Department of Medicine, Division of Nephrology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103
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ABSTRACT |
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Reactive oxygen species are recognized as
important mediators of biological responses. Hyperglycemia promotes the
intracellular generation of superoxide anion and hydrogen peroxide. In
several cell lines, oxidant stress has been linked to the activation of death programs. Here, we report for the first time that high ambient glucose concentration induces apoptosis in murine and human
mesangial cells by an oxidant-dependent mechanism. The signaling
cascade activated by glucose-induced oxidant stress included the
heterodimeric redox-sensitive transcription factor NF-B, which
exhibited an upregulation in p65/c-Rel binding activity and suppressed
binding activity of the p50 dimer. Recruitment of NF-
B and mesangial cell apoptosis were both inhibited by antioxidants, implicating oxidant-induced activation of NF-
B in the transmission of the death
signal. The genetic program for glucose-induced mesangial cell
apoptosis was characterized by an upregulation of the Bax/Bcl-2 ratio. In addition, phosphorylation of the proapoptotic protein Bad
was attenuated in mesangial cells maintained at high-glucose concentration, favoring progression of the apoptotic process. These
perturbations in the expression and phosphorylation of the Bcl-2 family
were coupled with the release of cytochrome c from mitochondria and caspase activation. Our findings indicate that in
mesangial cells exposed to high ambient glucose concentration, oxidant
stress is a proximate event in the activation of the death program,
which culminates in mitochondrial dysfunction and caspase-3 activation,
as the terminal event.
mesangial cell; reactive oxygen species; superoxide
anion; nuclear factor-B; mitochondria
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INTRODUCTION |
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CELL DEATH BY
APOPTOSIS IS a tightly orchestrated event under the
control of genetic programs, which have been highly conserved during
the evolutionary process (16). The identification of extracellular stimuli that promote cell death by apoptosis has become an area of intense investigation. Importantly, recent evidence that high-glucose concentration triggers the generation of reactive oxygen species (ROS) in mesangial cells (6, 18) raises
questions concerning the effect of oxidant stress on mesangial cell
survival. The enhanced production of free radicals has been linked to
increased mesangial matrix deposition, increased glomerular volume, and urinary transforming growth factor- excretion (6).
These alterations in the growth phenotype and biochemical properties of
mesangial cells are suppressed in genetically engineered mice
(6) with constitutively activated SOD. ROS have also been
implicated in the activation of death programs (42) and
ischemic preconditioning (10, 48). The late phase
of diabetic glomerulopathy is characterized by the loss of resident
glomerular cells, sclerosis of glomeruli, and occlusion, events that
correlate strongly with the decline in glomerular filtration rate
(29). Cell death may occur by necrosis or
apoptosis (4, 16). Necrosis is a process by which irreversible injury to the cell membrane results in the loss of structural integrity and the release of intracellular contents. Apoptosis, which is frequently not detected morphologically, is characterized by nuclear condensation, membrane blebbing, and the
formation of apoptotic bodies (4, 16, 45). Mesangial cells possess the genetic program for apoptosis (33,
35-37), and this mechanism of cell death has been reported
during the resolution phase of inflammatory glomerular lesions
(2, 26). An important question concerns the effect of
glucose-induced oxidant stress on mesangial cell survival and whether
ROS generation by this mechanism activates the genetic program for
apoptosis. Recruitment of the redox-sensitive transcription
factor NF-
B, plays a pivotal role in the regulation of cell survival
(19, 20, 22, 32, 39, 44, 46). Hyperglycemia has been
reported to promote nuclear translocation and activation of NF-
B in
several cell lines (27, 46). Presently, there are no data
available concerning the effect of glucose-induced NF-
B activation
on mesangial cell survival. Alternatively, the generation of ROS has
been implicated in the activation of cell death programs
(48), providing a rationale to examine the effect of
oxidant stress on the fate of mesangial cells exposed to a high-glucose environment.
In the present study, we have employed an SV40-transformed murine
glomerular mesangial cell (MMC) line and normal human mesangial cells
(NHMCs) to investigate the effect of high glucose on mesangial cell
survival. SV40 murine mesangial cells retain the phenotype and
biochemical characteristics observed in wild-type mesangial cells
(24, 43). NHMCs were harvested from normal human kidney tissue (BioWhittaker). To document the presence of oxidative stress, we
first measured the activity of the antioxidant enzymes, SOD, and
catalase in MMCs maintained at 5 and 25 mM glucose. To detect the
presence of intracellular OB activation protects against glucose-induced mesangial cell apoptosis.
