1 Division of Pediatric Nephrology, Yale University School of Medicine, New Haven, Connecticut 06520; and 2 Division of Pediatric Nephrology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York 10467
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ABSTRACT |
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Brief periods of in vitro hypoxia/ischemia induce
apoptosis of cultured renal epithelial cells, but the underlying
mechanisms remain unknown. We show that partial ATP depletion
(10-65% of control) results in a duration-dependent induction
of apoptosis in Madin-Darby canine kidney (MDCK) cells, as evidenced by
internucleosomal DNA cleavage (DNA laddering and in situ nick end
labeling), morphological changes (cell shrinkage), and plasma membrane
alterations (externalization of phosphatidylserine). The ATP-depleted
cells display a significant upregulation of Fas, Fas ligand, and the
Fas-associating protein with death domain (FADD). Exogenous application
of stimulatory Fas monoclonal antibodies also induces apoptosis in
nonischemic MDCK cells, indicating that they retain Fas-dependent
pathways of programmed cell death. Furthermore, cleavage of
poly(ADP)ribose polymerase (PARP) is evident after ATP depletion,
indicating activation of caspases. Indeed, the apoptotic cells display
a significant increase in caspase-8 (FLICE) activity. Finally,
apoptosis induced by ATP depletion is ameliorated by pretreatment with
inhibitors of caspase-8 (IETD), caspase-1 (YVAD), or caspase-3 (DEVD)
but is not affected by inhibitors of serine proteases (TPCK). Our results indicate that partial ATP depletion of MDCK cells results in
apoptosis and that Fas- and caspase-mediated pathways may play a
critical role.
annexin; chemical anoxia; caspase inhibitor; necrosis; Madin-Darby canine kidney
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INTRODUCTION |
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APOPTOSIS, or programmed cell death, is characterized by distinct morphological changes consisting of cell shrinkage, nuclear condensation, and internucleosomal DNA fragmentation (19) and has been shown to play a significant role in the normal development of the kidney (20, 21). Apoptosis has also been observed in an increasing array of renal disorders (4, 23, 34) and particularly mediates renal tubule cell death in autosomal dominant and recessive polycystic kidney disease (41), ureteral obstruction (6), and renal transplant rejection (27). Perhaps most significantly, brief periods of in vivo renal ischemia are consistently associated with apoptosis of tubule cells (29, 33, 35, 43, 44). Induction of apoptosis in these situations may be associated with the activation of stress kinases and endonucleases (39, 42). The purpose of apoptosis in renal ischemia is controversial and intriguing. On the one hand, apoptosis during the late recovery period may be an important mechanism by which previously damaged cells are removed, allowing for tubule regeneration (36). Alternatively, apoptosis during the early periods of ischemia/reflow may represent a direct pathogenic mechanism by which tubule cells are damaged after an ischemic insult, and downregulation of apoptosis may offer a unique and powerful therapeutic approach to the amelioration of ischemic renal injury (1, 33, 39). This possibility has contributed to the interest in characterizing apoptosis in renal epithelial cells in vitro. Indeed, ischemic injury produced by intracellular ATP depletion has recently been shown to induce apoptosis in LLC-PK1 (porcine proximal tubule) cells (13-15) and in primary cultures of mouse proximal tubule cells (24). However, the mechanisms and pathways involved in the stimulus recognition and signal transduction leading to renal cell apoptosis after ischemic injury or ATP depletion are unknown.
The most thoroughly studied (and most physiologically relevant) apoptotic pathways result from Fas- or tumor necrosis factor (TNF) receptor-dependent protein-protein interactions, facilitated by molecules that possess a conserved "death domain" (11, 28, 40). Both Fas receptors and TNF receptors are integral membrane proteins that possess the characteristic death domain, enjoy a wide tissue distribution, and participate in the following two models for the induction of apoptosis in a variety of cell types. In the first model, an activated Fas receptor binds the Fas-associating protein with death domain (FADD), with resultant activation of caspase-8 (FLICE). This pathway can be stimulated in vitro by monoclonal antibodies to Fas (8). In the second model, activated TNF receptor 1 (TNFR1) interacts with TRADD (TNFR1-associated death domain protein), which, in turn, recruits FADD. Both pathways can stimulate the caspase-1 family (ICE) of proteases (2, 7) and finally result in the activation of caspase-3 (CPP32/Yama/Apopain). Caspase-3 cleaves several substrates, including poly(ADP)ribose polymerase (PARP), lamins, and actins, with resultant chromosomal DNA degradation and cellular morphological changes characteristic of apoptosis. Promising new findings suggest that inhibition of caspases protects against hypoxia/reperfusion-induced apoptosis in neurons (16), endothelial cells (17), and hepatocytes (37).
