediterranea, Naples, Italy;4Department of Biochemistry and Medical Biotechnology, Federico II
University of Naples, Naples, Italy;5Arterra Bioscience, Naples, Italy and6Dipartimento di Scienze Biomorfologiche e Funzionali, Universita ` di Napoli Federico II,
Naples, Italy
*Corresponding authors: C Briguori, Laboratory of Interventional Cardiology and Department of Cardiology, Clinica Mediterranea, Via Orazio, 2, Naples I-80121, Italy.
Tel: þ39 081 7259 764; Fax: þ39 081 7259 777; E-mail: carlobriguori@clinicamediterranea.it
or G Condorelli, Department of Cellular and Molecular Biology and Pathology, ‘Federico II’ University of Naples, Via Pansini, 5, Naples I-80121, Italy and IEOS, CNR
Naples, Naples, Italy.
Tel: þ39 081 746 4416; Fax: þ39 081 746 3308; E-mail: gecondor@unina.it
Keywords: contrast media; kidney; apoptosis; prevention
Abbreviations: CM, contrast media; CI-AKI, contrast-induced acute kidney injury; ROS, reactive oxygen species; JNK 1/2, Jun N-terminal kinases 1 e 2; NAC,
N-acetylcysteine; AKI, acute kidney injury; MAPK, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinases; MKK, MAP kinase kinases; Gal-3,
galactine-3; CK7, cytokeratin 7; LOCM, low-osmolar contrast media; IOCM, iso-osmolar contrast media; PKC, protein kinase C; SAPK, stress-activated protein kinase;
Bcl-2, B-cell lymphoma 2; MDCK, Madin–Darby canine kidney; CKD, chronic kidney disease; EGTA, ethylene glycol tetraacetic acid; PBS, phosphate-buffered saline
Citation: Cell Death and Disease (2011) 2, e155; doi:10.1038/cddis.2011.38
& 2011 Macmillan Publishers Limited All rights reserved 2041-4889/11
www.nature.com/cddis

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irradiation, hypoxia, and proinflamatory cytokines.18Dual
specificity kinases activating JNK are MAP kinase kinases
(MKK) 4 and MKK7, whereas MKK3 and MKK6 were proved
to activate p38MAPK.
In this study, we investigated (1) the in vivo occurrence of
CM-induced tubular renal cells apoptosis; (2) the in vitro and
in vivo effects of CM on stress kinases and apoptotic
pathways; (3) the in vitro effects CM on ROS production in
renal tubular cells; and (4) the in vitro effects of stress kinases
inhibition by different approaches in preventing the contrast-
induced cell damage.
Results
In vivo assessment of the apoptotic pathway. The
characteristics of the 10 patients enrolled in this study are
summarized in Table 1. Epithelial tubular cells were
observed in all cases at both 24 and 48h following CM
exposure. Tubular cells occurred in clusters and casts, and
showed clear or vacuolated cytoplasm, intracytoplasmic
pigmented granules, and nuclear changes. The presence of
epithelial tubular cells was confirmed by both morphological
andimmunocytochemical criteria.
evaluated on cytospin preparations stained by standard
Papanicolaou or hematoxylin–eosin staining method (Figures
1a and b); the latter were assessed by immunostaining for the
galactine-3 (Gal-3; Figure 1c) and cytokeratin 7 (CK7) tubular
cell markers (Figure 1d). In all these patients, we observed
in vivo, the activation of the apoptotic process by the
assessment of caspase 3 activation was analyzed either by
immunocytochemistry (Figures 1e and f) or by western blot
(Figure 2c).
Theformer were
CM and stress kinases. We then evaluated the activation
of JNK1/2 (with the use of specific antibodies that recognize
the phosphorylated (activated) form of the kinases), and the
expression of the anti-apoptotic protein BAK in epithelial tubular
cells collected from these patients. In all these patients, we
observed a significantly increase of JNK phosphorylation
(Figure 2a) and an increase of BAK expression levels
(Figure 2b).
In the in vitro model, all tested CM induced a dose-
dependent phosphorylation of JNK1/2 and p38. Indeed,
although at low level, this activation was observed even with
low dose (50mgI/ml) of CM (Figure 3). Furthermore, this effect
was time-dependent and reached the maximum level at 1h.
N-acetylcysteine (NAC) pre-incubation induced a significant
reduction of phosphorylation levels of JNK1/2 (Figures 4a–c).