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METHODS |
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Reagents.
NAC, DPI chloride, ascorbic acid, HEPES, penicillin, streptomycin,
chelerythrine, and D-glucose were purchased from Sigma. Calyculin A, leupeptin, PMSF, and protease inhibitor cocktail were
purchased from Calbiochem-Novabiochem. [-32P]ATP was
purchased from PerkinElmer Lifesciences. All culture media were
procured from GIBCO-BRL and BioWhittaker.
MMC culture. SV40-transformed MMCs (MES-13) were obtained from the American Type Culture Collection and maintained in a 3:1 mixture of DMEM and Ham's F-12 medium containing 5% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), HEPES (14 mM), and glucose (100 mg/dl) at 37°C in an atmosphere containing 5% CO2-95% air. Cultures were passaged twice a week at 1:8 split with the above-mentioned medium. Under these conditions MES-13 exhibit the phenotypic characteristics of mesangial cells, including staining for desmin, vimentin, Thy I, and types I and IV collagen by immunofluorescence (31, 43). After reaching 80% confluence, cells were plated in serum-free medium (SFM) with a composition identical to that described above, with the exception of 0.2% BSA in place of FBS. MMCs were subsequently incubated for 12 h and utilized immediately in the protocols outlined below.
To determine the effect of high-ambient glucose concentration on MMC survival, cells were exposed to either 5 or 25 mM glucose for 16 h. The percentage of cells undergoing apoptosis was determined by ELISA cell death detection assay and confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). In separate studies, MMCs were maintained in SFM containing 5 mM glucose+20 mM mannitol for 16 h to control for osmolar-induced cytotoxicity. A similar protocol was followed to assess the effects of antioxidants and chelerythrine on mesangial cell apoptosis. In these studies, MMCs were pretreated with NAC (50 µM), DPI (10 µM), ascorbic acid (100 µM), and chelerythrine (2 µM) for 1 h and placed in 25 mM glucose containing the same inhibitor for 16 h. For other biochemical measurements, cell preparations were obtained as described above.NHMC culture. To determine whether primary mesangial cells undergo apoptosis when exposed to 25 mM glucose, experiments were performed with NHMCs. The cell line, isolated from normal human tissue, was obtained from BioWhittaker. Culture conditions were as follows; NHMCs were maintained in mesangial cell basal medium (MsGM, BioWhittaker), supplemented with 5% FBS, 30 mg/l gentamicin, and 15 µg/l amphotericin-B in a humidified incubator at 37°C and 5% CO2-95% air. Cultures were allowed to reach 80% confluence before passage. For experimental studies, 70% confluent primary NHMC cultures were incubated in SFM (0.2% BSA in place of FBS in the above medium) for 12 h. NHMCs were then exposed to medium containing 5 or 25 mM glucose in the presence of inhibitors for 16 h. All experiments were performed with NHMCs from five to six passages.
Analysis of DNA fragmentation by ELISA. Histone-associated DNA fragments were quantified by the Cell Death Detection ELISA kit (Roche Diagnostic) according to the manufacturer's instructions. In brief, MMCs and NHMCs were plated in 24-well cell culture plates (Corning), and, after experimental treatments, cells (2 × 104) were washed with PBS (pH 7.4). Attached MMCs and NHMCs were incubated with a cell lysis buffer (Roche Diagnostic) and centrifuged, and the resultant supernatant (20 µl; 2 mg/ml protein), which contained cytoplasmic histone-associated DNA fragments characteristic for apoptosis, was applied onto a streptavidin-coated microtiter plate. A mixture of biotin-labeled anti-histone antibody and peroxidase-conjugated anti-DNA antibody was added, followed by 2-h incubation at room temperature. Anti-histone antibody binds to the histone component of the nucleosomes and simultaneously fixes the immune complex to the streptavidin-coated microtiter plate. The peroxidase-conjugated anti-DNA antibody reacts with the DNA component of nucleosomes. After removal of unbound antibodies by washing, the amount of nucleosomes was quantified by the peroxidase retained in the immune complex. The activity of peroxidase was determined photometrically with 2,2-azino-di-[3-ethylbenzthiazoline sulfonate] as a substrate. The values from quadruplet absorbance (at 405 nm) measurements were averaged. The values were normalized and data are presented as ratios of experimental/control.