In this study, we have modified a previously described protocol of in
vitro ischemia (3, 5, 23, 25, 38) to achieve graded levels of
ATP depletion in Madin-Darby canine kidney (MDCK) collecting tubule
cells. We show that complete ATP depletion (<5% of control) results
in necrosis. Partial ATP depletion (10-65% of control) induces
apoptosis, as evidenced by internucleosomal DNA cleavage, changes in
cellular morphology, and alterations in the plasma membrane. This study
demonstrates, for the first time, that partial ATP depletion also
results in a significant upregulation of at least three molecules that
have been implicated in the early stimulus detection and transduction
phases of the apoptotic process in other cell types, namely Fas, Fas
ligand, and FADD. Exogenous application of stimulatory Fas monoclonal antibodies also induces programmed cell death in MDCK cells not depleted of ATP, indicating that they retain Fas-dependent apoptotic pathways. Furthermore, cleavage of PARP is evident after partial ATP
depletion, indicating the activation of caspases. Indeed, the apoptotic
cells display a significant increase in caspase-8 (FLICE) activity.
Finally, apoptosis induced by ATP depletion is ameliorated by
pretreatment with inhibitors of caspase-8 (IETD), caspase-1 (YVAD), or
caspase-3 (DEVD) but is not affected by inhibitors of serine proteases
(TPCK). Our results indicate that partial ATP depletion of MDCK cells
results in apoptosis and that the Fas-FADD axis and caspases
(specifically caspase-8) may play a critical role in the process.
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METHODS |
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Cell culture. MDCK type II cells, obtained from American Type Culture Collection (Manassas, VA), were passaged in DMEM with 10% fetal bovine serum (GIBCO BRL, Gaithersburg, MD) and analyzed within 1 to 2 days of reaching confluence.
ATP depletion.
We modified previously described protocols (3, 5, 24, 25, 38) to
achieve graded levels of ATP depletion in MDCK cells. The first set of
experiments utilized a combination of glycolytic
(2-deoxy-D-glucose; Sigma, St.
Louis, MO) and oxidative (antimycin A; Sigma) inhibitors. Confluent
monolayers of MDCK cells grown on six-well tissue culture-treated
polystyrene plates (Corning, NY) were washed with PBS and incubated in
Dulbecco's PBS with 1.5 mM CaCl2,
2 mM MgCl2, 10 µM antimycin A,
and 2 mM 2-deoxy-D-glucose for
various periods of time. For microscopy, cells were grown
directly on coverslips placed within the six-well plates. Control cells
were incubated in regular medium. ATP measurements were performed with
a luciferase-based assay kit (Sigma). Briefly, any nonadherent cells
were pelleted, washed with PBS, and solubilized in 500 µl of somatic
cell ATP-releasing agent, and the mixture was added to the washed
adherent cells. After a 5-min incubation period, the entire sample was
cleared of insoluble material by centrifugation, and equal volumes of
the supernatant were added to an equal volume of ATP assay
mix. ATP levels were measured with a luminometer and
expressed as a percentage of control values. In preliminary
experiments, we noted that inhibition of both glycolysis and oxidative
phosphorylation resulted in a very rapid and profound depletion of ATP
levels to <4% of controls (Fig. 1), and
that the cells displayed signs of necrotic death within 6 h of
treatment. Therefore, for the second set of experiments, we subjected
cells to Dulbecco's PBS with 1.5 mM
CaCl2, 2 mM
MgCl2, and 10 µM antimycin A
alone. This resulted in a duration-dependent, partial depletion of
intracellular ATP. Finally, measurements were also made with a third
set of conditions, during which cells previously subjected to 4 h of
partial ATP depletion were allowed to recover in regular medium for
varying times.
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Other experimental conditions.
To examine the effects of Fas and TNFR1 activation, for 4 or 8 h, we
exposed confluent MDCK cells to stimulatory Fas monoclonal antibody
(clone DX2; Clontech, La Jolla, CA) at 200 ng/ml, human TNF-
(Clontech) at 200 ng/ml, cycloheximide (Sigma) at 10 µg/ml, or a
combination of TNF-
and cycloheximide. To assess the role of
caspases and serine proteases, we pretreated cells for 2 h with an
inhibitor of caspase-1 (ICE), YVAD-cmk (50 µM; Clontech); an
inhibitor of caspase-8 (FLICE), IETD-fmk (10-50 µM; Clontech); an inhibitor of caspase-3 (CPP32/Yama/Apopain), DEVD-CHO (50 µM; Clontech); or with the serine protease inhibitor, TPCK (100 µM; Sigma). Cells were then subjected to 8 h of partial ATP
depletion with Dulbecco's PBS containing 1.5 mM
CaCl2, 2 mM
MgCl2, and 10 µM antimycin
A in the continued presence of the respective caspase inhibitor.