Moreover, this effect was associated with a reduction of
caspase 3 activation, as assessed by caspase 3 assay
(Figure 4d). CM also induced an increase in the expression
levels of the B-cell lymphoma 2 (Bcl2) family pro-apoptotic
proteins, namely, BAX, BAK, and BAD. Interestingly, the
pre-treatment with JNK 1/2 inhibitor prevented this effect
(Figure 5).
Pre-treatment with stress kinases inhibitors decreases
apoptosis. To establish a more direct link of JNK 1/2 and
p38 with CM-induced apoptosis, we used two different
approaches. First, we investigated the effect of specific
stress kinase inhibitors. Pre-treatment of renal cells with two
different JNK 1/2 inhibitors (SP600125 and AS601245;
Figure 6) and with a p38 inhibitor (SB203380; Figure 7a)
strongly attenuated CM-induced renal cell apoptosis. On the
contrary, inhibitors toward other kinases, such as ERK and
protein kinase C (PKC), did not impact on CM-induced
apoptosis (Figures 7c and d). Second, we thought to
transfect the cells with kinase-death mutants of the
upstream p38 and JNK1/2 kinases, MKK6-KR, and MKK4-
KR, respectively. As shown in Figure 7b, the kinase-death
mutants attenuated the CM-induced cell death. This effect
was even stronger on co-transfection of both constructs.
Production of ROS. To assess the molecular pathways
leading to CM apoptosis activation, we determined the
effects of CM on the formation of ROS. Renal cells were
incubated in the presence of different CM concentrations (50,
100, and 200mgI/ml) and ROS subsequently quantified. As
shown in Figure 8a, both low-osmolar contrast media
(LOCM) and iso-osmolar contrast media (IOCM) treatment
induced a dose–response increase of ROS. This effect on
ROS was significantly attenuated by NAC pre-treatment
(Figure 8b).
Discussion
Thisstudyclearlydemonstratesthat(1)CM-inducedepithelial
tubular renal cells apoptosis represents a key mechanisms of
CI-AKI; (2) CM induces apoptotic cell death via three
Table 1 Clinical characteristics
N¼10
Age (years)
Male (%)
Weight (kg)
Height (m)
Body mass index (kg/m2)
70±9 (41–90)
9 (90)
76±12
1.68±0.6
27±3
Blood pressure (mmHg)
Systolic
Diastolic
Mean
150±19
80±8
102±10
Left ventricular ejection fraction (%)
Systemic hypertension (%)
Diabetes mellitus (%)
50±10
8 (80)
4 (40)
Serum creatinine, median (IQR; mg/dl)
Baseline
After 24h
After 48h
1.64 (1.51–1.90)
1.70 (1.50–1.99)
2.01 (1.85–6.89)
eGFR (ml/min per 1.73m2)4110
Performed procedure
Coronary angiography (%)
PCI (%)
Coronary angiography and ad hoc PCI (%)
4 (40)
2 (20)
4 (40)
Volume of contrast media (ml)165±125
Abbreviations: eGFR, estimated glomerular filtration rate; IQR, interquartile
range; PCI, percutaneous coronary intervention. Continuous values are
expressed ad mean±S.D.; categorical values are expressed as a total number
and as a percentage of the global population (in parenthesis)
Contrast media and apoptosis
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important signaling pathways, namely, (a) ROS pathway,
(b) stress kinase pathway, and (c) intrinsic apoptotic path-
ways, which are triggered by CM in this sequence; and (3)
NACand/orstresskinaseinhibitionmaypreventthetriggering
of this cascade.
CM-induced
demonstrated for the first time that the proposed in vitro
apoptotic pathway inducing kidney damage was also
appreciatedin vivo. In vitro
pathophysiology of CM-induced
usually criticizedbecause
including (1) theassessment
mechanism of the CM-induced renal cell damage in the
absence of confounding variables that can be found in vivo
(e.g., hypoxia due to hemodynamic changes or other
systemic mechanisms); (2) the exposure to a constant
apoptosisand CI-AKI. This study
studies
apoptosis
the several
of only
addressing
have
the
been
oflimitations,
potentialone
concentration CM to all cells line, whereas in vivo, the
more distal epithelial tubular cells are exposed to much
higher concentration; (3) the potentially high dose of CM. Our
in vivo demonstration of CM-induced tubular renal cells
apoptosis confirms the crucial role of this mechanism in the
pathogenesis of CI-AKI. Prophylactic strategies aimed to
prevent contrast-induced renal cells apoptosis should be,
therefore, investigated as novel therapeutic approaches to
prevent CI-AKI.