In situ terminal deoxynucleotidyl transferase (TUNEL) assay. A TUNEL assay was performed to study the double-stranded cleavage of DNA in MMCs. Briefly, cells grown on chambered culture slides at 5 and 25 mM glucose for 16 h were washed in PBS, fixed with 10% neutral buffered formalin for 1 h at room temperature, and incubated with proteinase K (20 µg/ml) for 30 min in a moist chamber at room temperature. Mesangial cells were washed again with PBS and covered with 50 µl of terminal deoxynucleotidyl transferase reaction mixture containing 5 units of terminal deoxynucleotidyl transferase, 1.5 mM CoCl2, and 0.5 mM 2'-deoxyuridine-5'-triphosphate coupled to biotin (biotin-16-dUTP). All reagents were purchased from Boehringer Mannheim Biochemicals. Cultures were incubated in this solution for 60 min at 37°C in a humidified chamber, washed in PBS, and then incubated in staining buffer [4× concentrated SSC buffer and 5% (wt/vol) nonfat dry milk] for 30 min at room temperature in a moist chamber. After incubation, the cells were exposed to the staining solution containing 5 µg/ml of FITC-labeled Extravidin (Sigma), 4× concentrated SSC buffer, and 5% nonfat dry milk for 30 min in a moist chamber, washed with PBS, and finally mounted with Vectashield (Vector Labs) containing 4',6' diamidino-2-phenylindole dye to visualize nuclei. The staining was performed in quadruplets for each group, and 30 random fields (average 600 nuclei) were studied in each replicate. Double-stranded cleavage of DNA was determined by green (FITC) fluorescence in the nuclei.
Catalase and SOD activity. Cells (2 × 109) were lysed in 0.5 ml of buffer (M-PER Mammalian Extraction reagent; Pierce) containing 0.1 mM Na3VO4, 10 mM NaF, 0.5 mM PMSF, 1% Nonidet P-40, and protease inhibitor cocktail set I (Calbiochem-Novabiochem). The lysate samples were kept on ice for 1 h and centrifuged at 10,000 rpm for 20 min, and the supernatants from different groups were used for catalase and SOD activities. A sample volume was normalized for an equal amount of proteins in cells cultured in 5 and 25 mM glucose for 16 h. Catalase activity was determined at 520 nm by using a catalase assay kit (Calbiochem-Novabiochem) in triplicate; enzymatic activity was calculated by using a catalase standard curve and expressed as catalase units per milligram protein. SOD activity was determined by employing an SOD assay kit (Calbiochem-Novabiochem). The SOD activity was calculated at 525 nm from the ratio of the autooxidation rate of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorine in the presence and absence of lysates.
Immunofluorescent detection of glycooxidative stress.
Glucose-mediated oxidative stress in MMCs and NHMCs was studied by
trafficking of 2,3,4,5,6-pentafluorodihydrotetramethylrosamine (PF-H2TMRos or Redox Sensor red CC-1, Molecular Probes)
using fluorescence microscopy. MMCs and NHMCs were maintained for
16 h under one of the following conditions: SFM+5 mM glucose,
SFM+25 mM glucose, or SFM+25 mM glucose+NAC (50 µM). At the end of
the incubation, cells were loaded at 37°C for 20 min with Redox
Sensor red CC-1 (1 µM) and a mitochondria-specific fluorescent dye,
MitoTracker green FM (50 nM; Molecular Probes). Redox Sensor red CC-1
is oxidized in the presence of O
EMSA for NF-B activity.
