High glucose promotes mesangial cell apoptosis byoxidant-dependent mechanism - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:34 |只看该作者 |倒序浏览 |打印

作者：Barinder P. S.Kang, StanleyFrencher, VenkateshReddy, AlexKessler, AshwaniMalhotra,  Leonard G.Meggs作者单位：Department of Medicine, Division of Nephrology, University ofMedicine and Dentistry of New Jersey-New Jersey Medical School,Newark, New Jersey 07103 7 D# ~5 u. v8 {- J# B


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      【摘要】" P" d- C5 c" c3 S& c
   Reactive oxygen species are recognized asimportant mediators of biological responses. Hyperglycemia promotes theintracellular generation of superoxide anion and hydrogen peroxide. Inseveral 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 humanmesangial cells by an oxidant-dependent mechanism. The signalingcascade activated by glucose-induced oxidant stress included theheterodimeric redox-sensitive transcription factor NF- B, whichexhibited an upregulation in p65/c-Rel binding activity and suppressedbinding 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 deathsignal. The genetic program for glucose-induced mesangial cellapoptosis was characterized by an upregulation of the Bax/Bcl-2 ratio. In addition, phosphorylation of the proapoptotic protein Badwas attenuated in mesangial cells maintained at high-glucose concentration, favoring progression of the apoptotic process. Theseperturbations in the expression and phosphorylation of the Bcl-2 familywere coupled with the release of cytochrome c from mitochondria and caspase activation. Our findings indicate that inmesangial cells exposed to high ambient glucose concentration, oxidantstress is a proximate event in the activation of the death program,which culminates in mitochondrial dysfunction and caspase-3 activation,as the terminal event. 6 L4 F* D! |# k' b/ o  D7 Z
      【关键词】 mesangial cell reactive oxygen species superoxideanion nuclear factor B mitochondria
   INTRODUCTION

CELL DEATH BY APOPTOSIS IS a tightly orchestrated event under thecontrol of genetic programs, which have been highly conserved duringthe 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 ) raisesquestions concerning the effect of oxidant stress on mesangial cellsurvival. The enhanced production of free radicals has been linked toincreased mesangial matrix deposition, increased glomerular volume, and urinary transforming growth factor- excretion ( 6 ).These alterations in the growth phenotype and biochemical properties ofmesangial cells are suppressed in genetically engineered mice( 6 ) with constitutively activated SOD. ROS have also beenimplicated in the activation of death programs ( 42 ) andischemic preconditioning ( 10, 48 ). The late phaseof diabetic glomerulopathy is characterized by the loss of residentglomerular cells, sclerosis of glomeruli, and occlusion, events thatcorrelate strongly with the decline in glomerular filtration rate( 29 ). Cell death may occur by necrosis orapoptosis ( 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 theformation 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 reportedduring the resolution phase of inflammatory glomerular lesions( 2, 26 ). An important question concerns the effect ofglucose-induced oxidant stress on mesangial cell survival and whetherROS generation by this mechanism activates the genetic program forapoptosis. Recruitment of the redox-sensitive transcriptionfactor NF- B, plays a pivotal role in the regulation of cell survival( 19, 20, 22, 32, 39, 44, 46 ). Hyperglycemia has beenreported to promote nuclear translocation and activation of NF- B inseveral cell lines ( 27, 46 ). Presently, there are no dataavailable concerning the effect of glucose-induced NF- B activationon mesangial cell survival. Alternatively, the generation of ROS hasbeen implicated in the activation of cell death programs( 48 ), providing a rationale to examine the effect ofoxidant stress on the fate of mesangial cells exposed to a high-glucose environment.+ i- `6 A8 J( z) @! s5 l) ]8 }" n
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In the present study, we have employed an SV40-transformed murineglomerular mesangial cell (MMC) line and normal human mesangial cells(NHMCs) to investigate the effect of high glucose on mesangial cellsurvival. SV40 murine mesangial cells retain the phenotype andbiochemical 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, wefirst measured the activity of the antioxidant enzymes, SOD, andcatalase in MMCs maintained at 5 and 25 mM glucose. To detect thepresence of intracellular O 2 − · and H 2 O 2, MMCs and NHMCs were loaded with theoxidant-sensitive dye 2,3,4,5,6 pentafluorodihydrotetramethylrosamine(Redox Sensor red CC-1) ( 15 ). The cytotoxic effect ofglucose-induced ROS generation was evaluated in the presence andabsence of the free radical scavengers N -acetyl L -cysteine (NAC) and diphenyleneiodonium (DPI). To assessthe effect of the prooxidant environment on the integrity ofmitochondria, immunoblots were performed to evaluate the subcellulardistribution of cytochrome c. Finally, we demonstrate thatinhibition of ROS-dependent NF- B activation protects against glucose-induced mesangial cell apoptosis.