ROS pathway. In this study, we demonstrate that CM
induces an increase in ROS production. This leads to
eventual activation of the stress kinases JNK1/2 and p38
but not to ERK or PKC. It is well known that ROS can
effectively activate stress kinases.19,20Lee et al.21recently
reported that CM induce a time-dependent activation of
JNK1/2. Our findings confirm and extend this observation.
Figure 1
(regular elements with vacuolated and clear cytoplasm with eccentric nuclei) can be appreciated (Papanicolaou staining, ?400). (b) Urine cytological cell block preparation.
This specimen type was used to perform specific tubular cell marker immunostaining. (Hematoxylin and eosin staining, ?400). (c) The presence of tubular cells was
confirmed by a specific staining for the Gal-3 marker. (Haematoxylin counterstained, ?400). (d) The presence of distal tubular cells was confirmed by a specific staining for
the CK7 marker. (Haematoxylin counterstained, ?400). (e) Active caspase 3 staining of samples from untreated patient. (f) Active caspase 3 staining from samples of
patients upon 48h of CM
Immunohistochemistry of kidney tubular cells. (a) Cluster of tubular cells in an inflammatory background. The typical morphological features of tubular cells
Contrast media and apoptosis
C Quintavalle et al
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This study indeed clarifies that ROS induce stress kinases
activation following CM incubation. The observation that
ROS activation is dose- and time-dependent underscores
two important aspects of contrast-induced kidney damage
well known both in experimental4and clinical22models.
Stress kinases pathway. Our study demonstrates that CM
induces renal cell apoptosis by the activation of the JNK1/2
and the p38 pathways (but not ERK and PKC) via an
upregulation of the intracellular levels of ROS. JNK1/2, p38,
and ERK are well-characterized subgroups of a large MAPK
family. Although the ERK pathway is most commonly linked
to the regulation of cell proliferation, the JNK1/2 and p38
pathways are primarily activated by various types of
environmental stress:osmotic
oxidative stress,protein
proinflammatory cytokines.23Therefore, JNK1/2 and p38
are often grouped together and are referred as stress-
activated protein kinases (SAPKs). ERK, JNK1/2, and p38
have all been shown to be activated in response to the
shock,UV
inhibitors,
irradiation,
synthesis and
57 kDa
46.5 kDa
46 kDa
28 kDa
37 kDa
Pre
Patient #1
Patient #1Patient #2Patient #3 Patient #4
Patient #2
Patient #3
Patient #4
Post
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24hr
Pre
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Patient #1
Patient #2Patient #3Patient #4
37 kDa
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Caspase 3
β-Actin
Post
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PrePost
24hr
PrePre
p-JNK
JNK
BAK
β-Actin
Post
48hr
PrePost
48hr
PrePost
48hr
Post
48hr
Figure 2
before and at 24 and 48h after the contrast media exposure. (c) CM induced also an activation of caspase 3. Representative of four patients was shown
Effect of CM on renal cells in vivo. (a) JNK phosphorylation and BAK (b) expression levels were increasedfrom epithelial tubular cells collected from 10 patients
57 kDa
46.5 kDa
MDCKMDCKMDCK
MDCKMDCK
42 kDa
46 kDa
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lobitridol
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46.5 kDa
MDCK
p-JNK
p-p38
JNK
p38
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mgl/ml
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p38
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p-JNK
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- 50100 200lodixanol
mgl/ml
lopamidol
mgl/ml
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lodixanol-15’30’1hr3hr
p38
p38
JNK
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p-JNK
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lopamidol- 3hr1hr 15’30’
p38
JNK
p-p38
p-JNK
42 kDa
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- 50 100200
p38
JNK
p-p38
p-JNK
57 kDa
46.5 kDa
Figure 3
iobitridol (a), iodixanol (b), and iopamidol (c). Cell extracts were analyzed by western blot for phospho-JNK, phospho-p38, JNK expression, and p38 expression