Nuclear extracts of mesangial cells were prepared with an NE-PER kit
(Pierce). Approximately 5 × 106 cells were used for
each determination. Nuclear proteins were assayed for NF-
B p50 and
p65/c-Rel DNA binding activity by using NF-
B/c-Rel gelshift plus
assay kit (Geneka Biotechnology). The NF-
B and Rel ready-to-label
wild-type double-stranded oligo probes were also supplied by Geneka
Biotechnology. The oligonucleotides were labeled with
[
-32P]ATP and further purified by using a NUCTRAP
probe purification column (Stratagene). For the oligonucleotide-protein
complex (bandshift), nuclear extracts and purified hot probes were
premixed and incubated at 10°C for 20 min. Unlabeled wild-type and
unlabeled mutant oligonucleotides were included to determine the
specificity of the competition bandshifts. The oligonucleotide-protein
complexes were loaded onto 5% native polyacrylamide gels (38:2)
precooled at 4°C in 1× Tris-borate-EDTA (pH 8.0) buffer, and the
gels were electrophoresed at 100 V for 2 h, dried, and exposed for
autoradiographic visualization. The bands were quantified by using the
computerized image analysis software Un-Scan-IT (Automated Digitizing System).
Immunoelectrophoresis analysis. MMCs were homogenized in lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 7.5), 0.1 mM Na3VO4, 1 mM NaF, 0.5 mM PMSF, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, 0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin. Samples were separated by 8% (wt/vol) SDS-PAGE for p53 and phospho-p53 Ser392 or 12% (wt/vol) acrylamide gels for Bax, Bcl-2, Bad, phospho-Bad Ser112, and phospho-Bad Ser136. Proteins were transferred onto nitrocellulose membranes by using a semidry transfer cell apparatus (Bio-Rad). Primary rabbit polyclonal antibodies for Bax and Bcl-2 (1:800; Santa Cruz Biotechnology); Bad, phospho-Bad Ser112, phospho-Bad Ser136 (1:800; Cell Signaling Technology), and mouse monoclonal antibody for p53 (1:800; Santa Cruz Biotechnology); and phospho-p53 Ser392 (1:800; Calbiochem-Novabiochem) were used. For Bax, Bcl-2, p53, and phospho-p53 Ser392, secondary antibody was used at a dilution of 1:5,000. For Bad, phospho-Bad Ser112, and phospho-Bad Ser136, a dilution of 1:3,000 of secondary antibody was used. The blots were developed by using the ECL kit (Amersham-Pharmacia), and the bands were scanned and quantified as described above.
Preparation of subcellular fractions. MMCs were harvested from the cultures and fractionated into cytosol and mitochondria by using an ApoAlert cell fractionation kit (Clontech Laboratories). Briefly, cells were incubated with fractionation buffer (Clontech Laboratories) on ice for 10 min, homogenized in an ice-cold Dounce tissue grinder, and centrifuged at 700 g for 10 min at 4°C. The pellet was discarded and the supernatant was further centrifuged at 10,000 g for 25 min at 4°C. The supernatant (cytosolic fraction) and pellet (mitochondrial fraction) were collected separately, and protein concentration in these fractions was determined by Bio-Rad assay. The fractions were subjected to SDS-PAGE (12% gels) to ascertain the separation of cytosolic and mitochondrial fractions. Anti-cytochrome c oxidase subunit IV (COX IV) antibody (1:1,000; Molecular Probes) was used to probe the protein COX IV as a marker in mitochondria and its absence in cytosol by Western blot analysis. For detection, horseradish peroxidase-linked secondary antibody was used at a dilution of 1:3,000. Fractions obtained from cytosolic and mitochondrial compartments were also probed with cytochrome c antibody by immunoelectrophoresis. Mouse monoclonal anti-cytochrome c antibody (BD Biosciences) was used at a dilution of 1:250, and the protein was detected by horseradish peroxidase-linked secondary antibody (1:3,000). Cytochrome c was detected and quantified in the cytosolic fraction from different groups as described above.
Cleaved caspase-3 expression. Lysates of MMC were subjected to 4-20% gradient gel electrophoresis, transferred onto nitrocellulose, and probed for the presence of cleaved caspase-3. For this, blots were incubated with rabbit polyclonal antibody (1:800; Cell Signaling Technology), washed, and treated with secondary antibody (1:5,000). The cleaved caspase-3 expression was quantified as described above.
Statistical analysis.
Data are expressed as means ± SE. Comparison between two values
was performed by unpaired Student's t-test. For multiple
comparisons among different groups of data, the significant differences
were determined by the Bonferroni method. Significance was defined at
P 0.05.
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RESULTS |
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High glucose promotes MMC apoptosis.