Apoptosis assays. Internucleosomal DNA fragmentation was detected primarily by DNA laddering (31). Briefly, nonadherent cells were pelleted, washed with PBS, and added to washed and scraped adherent cells. All cells were pelleted, resuspended in 500 µl of lysis buffer (1% SDS, 25 mM EDTA, and 1 mg/ml proteinase K, pH 8), and incubated overnight at 50°C. Ribonuclease A (10 mg/ml) was then added, for an additional 2-h incubation at 37°C. The chromosomal DNA was extracted with phenol/chloroform, precipitated with ethanol, and analyzed by agarose gel electrophoresis followed by staining with ethidium bromide to reveal the fragmentation pattern.
We confirmed DNA fragmentation in situ utilizing the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay (ApoAlert DNA Fragmentation Assay Kit; Clontech), by which fluorescein-dUTP incorporation at the free ends of fragmented DNA can be visualized by fluorescent microscopy (12, 32). Briefly, adherent cells, grown on coverslips and subjected to experimental conditions as above, were washed with PBS, fixed with 4% formaldehyde/PBS for 30 min at 4°C, permeabilized with 0.2% Triton X-100/PBS for 15 min at 4°C, and incubated with a mixture of nucleotides and TdT enzyme for 60 min at 37°C in a dark, humidified incubator. The reaction was terminated with 2× SSC, the cells were washed with PBS, and the coverslips were mounted on glass slides with Crystal/mount (Biomeda, Foster City, CA). Fluorescent nuclei were detected by visualization with a microscope equipped with fluorescein filters (IX70; Olympus). Because internucleosomal DNA cleavage may also be observed in necrotic cells, it was important to confirm the occurrence of apoptosis by additional assays (9, 23, 24). The characteristic morphological changes of apoptosis, including cell shrinkage and blebbing, were detected by direct light microscopy (24, 31). Because nuclear condensation is often seen during ATP depletion (independent of apoptosis), this morphological criterion was not used in this study. In addition, we used the annexin V-FITC cell membrane labeling assay (ApoAlert Annexin V Kit; Clontech) to detect translocation of phosphatidylserine from the inner face of the plasma membrane to the cell surface, where it binds an annexin V-FITC conjugate and serves as an early marker of apoptosis (26). Nonadherent cells, or cells grown on coverslips, were incubated with annexin V-FITC for 15 min at room temperature in the dark, and visualized by fluorescent microscopy with fluorescein filters, as before. In some cases, the cells were also stained with propidium iodide (Clontech) and visualized using rhodamine filters.Other methods. For SDS-PAGE, both adherent and nonadherent cells were washed with PBS, solubilized in 2× SDS sample buffer, and boiled for 10 min. Monoclonal antibodies to Fas, Fas ligand, and FADD used for Western analysis were from Transduction Laboratories, and the PARP monoclonal antibody was from Clontech. Immunodetection of transferred proteins was done by enhanced chemiluminescence (Amersham). We detected caspase-8 activity using a fluorescent assay kit (Clontech).
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RESULTS |
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Metabolic inhibition results in a duration-dependent depletion of intracellular ATP. Confluent MDCK cells subjected to inhibition of glycolysis (with 2-deoxy-D-glucose) and oxidative phosphorylation (antimycin A) displayed a rapid and profound decrease in intracellular ATP levels to <4% of controls within 2 h (Fig. 1; AA + deoxyglucose) and showed signs of necrotic death within 6 h of treatment (data not shown). Others have reported similarly severe ATP depletion under these conditions (38). However, cells subjected to only antimycin A showed a duration-dependent, partial depletion of intracellular ATP (Fig. 1; AA only). Thus ATP levels were 28 ± 4.7%, 15.5 ± 1.6%, and 9.6 ± 1.5% of controls (means ± SD from 3 separate experiments) at 2, 4, and 6 h, respectively. When cells previously subjected to 4 h of partial ATP depletion were then allowed to recover in regular medium, a significant elevation of ATP levels was evident, to 48.7 ± 3.3% and 65.3 ± 4.1% of controls (means ± SD from 3 separate experiments) at 4 and 8 h of recovery, respectively (Fig. 1; AA + recovery). Thus a spectrum of intracellular ATP depletion from ~10-65% of control was reproducibly achieved, depending on the duration of antimycin A treatment and the period of recovery.