Activation of JNK and p38 after contrast media exposure. Dose–response and time-course of JNK and p38 activation induced by treatment of MDCK cells with
Contrast media and apoptosis
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57 kDa
46.5 kDa
MDCK
p-JNK
57 kDa
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57 kDa
46.5 kDa
MDCK
MDCK
p-JNK
JNK
β-Actin
46 kDa
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37 kDa
37 kDa
-
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+
lodixanol
NAC
5
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Caspase 3 activity
(Fold increase over control)
1
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lobitridol
lodixanol lopamidol
NAC
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lopamidol
NAC
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lobiridol
NAC
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Figure 4
and iopamidol (c). (d) Caspase 3 assay in renal cells incubated with contrast media and pretreated with NAC. *P40.05 versuscontrol;wPo0.030 versus control;zPo0.005
versus column without NAC
NAC effects on JNK activation and on apoptosis in renal cells. Western blot of the effects of NAC pretreatment on JNK activation by iobitridol (a), iodixanol (b),
27 kDa
BAD
27 kDa
27 kDa
BAD
BAK
BAX
28 kDa
28 kDa
21 kDa
21 kDa
37 kDa
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BAK
lopamidol
SP600125
BAX
β-Actin
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BAD
BAK
BAX
β-Actin
28 kDa
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+
lobitridol
SP600125
lodixanol
SP600125
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+
Figure 5
BAK that was reverted by the pre-treatment with SP600125
Effect of CM-inducedstress kinasesactivation on Bcl2pro-apoptoticfamily members.CM inducedan increaseof pro-apoptoticfamilymembers BAX,BAD, and
14
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Figure 6
apoptosisevaluatedbyannexinVstaining.(d)Caspase3assayinrenalcellsincubatedwithcontrastmediaandpretreatedwithSP600125andAS601245.*Po0.001versus
control;wP40.05 versus control
Effects of JNK inhibitors on renal cells apoptosis. SP600125 and AS601245 strongly reduced iodixanol (a), iobitridol (b), and iopamidol (c) induced renal cell
Contrast media and apoptosis
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intracellular redox state and oxidative stress, and potentially
contribute to influencing cell survival or cell death. ERK and
JNK/p38 have opposing functions, whereas ERK are
generally
modification of MAPK signal transduction pathway by ROS
generates a great variety of biological responses. JNK1/2
phosphorylate and release two Bcl-2-related proteins that are
normally sequestered within the cell.20,24The release of
these key proteins can directly activate Bax by causing
dissociation from its cytoplasmic anchor. Bax is then free to
translocatetothemitochondria,
oligomerization and initiates the release of cytochrome c
and other pro-death mediators into the cytosol. JNK1/2 are
also capable to enhanced Bax-to Bcl2 expression ratio, loss
of mitochondrial membrane potential cytochrome c release,
and caspase cascade reaction.25Our data confirm previous
findings, demonstrating that CM are able to induce an
increase of some of the Bcl2-family pro-apoptotic members
(BAD, BAK, and BAX), and that JNK1/2 inhibitor was able to
prevent this effect.
pro-survival andSAPKspro-apoptotic.The
whereitundergoes
Intrinsic apoptotic pathway. Previous studies demonstrated
that CM induces renal cells apoptosis through the activation of
the intrinsic or mitochondrial pathway.4In this study, we clarify
that this effect is triggered by stress kinases JNK1/2 and p38
activation. Indeed we observed that pre-treatment with JNK1/2
and p38 inhibitors prevent (1) CM-induced increase of the
Bcl2-family pro-apoptotic members (BAD, BAK, and BAX), and
(2) CM-mediated caspase 3 activation. These effects act in
concert to attenuate CM-induced renal cell apoptosis. The
essential role of JNK1/2 during the apoptotic process of CM
confirms that CM uses the intrinsic apoptotic signaling rather
than the extrinsic or death receptor pathway. The intrinsic or
mitochondrial pathway was originally identified as the main
mediator of apoptosis signals initiated by stress or toxic stimuli.