To determine the effect of high ambient glucose concentration on MMC
survival, cells were plated and cultured in SFM containing 5 or 25 mM
glucose for 16 h. Apoptotic cell death was detected by ELISA
cell death assay, which detects histone-associated DNA fragments within
the cytoplasmic fraction of cells with high specificity. As shown in
Fig. 1, MMCs maintained in SFM+5 mM
glucose for 16 h exhibited a detectable baseline level of
apoptosis. A 50% increase in apoptotic
cell death was detected when the glucose concentration in the media was
increased to 25 mM (P 0.01). To control for the potential
effect of osmolarity on MMC apoptosis, in separate studies,
cell death was assessed under osmolar equivalent conditions of 5 mM
glucose+20 mM mannitol for 16 h. The percentage of apoptotic MMCs was similar to baseline values detected in cells maintained in
SFM+5 mM glucose. As shown in Fig. 2,
TUNEL staining confirmed the increased number of apoptotic nuclei
in cells maintained in SFM+25 mM glucose. Taken together, these results
indicate that high ambient glucose concentration activates the death
program in MMCs.
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High glucose promotes intracellular ROS generation.
High ambient glucose concentration has been reported to alter the redox
status of cells through the overproduction of ROS (27,
31). H2O2 is a toxic product of both
aerobic metabolism and pathogenic ROS production. The heme-containing
enzyme catalase metabolizes H2O2 by
dismutation, resulting in the formation of O2+2
H2O. O 0.01), whereas
SOD activity increased twofold (P
0.01). To provide
a more direct assessment of oxidative stress, we performed additional
experiments with the redox-sensitive dye Redox Sensor red CC-1. These
studies were also performed in NHMCs to determine whether glucose
promotes oxidative stress in this primary mesangial cell line. MMCs
(Fig. 4, A-C) and NHMCs
(Fig. 4, D-F) were loaded with Redox Sensor red CC-1
and the mitochondria-specific dye MitoTracker green FM. Redox Sensor
red CC-1 is oxidized in the presence of O
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Role of ROS in glucose-induced apoptosis.
H2O2 has been reported to promote
oxidant-induced apoptosis in rat mesangial cells
(17). The prooxidant environment of MMCs and NHMCs
maintained at 25 mM glucose implicates oxidant stress as a potential
trigger for apoptosis. To determine whether glucose-induced intracellular ROS generation activates the death program, ascorbic acid
and the cell permeable thiol antioxidants NAC and DPI were added
separately to the culture media. As shown in Fig.
5A, NAC, DPI, and ascorbic
acid reduced MMC apoptosis to baseline values, whereas the PKC
inhibitor chelerythrine had no detectable effect on glucose-induced
cell death. Figure 5B shows identical experiments at 5 mM
glucose, indicating the absence of cytotoxic effects of the
antioxidants and chelerytherine at the concentrations used. To
determine whether NHMCs exhibit a similar pattern of glycooxidative stress, an identical experimental protocol was performed. As shown in
Fig. 6, 25 mM glucose increased NHMC
apoptosis by ~50% (P 0.001). The
glucose-induced component of apoptosis is markedly attenuated
by the antioxidants NAC, DPI, and ascorbic acid (P
0.001). Similar to MMCs, chelerytherine has no detectable effect on
glucose-induced NHMC apoptosis. These findings strongly suggest that 25 mM glucose induces MMC and NHMC apoptosis by an
oxidant-dependent mechanism.
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ROS-dependent activation of NF-B.
In several cell lines, NF-
B has been identified as a target for
ROS-dependent signals (27, 46). In the inactive state, NF-
B is sequestered in the cytoplasm, which is associated with an
endogenous inhibitor protein of the I
B family (19).
Diverse stimuli activate NF-
B through the phosphorylation of IKK.
The NF-
B-I
B complex is phosphorylated by IKK, resulting in
ubiquination and proteosomal degradation of I
B, promoting nuclear
translocation of NF-
B. To determine whether NF-
B is activated by
ROS-dependent signals in MMCs maintained in SFM+25 mM glucose, gel
shift assays were performed with nuclear proteins and an NF-
B
binding site-specific probe. As shown in Fig.