Partial ATP depletion induces apoptosis.
We performed a series of apoptosis assays on confluent MDCK cells
subjected to varying degrees of intracellular ATP depletion. Approximately 80% and 60% of cells remained adherent after 6 and 8-12 h of ATP depletion, respectively. Both adherent and
nonadherent cells displayed evidence of activation of programmed cell
death. Internucleosomal DNA fragmentation was detectable by the
characteristic 180-bp laddering pattern in cells subjected to 6 h of
ATP depletion, and was obvious after 8-12 h of ATP depletion or
after 4 h of ATP depletion and either 4 or 8 h of recovery (Fig.
2). Because DNA laddering is a late
indicator of apoptosis, these results suggest that the apoptotic
cascade was activated during the early hours of treatment, when ATP
depletion was only partial.
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Partial ATP depletion results in upregulation of
Fas, Fas ligand, and FADD, and cleavage of PARP.
Because the Fas-FADD axis is thought to be an important mechanism for
the recognition and transduction of apoptotic stimuli, it was of
interest to determine whether these molecules may participate in the
programmed cell death induced by ATP depletion. Confluent MDCK cells
subjected to varying periods of partial ATP depletion were analyzed by
SDS-PAGE and Western blotting with monoclonal antibodies against Fas,
Fas ligand, or FADD. All three proteins were detected in low abundance
in control MDCK cells, and were significantly upregulated after 4 or 6 h of partial ATP depletion (Fas at 45 kDa, Fas ligand at 37 kDa, and
FADD at 24 kDa; Fig. 4).
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MDCK cells retain Fas-dependent apoptotic
pathways.
Given the above results, it was important to establish that MDCK cells
retain pathways required for Fas-mediated apoptosis. Application of
stimulatory monoclonal Fas antibodies induced apoptosis in cells not
depleted of ATP, as detected by positive annexin V staining after 4 h,
and by characteristic DNA laddering, as well as by in situ TUNEL assay
after 8 h of treatment (Fig. 5). Interestingly, MDCK cells were relatively resistant to the proapoptotic properties of either TNF- or cycloheximide alone. Programmed cell
death could be induced only by a combination of TNF-
and cycloheximide, suggesting that Fas-dependent pathways of apoptosis may
predominate in this cell line.
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Partial ATP depletion results in stimulation of
caspase-8 activity.
Because partial ATP depletion of MDCK cells results in upregulation of
the Fas-FADD axis, it was of interest to examine the activity of
caspase-8 (FLICE), which is the most proximal member of the caspase
family that is activated in Fas-dependent apoptotic pathways. Indeed,
we detected an increase in specific caspase-8 activity within 2 h of
ATP depletion, as determined by fluorescent assays (Fig.
6). This increase in caspase-8 activity was
blocked by preincubation of cells for 2 h with 10 µM IETD-fmk
peptide, a specific inhibitor of caspase-8 (Fig. 6).
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Caspase inhibitors ameliorate apoptosis induced by
partial ATP depletion.
Because our results indicated that partial ATP depletion in MDCK cells
induced apoptosis via caspase activation, it was of significant
interest to examine the effects of caspase inhibition. Pretreatment and
maintenance of cells with inhibitors of caspase-8 (IETD-fmk), caspase-1
(YVAD-cmk), or caspase-3 (DEVD-CHO) significantly ameliorated the DNA
laddering typically seen after 8 h of ATP depletion, whereas the serine
protease inhibitor TPCK did not have a protective effect (Fig.
7).
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DISCUSSION |
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These studies show that in vitro ischemia induced by partial ATP depletion results in apoptosis of MDCK cells, according to morphological, biochemical, and molecular criteria. We have demonstrated, for the first time, that this is associated with a marked overexpression of Fas, Fas ligand, and FADD, and cleavage of PARP, indicating activation of caspases. Stimulatory Fas monoclonal antibodies also induced programmed cell death in MDCK cells not depleted of ATP, confirming the presence of Fas-dependent apoptotic pathways. We have also shown, for the first time, that apoptosis induced by partial ATP depletion is accompanied by increased activity of caspase-8 and is ameliorated by pretreatment with inhibitors of caspase-8. These results indicate that Fas- and caspase-mediated pathways may play a critical role in the apoptosis induced by partial ATP depletion in MDCK cells.