The major participants in this kinase cascade are two members
of the MAPKs, JNK1/2, and p38MAP kinase, as well as their
upstream kinases such as MKKs.17
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Figure 7
mediainducedapoptosis(b).However,MEK1/2inhibitor(c)andPKCinhibitor(d)didnotimpactiobitridol-,iodixanol-,andiopamidol-inducedrenalcellapoptosisevaluatedby
annexin V staining. *Po0.001 versus control;wP40.05 versus control
Effects of p38, MAPK, and PKC inhibitors on renal cells apoptosis. P38 inhibitor, SB203580 (a) or MKK4-KR, and MEKK6-KR transfection reduced contrast
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100 200
100
Figure 8
treatment. MDCK cells were treated for 3h with 50, 100 or 200mgI/ml of iodixanol,
iobitridol, iopamidol, and with 400mM of H2O2(positive control). (a) ROS formation
on different CM. By ANCOVA test, ROS production was significantly increased in
the presence of medium (100mgI/ml) and high (200mg/ml), but not at low (50mgI/
ml) dose of contrast media. *P40.05 versus control;wPo0.030 versus control;
zPo0.001 versus control. (b) Effects of 2h NAC (100mM) pretreatment on ROS
formation following contrast media exposure.wPo0.001 versus control;zPo0.006
versus column without NAC
Production of reactive oxygen species (ROS) after contrast media
Contrast media and apoptosis
C Quintavalle et al
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Potential strategies for preventing CM-induced renal cell
apoptosis. In this study, we demonstrated that strategies
inhibiting the CM-signaling pathways may prevent renal cell
apoptosis. In particular, we tested two approaches inducing
an upstream (NAC) or a downstream (JNK1/2 and p38
inhibitors) CM signaling pathway blockage. The positive
results obtained by both these approaches represent a
further confirmation of our proposed CM signaling pathway
(Figure 8). We have already demonstrated that NAC pre-
treatment is capable to prevent apoptosis in renal cells.4,26In
this study, we observed that NAC prevents CM-induced ROS
production and therefore inhibits JNK1/2 and p38 activation
as well as apoptosis, suggesting the existence of a specific
target for NAC upstream to the apoptosis-executing stress
kinases in the CM-activated signaling pathway. Indeed, the
increase of intracellular ROS by CM and/or H2O2was almost
completely abolished by NAC.
Study limitations. Additional data are necessary to address
the issue of which CM component and chemical properties
(such as viscosity) cause ROS production. The investigators
who evaluated the cells damage were not blinded to the CM
and the protective strategy attempted. We did not use
proximal tubular cells. We selected Madin–Darby canine
kidney (MDCK) cells for two reasons: (1) distal tubular cells
are more affected by CM damage, and (2) handling of the
MDCK cells is easier than other kidney cell lines (such as,
porcine proximal renal tubular LLC-PK1).
Conclusions. In this work, we were able to demonstrate
that CM induces apoptotic cell death via three important
signaling pathways, (a) ROS pathway, (b) JNK/p38 pathway,
and (c) intrinsic apoptosis pathway, which are triggered by
CM in this sequence. The relationship between these three
sequential pathways was strongly suggested and supports
novel therapeutic approaches to prevent CI-AKI.
Patients and Methods
Patients’ population and urine cells collection. The urine of patients
with chronic kidney disease (CKD) who experienced contrast-induced AKI (CI-AKI)
were collected the day before and through the 24 and 48h following CM exposure.
CKDwasdefinedasanestimatedglomerularfiltrationrateo60ml/minper1.73m2,
calculated by applying the Levey-modified Modification of Diet in renal Disease
formula: (186.3?serum creatinine?1.154)?(age–0.203)?(0.742 if female).27
CI-AKIwasdefinedasan increaseintheserumcreatinineconcentration,Z0.3mg/
dl, from the baseline value at 48h after CM administration or the need for dialysis
(CritCare2007;11:R31).Inallinstancestheiodixanol(Visipaque,320mgiodine/ml,
GE Healthcare Europe, Buckinghamshire, UK), a non-ionic, iso-osmolar CM was
used. Exfoliated cell pellets from the urine of the patients were collected by
centrifugation at 1200r.p.m. for 25min. A fraction of urine samples was sent to the
pathologist for cytological analysis and a fraction to the laboratory for in vitro assay.
All samples were stored at ?801C for a maximum of 2 months.