7, A and B, NF-
B
binding complexes were detected in MMCs maintained at 5 and 25 mM
glucose. The identity of the bands was determined by competitive
bandshift analysis using unlabeled consensus or mutant oligonucleotide
(Fig. 7, C and D). As shown in the densitometric
analysis (Fig. 7F), nuclear proteins from mesangial cells
maintained at 25 mM glucose exhibited an upregulation of p65/c-Rel
binding (P
0.001). This upregulation in p65/c-Rel binding
activity was suppressed by the antioxidants NAC and DPI (P
0.001). Conversely, as shown in Fig. 7E, p50 DNA
binding activity was inhibited by 25 mM glucose (P
0.001). Inclusion of antioxidants NAC and DPI restored p50 binding
activity to baseline. NAC had no detectable effect on binding
activities of p50 or p65/c-Rel dimers at 5 mM glucose. Taken together,
the aggregate data indicate that in serum-starved MMCs, 25 mM glucose selectively activates the p65/c-Rel dimer of NF-
B by an
oxidant-dependent mechanism. ROS-dependent signals appear to exert a
deleterious effect on p50 binding, which can be reversed in the
presence of antioxidants.
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Expression of apoptosis-related factors: Bcl-2 family of
proteins.
To determine whether MMC apoptosis in response to 25 mM glucose
is characterized by alteration in the Bax/Bcl-2 ratio and phosphorylation status of Bad and p53, immunoblot analyses were performed. As shown in Fig.
8A, the ratio of Bax/Bcl-2 was
increased in lysates of mesangial cells maintained at 25 mM glucose.
The upregulation of Bax/Bcl-2 ratio was completely prevented by NAC (P 0.001). Interestingly, NAC has an inhibitory effect on
Bax/Bcl-2 ratio at 5 mM glucose as well. The phosphorylation status of
Bad (Fig. 8, B and C) was also altered in MMCs
maintained at 25 mM glucose. Phosphorylation at serine residues is
recognized as a mechanism of inactivating the proapoptotic function
of Bad (16, 30). As shown in Fig. 8, B and
C, phosphorylation at Ser112 and Ser136 was markedly
attenuated in MMCs exposed to 25 mM glucose. The inclusion of NAC in
the culture media of MMCs at 25 mM glucose enhanced the phosphorylation
at Ser112 and Ser136 of the Bad protein. Because NF-
B is known to
regulate p53 expression (22, 44) and Bax is a target gene
for p53 (23, 47), we examined the phosphorylation status
of the p53 protein. Ser392 is located at the COOH terminus of p53 and
linked to transcriptional activation (7). Phosphorylation
of Ser392 was upregulated in MMCs exposed to 25 mM glucose (Fig.
8D), implicating p53 in the alterations of Bax and Bcl-2
expression (7). This upregulation of Ser392 phosphorylation was blocked by addition of NAC to the culture medium.
Taken together, the data indicate that in MMCs maintained at 25 mM
glucose, oxidative stress promotes directional shifts in the expression
and phosphorylation status of the Bcl-2 family of proteins that favors
progression of the apoptotic process.
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Cytochrome c release and caspase activation.
To determine whether perturbations in the Bcl-2 family of proteins are
coupled with the release of cytochrome c from the
mitochondrial compartment and caspase activation, immunoblots were
performed on cytosolic- and mitochondria-enriched fractions of MMCs. As shown in Fig. 9A,
top, 25 mM glucose (H) increased the release of cytochrome
c from mitochondria compared with MMCs maintained at 5 mM
glucose (C). To control for possible contamination of cytosol by
mitochondrial proteins, immunoblots were probed with an antibody
against COX IV. To control for variations in loading conditions,
Coomassie blue-stained gels (Fig. 9B) are shown that document equal loading conditions in cytosolic and mitochondrial fractions. As shown in Fig. 9A, bottom, COX IV
was not detected in the cytosol of MMCs maintained at 5 or 25 mM
glucose. The inclusion of NAC in MMC cultures maintained at 25 mM
glucose markedly attenuated cytochrome c release (Figs. 9,
A and C). Immunoblot analysis of the cytochrome
c enriched mitochondrial fraction did not detect changes in
cytochrome c content among the different groups.
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DISCUSSION |
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In the present study, we demonstrate that in vitro exposure of
MMCs and NHMCs to 25 mM glucose activates the genetic program for
apoptosis. Glucose-induced apoptosis was independent of
mechanical strain and osmolar forces and triggered by oxidant stress.