METHODS

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 werepurchased from Calbiochem-Novabiochem. [ - 32 P]ATP waspurchased from PerkinElmer Lifesciences. All culture media wereprocured from GIBCO-BRL and BioWhittaker., G1 @0 C- K- o. ]2 l6 h

MMC culture. SV40-transformed MMCs (MES-13) were obtained from the American TypeCulture Collection and maintained in a 3:1 mixture of DMEM and Ham'sF-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 anatmosphere containing 5% CO 2 -95% air. Cultures werepassaged twice a week at 1:8 split with the above-mentioned medium.Under these conditions MES-13 exhibit the phenotypic characteristics ofmesangial cells, including staining for desmin, vimentin, Thy I, andtypes 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 theexception of 0.2% BSA in place of FBS. MMCs were subsequently incubated for 12 h and utilized immediately in the protocolsoutlined below.( b: L5 K5 X/ j3 ]

To determine the effect of high-ambient glucose concentration on MMCsurvival, 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 mMglucose 20 mM mannitol for 16 h to control for osmolar-induced cytotoxicity. A similar protocol was followed to assess the effects ofantioxidants and chelerythrine on mesangial cell apoptosis. Inthese studies, MMCs were pretreated with NAC (50 µM), DPI (10 µM),ascorbic acid (100 µM), and chelerythrine (2 µM) for 1 h andplaced in 25 mM glucose containing the same inhibitor for 16 h. For other biochemical measurements, cell preparations were obtained as described above.0 D3 J8 `# S* T: `, {5 M/ {

NHMC culture. To determine whether primary mesangial cells undergo apoptosiswhen exposed to 25 mM glucose, experiments were performed with NHMCs.The cell line, isolated from normal human tissue, was obtained fromBioWhittaker. 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/lamphotericin-B in a humidified incubator at 37°C and 5%CO 2 -95% air. Cultures were allowed to reach 80%confluence before passage. For experimental studies, 70% confluentprimary NHMC cultures were incubated in SFM (0.2% BSA in place of FBSin the above medium) for 12 h. NHMCs were then exposed to mediumcontaining 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 DeathDetection ELISA kit (Roche Diagnostic) according to the manufacturer'sinstructions. In brief, MMCs and NHMCs were plated in 24-well cellculture plates (Corning), and, after experimental treatments, cells(2 × 10 4 ) were washed with PBS (pH 7.4). AttachedMMCs and NHMCs were incubated with a cell lysis buffer (RocheDiagnostic) and centrifuged, and the resultant supernatant (20 µl; 2 mg/ml protein), which contained cytoplasmic histone-associated DNAfragments characteristic for apoptosis, was applied onto astreptavidin-coated microtiter plate. A mixture of biotin-labeledanti-histone antibody and peroxidase-conjugated anti-DNAantibody was added, followed by 2-h incubation at room temperature.Anti-histone antibody binds to the histone component of the nucleosomesand simultaneously fixes the immune complex to the streptavidin-coatedmicrotiter plate. The peroxidase-conjugated anti-DNA antibody reactswith the DNA component of nucleosomes. After removal of unboundantibodies by washing, the amount of nucleosomes was quantified by theperoxidase retained in the immune complex. The activity of peroxidasewas determined photometrically with2,2-azino-di-[3-ethylbenzthiazoline sulfonate] as a substrate. Thevalues from quadruplet absorbance (at 405 nm) measurements wereaveraged. The values were normalized and data are presented as ratiosof experimental/control.