The presence of tubular cells was assessed by using morphological and
immunocytochemical criteria. The former were evaluated on cytospin preparations
stained by standard Papanicolaou staining method; the latter were assessed by
immunostaining for the Gal-3, CK7 tubular cell markers, and active caspase 3. To
this end, cell block preparations were used. To ensure their adequacy, cell blocks
were stained with hematoxylin and eosin. CK7 was immunostained by mouse
monoclonal antibody (clone OV-TL12/30; Dako, Milano, Italy); the caspase 3
expression was detected by rabbit polyclonal antibody (Cell Signaling 9661, Cell
Signaling, Danvers, MA, USA). For CK7 and caspase 3, signal was developed by
the polyvalent LSAB-peroxidase Dako Kit (Dako). As far as, Gal 3 staining is
concerned, the galectin 3 tyrotest (Biocare Medical, Concord, CA, USA) was used
as previously described.28To extract protein from exfoliated cells, the pellet
obtained on centrifugation was resuspended in ice-cold TRAP buffer (tris(idrossi-
metil)amminometano cloridrato (ph 7.5) 10mM, MgCl21mM, ethylene glycol
tetraacetic acid (EGTA) 1mM, phenyl methylsulfonyl 0.1mM, b-mercaptoethanol
5mM,CHAPS0.5%,andglycerol10%)andincubatedonicefor1h.Thelysatewas
centrifuged for 20min a 13.200r.p.m. at 41C. The supernatant was collected.
Culture conditions and reagents. Canine Madin–Darby proximal renal
tubular (MDCK) cells were grown in a 5% CO2atmosphere in Dulbecco’s modified
Eagle’s medium containing 10% heat-inactivated fetal bovine serum, 2mM
L-glutamine and 100U/ml penicillin–streptomycin. Cells were routinely passaged
when 80–85% confluent. Media, sera, and antibiotics for cell culture were from Life
Technologies Inc. (Grand Island, NY, USA). Protein electrophoresis reagents were
from Bio-Rad (Richmond, VA, USA) and western blotting and ECL reagents (GE
Healthcare).PKCinhibitorRo-32-0432wasfromCalbiochem(Gibbstown,NJ,USA)
and LY294002 was from Cell Signaling). JNK inhibitors AS601245 and SP 60125
were, respectively, from Alexis (Florence, Italy) and Sigma (St. Louis, MO, USA).
P38 inhibitor, SB203380, and MEK1/2 inhibitor, V0126 were from Calbiochem. All
other chemicals were from Sigma. The following antibodies were used for
immunoblotting: anti-b actin (Sigma), anti-P-JNK and P-p38, p38 (Cell Signaling),
anti-JNK(BDBioscience,FranklinLakes,NJ,USA),BAKandBADwerefromSanta
Cruz Biotechnology (Santa Cruz, CA, USA), BAX was from BD Bioscience.
Contrast media. Several CM were tested: (1) iodixanol (Visipaque, 320mg
iodine/ml, GE Healthcare, Europe) non-ionic, IOCM (290mOsm/kg of water); (2)
iobitridol (Xenetix, 250mg iodine/ml, Guerbet, Roissy CDG, France) non-ionic,
LOCM (915mOsm/kg of water); (3) iopamidol (Iomeron, 350mg iodine/ml, Bracco,
Milano, Italy) non ionic, LOCM (796mOsm/kg of water).
Cell transfection. To inhibit p38or JNK1/2activation,we transfected thecells,
respectively, with the kinase-dead mutant of the upstream p38 kinase, MKK6
(pCEFL GST MKK6-KR), or with the kinase-dead mutant of the upstream JNK1/2
kinase, MKK4-KR (pcDNA3 MKK4-KR) MDCK cells were cultured to 80%
confluence, kept in antibiotic-free, serum-containing medium, and transiently
transfected using Lipofectamine and Plus Reagent (Invitrogen, Milano, Italy) with
5mg of MKK4-KR, MKK6-KR cDNAs or with control vector, as indicated. cDNA
plasmids were a kind gift of Dr. Mario Chiariello (Siena, Italy).
ROS determination. Formation of ROS was detected by the signal obtained
from the fluorescent reaction products dichlorofluorescein and BODIPY, a
fluorescent ratio probe for indexing peroxidation in membranes29by the use of
flow cytometry (FACSCalibur, BD Bioscience; Perkin Elmer; Waltham, MA, USA;
Cell Quest software, BD Bioscience). ROS production was evaluated upon 50, 100,
or 200mgI/ml.
To assess the effects of NAC on ROS production, MDCK cells were pretreated
for 2h with NAC (100mM), incubated with BODIPY for 45min and then exposed to
CM. ROS formation was valuated after 2h of CM exposure.