Direct evidence is also provided for perturbations in the pro- and
antiapoptotic members of the Bcl-2 family, culminating in the
release of cytochrome c from mitochondria and caspase
activation. Finally, we demonstrated that inhibition of the
redox-sensitive transcription factor NF-B prevents glucose-induced
MMC apoptosis.
High glucose promotes mesangial cell apoptosis. Hyperglycemia dominates the pathophysiology and clinical course of type 1 and type 2 diabetes. Compelling evidence from the Diabetes Control and Complications Trial indicates that rigorous control of blood glucose reduces the risk of long-term microvascular complications (6a). In the present study, we demonstrate for the first time that high ambient glucose concentration activates the genetic program for MMC and NHMC apoptosis. The cytotoxic property of high glucose was independent of osmolar forces, because the percentage of apoptotic MMCs with an osmolar equivalent glucose-mannitol media did not differ from control values. The latter property may serve to protect mesangial cells from transient elevations in osmolarity or reflect the unique ability of high glucose to trigger activation of intracellular signaling molecules involved in the expression of the death program. Hyperglycemia has recently been reported to induce apoptosis in cardiac myocytes (7, 8), whereas in vascular smooth muscle cells, hyperglycemia was found to protect against apoptotic cell death (14). These observations suggest that the cytotoxic properties of glucose vary across cell lines. Several factors may operate to limit detection of this form of cell death under in vivo conditions. First, cell loss is not a prominent characteristic during the early phases of diabetic glomerulopathy (38), in which thickening of the basement membrane and glomerular hypertrophy are the most characteristic deviations from normal (25). Second, during the late phases of this disorder, expansion of the mesangial matrix dominates the histological picture (28) along with sclerosis and occlusion of glomeruli (4, 29). Third, cell death by apoptosis does not result in sclerosis or residual scarring (16) and, in the absence of an immunocytochemical analysis, cannot be detected morphologically (7). Taken together, our finding that high glucose, independent of hemodynamic or physical forces, promotes mesangial cell apoptosis raises additional questions concerning the fate of mesangial cells in diabetic nephropathy.
High glucose, ROS, and mesangial cell apoptosis.
Multiple lines of evidence have established a role for ROS as important
mediators of cell biology (9, 21, 34).
O
Oxidant-dependent NF-B activation and mesangial cell
apoptosis.
NF-
B comprises an inducible family of transcriptional factors that
are important regulators of host immune and inflammatory responses. In
addition, NF-
B-dependent signaling pathways have been implicated in
cell survival (19, 20, 22, 32, 39, 44, 46). Stimulation of
the NF-
B signaling pathway occurs by phosphorylation and degradation
of the NF-
B inhibitory protein I
B, with subsequent translocation
of NF-
B to the nucleus (5, 19). Our results indicate a
differential effect of 25 mM glucose on NF-
B binding activity, which
is ROS dependent. The upregulation in p65/c-Rel activity was markedly
suppressed in the presence of NAC or DPI, implying that ROS
preferentially target this dimer. Interestingly, p50 binding activity,
which was downregulated by high glucose, was restored by NAC and DPI,
suggesting an inhibitory effect of ROS on this dimer. Previous studies
in other cell lines (27, 46) have documented an
association between glucose-induced ROS generation and NF-
B
activity, suggesting this transcription factor may modulate cellular
responses to a high-glucose environment. The observation that NAC and
DPI not only blocks the recruitment of NF-
B but also prevents MMC
apoptosis is consistent with such a hypothesis. The cumulative
results strongly suggest that hyperglycemia recruits NF-
B via
ROS-dependent signals and implicates oxidant-dependent activation
of NF-
B in the signaling cascade of glucose-induced MMC apoptosis.
Oxidant stress and proapoptosis gene program.
A growing body of evidence supports the cytotoxic potential of ROS
(12, 13, 42) and their direct participation in the activation of the death program (9, 17). The present study is the first report documenting that glucose-induced oxidant stress activates the genetic program for mesangial cell apoptosis.