In situ terminal deoxynucleotidyl transferase (TUNEL) assay. A TUNEL assay was performed to study the double-stranded cleavage ofDNA in MMCs. Briefly, cells grown on chambered culture slides at 5 and25 mM glucose for 16 h were washed in PBS, fixed with 10% neutralbuffered formalin for 1 h at room temperature, and incubated withproteinase K (20 µg/ml) for 30 min in a moist chamber at roomtemperature. Mesangial cells were washed again with PBS and coveredwith 50 µl of terminal deoxynucleotidyl transferase reaction mixturecontaining 5 units of terminal deoxynucleotidyl transferase, 1.5 mMCoCl 2, and 0.5 mM 2'-deoxyuridine-5'-triphosphate coupled to biotin (biotin-16-dUTP). All reagents werepurchased from Boehringer Mannheim Biochemicals. Cultures wereincubated in this solution for 60 min at 37°C in a humidifiedchamber, washed in PBS, and then incubated in staining buffer [4×concentrated SSC buffer and 5% (wt/vol) nonfat dry milk] for 30 minat room temperature in a moist chamber. After incubation, the cellswere exposed to the staining solution containing 5 µg/ml ofFITC-labeled Extravidin (Sigma), 4× concentrated SSC buffer, and 5%nonfat dry milk for 30 min in a moist chamber, washed with PBS, andfinally mounted with Vectashield (Vector Labs) containing 4',6'diamidino-2-phenylindole dye to visualize nuclei. The staining wasperformed in quadruplets for each group, and 30 random fields (average600 nuclei) were studied in each replicate. Double-stranded cleavage ofDNA was determined by green (FITC) fluorescence in the nuclei.$ j  o! C" O& ~9 A6 N3 ?/ P2 h

Catalase and SOD activity. Cells (2 × 10 9 ) were lysed in 0.5 ml of buffer (M-PERMammalian Extraction reagent; Pierce) containing 0.1 mMNa 3 VO 4, 10 mM NaF, 0.5 mM PMSF, 1% NonidetP-40, and protease inhibitor cocktail set I (Calbiochem-Novabiochem).The lysate samples were kept on ice for 1 h and centrifuged at10,000 rpm for 20 min, and the supernatants from different groups wereused for catalase and SOD activities. A sample volume was normalizedfor an equal amount of proteins in cells cultured in 5 and 25 mMglucose for 16 h. Catalase activity was determined at 520 nm byusing a catalase assay kit (Calbiochem-Novabiochem) in triplicate;enzymatic activity was calculated by using a catalase standard curveand expressed as catalase units per milligram protein. SOD activity wasdetermined by employing an SOD assay kit (Calbiochem-Novabiochem). TheSOD activity was calculated at 525 nm from the ratio of theautooxidation rate of5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzofluorine in thepresence and absence of lysates.
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Immunofluorescent detection of glycooxidative stress. Glucose-mediated oxidative stress in MMCs and NHMCs was studied bytrafficking of 2,3,4,5,6-pentafluorodihydrotetramethylrosamine (PF-H 2 TMRos or Redox Sensor red CC-1, Molecular Probes)using fluorescence microscopy. MMCs and NHMCs were maintained for16 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 ofthe incubation, cells were loaded at 37°C for 20 min with RedoxSensor red CC-1 (1 µM) and a mitochondria-specific fluorescent dye,MitoTracker green FM (50 nM; Molecular Probes). Redox Sensor red CC-1is oxidized in the presence of O 2 − · andH 2 O 2. Culture slides were washed and mountedwith PBS and visualized at ×40 magnification by using a Nikonfluorescence microscope (Nikon Eclipse E800) equipped with a triplefilter cube and charge-coupled device camera (Nikon DXM1200).The staining was performed in quadruplets for each group, and 30 randomfields (average 600 cells) were studied in each replicate. Images werecaptured by using Nikon ACT-1 (version 1.12) software and combined forpublishing format with Adobe Photoshop 6.0 software.4 W, c: A) o+ x1 _