Stresskinases. To addresswhetherthe ROSproductioninduces an activation
of stress kinases, MDCK cells were treated at different times and different doses of
CM(50,100,or200mgI/ml;withallthetestedCMatthesamedosereportedabove.
JNK and p38 phosphorylation (activation) was assessed with western blot with
specific anti-P-JNK or anti-P-38 antibodies (Cell Signaling), as described.30To
investigate whether inhibition of JNK1/2 was capable to prevent cell death, renal
cells were overnight pre-treated with 40mM of JNK1/2 inhibitors SP600125 or
AS601245 and then exposed to CM for a short (30min) or a longer (3h) period.
Similarly, inhibitors of other signaling pathways were used: MEK1/2 inhibitor V0126
(10mM incubated over night), PKC inhibitor (212mM incubated over night) and p38
inhibitor, SB203580 (10mM incubated overnight). The doses and the incubation
time of drug inhibitors used have been choose in order of not causing any
nonspecific effects on other signaling pathways. Furthermore, in order of reinforce
the role of JNK activation on CM-induced apoptosis, we used two different JNK
inhibitors (SP600125 and AS601245).
Caspase assay. The assay was performed using the Colorimetric CaspACE
Assay System, (Promega, Madison, WI, USA) as reported by instruction manual.
Briefly, MDCK cells were pre-treated with NAC (100mM) and then treated for 3h
with iodixanol, iobitridol, and iopamidol. Cells were harvest in caspase assay buffer,
and proteins were quantified by Bradford assay. Total protein used was 50mg.
Contrast media and apoptosis
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Protein isolation and western blotting. Cellular pellets were washed
twice with cold phosphate-buffered saline (PBS) and resuspended in JS buffer
(Hepes 50mM, NaCl 150nM, 1% glycerol, 1% Triton X100, 1.5mM MgCl2, and
5mM EGTA) containing Proteinase Inhibitor Cocktail (Roche, Milano, Italy).
Solubilized proteins were incubated for 1h on ice. After centrifugation at
13200r.p.m. for 10min at 41C, lysates were collected as supernatants. Sample
extract (80mg) were resolved on a 12% SDS-polyacrylamide gel using a mini-gel
apparatus (Bio-Rad Laboratories) and transferred to Hybond-C extra nitrocellulose
(GE Healthcare Europe). Membrane was blocked for 1h with 5% non-fat dry milk in
TBS containing 0.05% Tween-20 and incubated over night at 41C with specific
antibodies. Indicated antibodies were used for the immunoblotting. Washed filters
were then incubated for 45min with horseradish peroxidase-conjugated anti-rabbit
or anti-mouse secondary antibodies (GE Healthcare Europe) and visualized using
chemioluminescence detection (GE Healthcare Europe).
Cell death quantification. Cells were plated in 96-well plates in triplicate,
stimulated and incubated at 371C in a 5% CO2incubator. Iobitridol, iodixanol,
iopamidol, iohexol, and NAC were used in vitro at doses and time indicated.
Apoptosiswasanalyzedviapropidiumiodideincorporationinpermeabilizedcellsby
flowcytometryaspreviouslydescribed.31Briefly,thecells(2?105)werewashedin
PBS and resuspended in 200ml of a solution containing 0.1% sodium citrate, 0.1%
Triton X-100, and 50mg/ml propidium iodide (Sigma). Following incubation at 41C
for 30min in the dark, nuclei were analyzed with a Becton Dickinson FACScan flow
cytometer (BD Bioscience). Cellular debris was excluded from analyses by raising
theforwardscatterthreshold,andtheDNAcontentofthenucleiwasregisteredona
logarithmic scale. The percentage of elements in the hypodiploid region was
calculated.
Statistical analysis. Continuous variables are given as mean±1 S.D.
or median and interquartile ranges, when appropriate. Categorical variables were
reported as percentage. Continuous variables in the groups were analyzed by one-
way analysis of variance test. The level of statistical significance was o0.05.
Multiplicity issues were addressed using the Bonferroni adjustment. Data were
analyzed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA) for Windows.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements. This work was partially supported by funds from the
AIRC(AssociazioneItalianaRicercasulCancro)(No10620toGC)andFondazione
SDN. CQ is recipient of FIRC (Associazione Italiana Ricerca sul Cancro) fellowship.
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Contrast media and apoptosis
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