Until recently, the redox-sensitive transcription factor NF-B was
viewed as prosurvival and antiapoptotic (22, 44). It
is now recognized that NF-
B may also assume a proapoptotic
function through the regulation of apoptosis genes. The nuclear
transcription factor p53 regulates proapoptotic gene programs
(20, 32), and NF-
B has been reported to be a
prerequisite for the induction of p53-mediated apoptosis
(32). Although our data do not establish a cause and effect relationship between NF-
B and the genetic program for apoptosis, the marked attenuation of mesangial cell death in
association with p65/c-Rel inhibition is consistent with such a
hypothesis. Alternatively, we demonstrate phosphorylation of p53 at
Ser392, a modification of the p53 protein linked with transcriptional activation (7). Our results also indicate that MMC
apoptosis was accompanied by an increase in the Bax/Bcl-2
ratio, an alteration that favors progression of apoptosis
(16). High-glucose concentration attenuated
phosphorylation of Bad at Ser112 and Ser136. The latter finding
is consistent with the proapoptosis function of Bad (30). Of note, the antioxidant NAC restored phosphorylation of Bad to levels
detected at 5 mM glucose. The increase in p53 phosphorylation detected
at 25 mM glucose was prevented by the addition of NAC, as was the
increase in the Bax/Bcl-2 ratio. It seems reasonable to infer that
either oxidant stress directly activates p53 (33) or
NF-
B and p53 may cooperate to regulate the expression of Bax (20, 32, 44). Additional studies will be required to test this hypothesis.
Oxidant stress and mitochondrial dysfunction. The mitochondria are key determinants of cell death and cell survival (1, 11). Cytochrome c release by mitochondria and caspase activation are critical events in triggering oxidant-induced apoptosis (11). Anti- and proapoptotic proteins of the Bcl-2 family possess a COOH-terminal domain, which serves to target proteins to specific cell compartments (16). Several mechanisms are utilized by the antiapoptotic protein Bcl-2 to interrupt transmission of death signals, direct antioxidant effect, protein-protein interaction, and inhibition of cytochrome c release from mitochondria (16). This latter mechanism enables Bcl-2 to directly interfere with cytochrome c-dependent activation of caspases, a key event in the execution of the death signal. The antiapoptotic function of Bcl-2 may be neutralized by translocation of Bad and Bax from the cytosol to the mitochondria, with the subsequent formation of heterodimers (16, 42). Cytochrome c release is tightly regulated by protein-protein interactions among Bcl-2, Bax, and Bad (42). A growing body of evidence suggests that release of cytochrome c from mitochondria commits a cell to die by either apoptosis or necrosis (11). Cytochrome c was detected in the cytosolic fractions of MMC maintained at 25 mM glucose, strongly suggestive of an oxidant-induced mitochondrial dysfunction (1, 11, 42). Of note, we were unable to detect alterations in the cytochrome c content of mitochondrial fractions in experimental and control groups. This may be due to the enriched cytochrome c content of mitochondria compared with the relatively small fraction released to the cytosol. Moreover, the expression of cleaved caspase-3 was upregulated by 25 mM glucose. The antioxidants NAC and DPI markedly attenuated the glucose-induced upregulation of cleaved caspase-3 expression. Taken together, our data indicate that oxidant-induced perturbations in the Bcl-2 family of proteins facilitate the release of cytochrome c from mitochondria, initiating the terminal cascade of the death signal.
The present study has certain limitations. First, the duration of exposure to 25 mM glucose was brief compared with an in vivo model of hyperglycemia. Second, although beyond the scope of this investigation, infection of mesangial cells with constitutively active mutants of I ![]() |
APPENDIX A |
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ACKNOWLEDGEMENTS |
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We acknowledge Drs. G. P. Yang
and V. Gaussin (University of Medicine and Dentistry of New Jersey) for
help with the TUNEL assay. This work was partially supported by
American Heart Association Grant-in-Aid 9750856A (A. Malhotra), a
research grant from the Foundation of University of Medicine and
Dentistry of New Jersey Annual Grants Program (A. Malhotra), and
support from the Summit Area Public Foundation through the generosity of the Mrs. Elaine B. Burnett Fund (L. G. Meggs and S. Baskin).
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. G. Meggs, Div. of Nephrology and Hypertension, Dept. of Medicine, MSB I-524, Univ. of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103 (E-mail: meggslg{at}umdnj.edu).
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. Section 1734 solely to indicate this fact.
First published November 5, 2002;10.1152/ajprenal.00137.2002
Received 11 April 2002; accepted in final form 30 October 2002.
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