EMSA for NF- B activity. Nuclear extracts of mesangial cells were prepared with an NE-PER kit(Pierce). Approximately 5 × 10 6 cells were used foreach determination. Nuclear proteins were assayed for NF- B p50 andp65/c-Rel DNA binding activity by using NF- B/c-Rel gelshift plusassay kit (Geneka Biotechnology). The NF- B and Rel ready-to-labelwild-type double-stranded oligo probes were also supplied by GenekaBiotechnology. The oligonucleotides were labeled with[ - 32 P]ATP and further purified by using a NUCTRAPprobe purification column (Stratagene). For the oligonucleotide-proteincomplex (bandshift), nuclear extracts and purified hot probes werepremixed and incubated at 10°C for 20 min. Unlabeled wild-type andunlabeled mutant oligonucleotides were included to determine thespecificity of the competition bandshifts. The oligonucleotide-proteincomplexes were loaded onto 5% native polyacrylamide gels (38:2)precooled at 4°C in 1× Tris-borate-EDTA (pH 8.0) buffer, and thegels were electrophoresed at 100 V for 2 h, dried, and exposed forautoradiographic visualization. The bands were quantified by using thecomputerized image analysis software Un-Scan-IT (Automated Digitizing System).
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Immunoelectrophoresis analysis. MMCs were homogenized in lysis buffer containing 150 mM NaCl, 50 mMTris (pH 7.5), 0.1 mM Na 3 VO 4, 1 mM NaF, 0.5 mMPMSF, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, 0.5 µg/mlleupeptin, 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, andphospho-Bad Ser136. Proteins were transferred onto nitrocellulosemembranes 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-p53Ser392, 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 ofsecondary antibody was used. The blots were developed by using the ECLkit (Amersham-Pharmacia), and the bands were scanned and quantified asdescribed above.# W; {+ z4 i" L
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Preparation of subcellular fractions. MMCs were harvested from the cultures and fractionated into cytosol andmitochondria by using an ApoAlert cell fractionation kit (ClontechLaboratories). Briefly, cells were incubated with fractionation buffer(Clontech Laboratories) on ice for 10 min, homogenized in an ice-coldDounce tissue grinder, and centrifuged at 700 g for 10 minat 4°C. The pellet was discarded and the supernatant was furthercentrifuged at 10,000 g for 25 min at 4°C. The supernatant (cytosolic fraction) and pellet (mitochondrial fraction) were collectedseparately, and protein concentration in these fractions was determinedby 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 amarker in mitochondria and its absence in cytosol by Western blotanalysis. For detection, horseradish peroxidase-linked secondaryantibody was used at a dilution of 1:3,000. Fractions obtained fromcytosolic and mitochondrial compartments were also probed withcytochrome c antibody by immunoelectrophoresis. Mousemonoclonal anti-cytochrome c antibody (BD Biosciences) was used at a dilution of 1:250, and the protein was detected byhorseradish peroxidase-linked secondary antibody (1:3,000). Cytochrome c was detected and quantified in the cytosolic fraction fromdifferent groups as described above.; F7 K5 r2 \6 C9 F* N
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Cleaved caspase-3 expression. Lysates of MMC were subjected to 4-20% gradient gelelectrophoresis, transferred onto nitrocellulose, and probed for thepresence of cleaved caspase-3. For this, blots were incubated withrabbit 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., V! N; k& A- X7 l5 Y# P
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Statistical analysis. Data are expressed as means ± SE. Comparison between two valueswas performed by unpaired Student's t -test. For multiplecomparisons among different groups of data, the significant differenceswere 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 MMCsurvival, cells were plated and cultured in SFM containing 5 or 25 mMglucose for 16 h. Apoptotic cell death was detected by ELISAcell death assay, which detects histone-associated DNA fragments withinthe cytoplasmic fraction of cells with high specificity. As shown inFig. 1, MMCs maintained in SFM 5 mMglucose for 16 h exhibited a detectable baseline level ofapoptosis. A 50% increase in apoptoticcell death was detected when the glucose concentration in the media wasincreased to 25 mM ( P 0.01). To control for the potentialeffect of osmolarity on MMC apoptosis, in separate studies,cell death was assessed under osmolar equivalent conditions of 5 mMglucose 20 mM mannitol for 16 h. The percentage of apoptotic MMCs was similar to baseline values detected in cells maintained inSFM 5 mM glucose. As shown in Fig. 2,TUNEL staining confirmed the increased number of apoptotic nucleiin cells maintained in SFM 25 mM glucose. Taken together, these resultsindicate that high ambient glucose concentration activates the deathprogram in MMCs.