Renal tubule cell apoptosis has been consistently observed after brief
periods of in vivo renal ischemia (29, 33, 35, 43, 44) and may
represent a direct mechanism by which tubule cells are damaged.
Inhibition of apoptosis may, therefore, offer a unique approach to the
amelioration of ischemic renal injury (1, 33, 39). This possibility has
stimulated interest in the characterization of renal epithelial cell
apoptosis in vitro. Indeed, ischemic injury produced by intracellular
ATP depletion has recently been shown to induce apoptosis in
LLC-PK1 cells (14-16) and
primary cultures of mouse proximal tubule cells (24). The latter study
elegantly demonstrated that proximal tubule cells subjected to partial
ATP depletion (25-70% of control) died by apoptosis, whereas
ATP depletion below
15% of control resulted in necrosis. Our
findings in MDCK cells are in agreement with that study and emphasize
the emerging concepts that 1)
apoptosis is a process that requires energy and
2) intracellular ATP concentration plays a crucial role in the determination of cell death fate by apoptosis or necrosis (10, 22). In Jurkat cells, ATP has been shown to
be required for apoptotic signal transduction, both upstream and
downstream of caspase activation, although it appears to be most
crucial to the final DNA fragmentation stage (10, 22).
Although activation of endonucleases (39) has been implicated in the
final postmortem phase (40), the proximal pathways involved in the
stimulus recognition, signal transduction, and effector phases of renal
tubule cell apoptosis after ischemic injury or ATP depletion are
largely unknown. Because both Fas expression and apoptosis have been
shown to increase in mouse renal tubule cells after endotoxin treatment
(30), we examined the role of Fas in MDCK cell apoptosis resulting from
partial ATP depletion. We have shown, for the first time, a significant upregulation of Fas early in in vitro ischemic injury and a concomitant increase in Fas ligand and FADD expression. Furthermore, we have shown
that Fas alone was pro-apoptotic in nonischemic MDCK cells, indicating
that Fas-dependent pathways of apoptosis are present in this cell line.
TNFR1, the other well-known receptor for apoptotic stimuli, does not
appear to play a major role because MDCK cells were relatively
resistant to TNF- alone. Similar results have recently been reported
for LLC-PK1 cells, where the
apoptotic effects of TNF-
were pronounced only in the presence of
cycloheximide (31). Taken together, our data suggest that death
domain-containing proteins such as those implicated in the Fas-FADD
cascade (28) participate in the apoptotic signal transduction after ATP
depletion of MDCK cells.
Because the Fas-FADD cascades are thought to sequentially activate the caspase-8, caspase-1, and caspase-3 families of proteases, we sought evidence for such activation. Indeed, partial ATP depletion resulted in the cleavage of PARP to its proteolyzed products, a phenomenon that is well known to result from caspase-3 activation (7). Furthermore, we have demonstrated, for the first time, a significant and specific increase in caspase-8 activity in MDCK cells after partial ATP depletion. Pretreatment of MDCK cells with inhibitors of caspase-8 resulted in a marked amelioration of apoptosis induced by partial ATP depletion. Inhibitors of caspase-1 and caspase-3 were also effective in preventing apoptosis of MDCK cells after partial ATP depletion. Similar results have recently been reported in cultured LLC-PK1 cells (18). In contrast, pretreatment with serine protease inhibitors was not effective in preventing apoptosis. Caspase inhibitors have also recently been shown to protect from hypoxia-induced apoptosis in neurons (16), endothelial cells (17), and hepatocytes (37). Thus our results lend support to the notion that inhibition of apoptosis may offer a novel approach to cytoprotection of a variety of cell types from hypoxic-ischemic injury.
In summary, we have demonstrated that partial ATP depletion induces apoptosis via Fas- and caspase-dependent pathways. It will be important to confirm the role of the Fas-FADD axis and caspases in apoptosis after in vivo renal ischemia, to identify other death domain-containing molecules that may also be cooperatively involved, and to identify other factors that may have an inhibitory effect (such as Bcl-2 and growth factors). A better understanding of such stimulatory and inhibitory influences on renal tubule cell apoptosis may reveal clues for the rational design of novel therapeutic interventions for ischemic renal injury.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grant DK-47072 to P. Devarajan.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. Devarajan, Montefiore Medical Center, Albert Einstein College of Medicine, Pediatric Nephrology, 111 East 210th St., Bronx, NY 10467 (E-mail: pdevaraj{at}aecom.yu.edu).
Received 2 June 1998; accepted in final form 22 February 1999.
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