Fig. 1. Effect of 25 mM glucose on murine glomerular mesangialcell (MMC) apoptosis. MMCs were maintained under one of thefollowing conditions for 16 h: serum-free medium (SFM) 5 mMglucose, SFM 25 mM glucose, or SFM 5 mM glucose 20 mM mannitol. Abaseline level of apoptosis was detected in serum-starved MMCsunder euglycemic conditions (C) by using an ELISA cell death assay. Thehistone-associated DNA fragments are presented as optical density at405 nm relative to the control value. For each assay, 20 µl of lysate(2.0 mg/ml) were used. Values are means ± SE and represent8-9 independent experiments. The following abbreviations are usedthroughout the figures: C, SFM 5 mM glucose; H, SFM 25 mM glucose; M,C 20 mM mannitol; D, diphenyleneiodonium; N, N -acety L -cysteine; A, ascorbic acid; CL, chelerythrine; HN,H  N -acety L -cysteine; HD, H diphenyleneiodonium;CN, C  N -acety L -cysteine; CD,C diphenyleneiodonium; HA, H ascorbic acid; HCL, H chelerythrine; CCL,C chelerythrine. * P 0.001, C vs. H. # P 0.001, H vs. M.) i9 s( ^: O' z2 n9 L# B

Fig. 2. Terminal deoxynucleotidyl transferase-mediated dUTPnick-end labeling (TUNEL) staining of MMC apoptotic nuclei. MMCswere maintained in C or H for 16 h. A and D :phase-contrast images of MMCs maintained under C or H conditions,respectively. B and E : TUNEL-positive MMC nuclei. C and F : MMC nuclei labeled with 4',6'diamidino-2-phenylindole dye, a nuclear-specific counterstain.Arrows, apoptotic nuclei. Magnification, ×40.

High glucose promotes intracellular ROS generation. High ambient glucose concentration has been reported to alter the redoxstatus of cells through the overproduction of ROS ( 27, 31 ). H 2 O 2 is a toxic product of bothaerobic metabolism and pathogenic ROS production. The heme-containingenzyme catalase metabolizes H 2 O 2 bydismutation, resulting in the formation of O 2  2H 2 O. O 2 − · is the first product of theunivalent reduction of oxygen. SOD catalyzes the dismutation ofO 2 − · by conversion toH 2 O 2  O 2. To determine whether highambient glucose concentration induces a prooxidant environment in MMCs,catalase and SOD activity were measured 16 h after plating in SFMat 5 or 25 mM glucose. As shown in Fig. 3, A and B, 25 mMglucose increased the activities of both catalase and SOD. Theincrement in catalase activity was 25% ( P 0.01), whereasSOD activity increased twofold ( P 0.01). To providea more direct assessment of oxidative stress, we performed additionalexperiments with the redox-sensitive dye Redox Sensor red CC-1. Thesestudies were also performed in NHMCs to determine whether glucosepromotes 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-1and the mitochondria-specific dye MitoTracker green FM. Redox Sensorred CC-1 is oxidized in the presence of O 2 − · andH 2 O 2. As shown in Fig. 4, B and E, bright yellow-orange fluorescence was seen inmitochondria due to the colocalization of oxidized red CC-1 dye (redfluorescence) and MitoTracker green FM dye (green fluorescence). Thiseffect is inhibited by NAC (Fig. 4, C and F ). Thefindings indicate that exposure of MMCs and NHMCs to 25 mM glucosealters the intracellular redox status by increasing the production ofROS.

Fig. 3. Effect of 25 mM glucose on antioxidant enzymatic activity. MMCswere maintained in C or H for 16 h. Catalase ( A ) andSOD ( B ) activities were measured in C and H as an index ofoxidant stress. Values are means ± SE and represent 3-4independent experiments. * P 0.01, C vs. H.
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Fig. 4. Glucose-induced formation of ROS in mesangial cells. MMCs( A-C ) and normal human mesangial cells (NHMCs; D-F ) were maintained in C ( A and D ), H ( B and E ), and HN ( C and F ). MMCs and NHMCs were loaded with theoxidant-sensitive dye Redox Sensor red CC-1 and themitochondrial-specific dye MitoTracker green FM. Brightyellow-orange fluorescence was seen in mitochondria (arrows) due to thecolocalization of oxidized red CC-1 (red fluorescence) and Mitotrackergreen FM (green fluorescence). Increased oxidation (brightyellow-orange fluorescence) of Redox Sensor red CC-1 ( B and E ) and inhibition of red CC-1 oxidation by N ( C and F ) can be seen in this figure. Magnification,×40.

Role of ROS in glucose-induced apoptosis. H 2 O 2 has been reported to promoteoxidant-induced apoptosis in rat mesangial cells( 17 ). The prooxidant environment of MMCs and NHMCsmaintained at 25 mM glucose implicates oxidant stress as a potentialtrigger for apoptosis. To determine whether glucose-induced intracellular ROS generation activates the death program, ascorbic acidand the cell permeable thiol antioxidants NAC and DPI were addedseparately to the culture media. As shown in Fig. 5 A, NAC, DPI, and ascorbicacid reduced MMC apoptosis to baseline values, whereas the PKCinhibitor chelerythrine had no detectable effect on glucose-inducedcell death. Figure 5 B shows identical experiments at 5 mMglucose, indicating the absence of cytotoxic effects of theantioxidants and chelerytherine at the concentrations used. Todetermine whether NHMCs exhibit a similar pattern of glycooxidative stress, an identical experimental protocol was performed. As shown inFig. 6, 25 mM glucose increased NHMCapoptosis by ~50% ( P 0.001). Theglucose-induced component of apoptosis is markedly attenuatedby the antioxidants NAC, DPI, and ascorbic acid ( P 0.001). Similar to MMCs, chelerytherine has no detectable effect onglucose-induced NHMC apoptosis. These findings strongly suggest that 25 mM glucose induces MMC and NHMC apoptosis by anoxidant-dependent mechanism.; W( ~2 Q8 i2 K* S$ P
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Fig. 5. Effect of antioxidants and PKC inhibitor on MMC apoptosis.MMCs were maintained under one of the following conditions for 16 h: C, C inhibitor, H, or H inhibitor. Inhibitors were D (10 µM), N(50 µM), A (100 µM), and CL (2 µM). Apoptosis wasdetected by the ELISA cell death assay. For each assay, 20 µl oflysate (2.0 mg/ml) were used. A : C, H, and H inhibitors (HD,HN, HCL, and HA). *C vs. H or HCL, P 0.001. #Hvs. HD, HN, or HA, P 0.001. B : C andC inhibitors (CN, CD, CA, and CCL). Values are means ± SE andrepresent 5-8 independent experiments.

Fig. 6. Effect of antioxidants and PKC inhibitor on NHMCapoptosis. NHMCs were maintained under one of the followingconditions for 16 h: C, H, H inhibitor (HD, HN, HCL, and HA), andC inhibitor (CN). The inhibitors added to the culture media were D (10 µM), N (50 µM), A (100 µM ), and CL (2 µM ). Apoptosiswas detected by the ELISA cell death assay. For each assay, 20 µl oflysate (2.0 mg/ml) were used. Values are means ± SE and represent6-8 independent experiments. *C vs. HN ( P 0.05). ***C vs. H or HCL ( P 0.001). ###H vs. HN, HD,HA, or CN ( P 0.001).

ROS-dependent activation of NF- B. In several cell lines, NF- B has been identified as a target forROS-dependent signals ( 27, 46 ). In the inactive state, NF- B is sequestered in the cytoplasm, which is associated with anendogenous 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 inubiquination and proteosomal degradation of I B, promoting nucleartranslocation of NF- B. To determine whether NF- B is activated byROS-dependent signals in MMCs maintained in SFM 25 mM glucose, gelshift assays were performed with nuclear proteins and an NF- Bbinding site-specific probe. As shown in Fig. 7, A and B, NF- Bbinding complexes were detected in MMCs maintained at 5 and 25 mMglucose. The identity of the bands was determined by competitivebandshift analysis using unlabeled consensus or mutant oligonucleotide(Fig. 7, C and D ). As shown in the densitometricanalysis (Fig. 7 F ), nuclear proteins from mesangial cellsmaintained at 25 mM glucose exhibited an upregulation of p65/c-Relbinding ( P 0.001). This upregulation in p65/c-Rel bindingactivity was suppressed by the antioxidants NAC and DPI ( P 0.001). Conversely, as shown in Fig. 7 E, p50 DNAbinding activity was inhibited by 25 mM glucose ( P 0.001). Inclusion of antioxidants NAC and DPI restored p50 bindingactivity to baseline. NAC had no detectable effect on bindingactivities 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 anoxidant-dependent mechanism. ROS-dependent signals appear to exert adeleterious effect on p50 binding, which can be reversed in thepresence of antioxidants.

Fig. 7. Effect of glucose-induced ROS generation on NF- B bindingactivity. MMCs were maintained under one of the following conditionsfor 16 h: C, H, HN, HD, or CN. Nuclear extracts were prepared fromMMCs and analyzed for p50- ( A ) and p65/c-Rel-bindingactivity ( B ) by EMSA. C and D :competitive bandshift analysis confirming specificity of bindingcomplexes in A and B. C, labeled oligonucleotide;Wt, cold oligonucleotide; and Mu, mutant oligonucleotide. Jurkat cellswere used as positive control ( ) for p50 and p65 dimers. E and F : densitometric analyses of p50 and p65 DNA bindingactivity. Values are means ± SE and represent 3-4independent experiments. * P 0.05, *** P 0.001, C vs. H, HN, HD or CN. ### P 0.001, H vs. HN,HD, or CN.
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Expression of apoptosis-related factors: Bcl-2 family ofproteins. To determine whether MMC apoptosis in response to 25 mM glucoseis characterized by alteration in the Bax/Bcl-2 ratio and phosphorylation status of Bad and p53, immunoblot analyses were performed. As shown in Fig. 8 A, the ratio of Bax/Bcl-2 wasincreased 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 onBax/Bcl-2 ratio at 5 mM glucose as well. The phosphorylation status ofBad (Fig. 8, B and C ) was also altered in MMCsmaintained at 25 mM glucose. Phosphorylation at serine residues isrecognized as a mechanism of inactivating the proapoptotic functionof Bad ( 16, 30 ). As shown in Fig. 8, B and C, phosphorylation at Ser112 and Ser136 was markedlyattenuated in MMCs exposed to 25 mM glucose. The inclusion of NAC inthe culture media of MMCs at 25 mM glucose enhanced the phosphorylationat Ser112 and Ser136 of the Bad protein. Because NF- B is known toregulate p53 expression ( 22, 44 ) and Bax is a target genefor p53 ( 23, 47 ), we examined the phosphorylation statusof the p53 protein. Ser392 is located at the COOH terminus of p53 andlinked to transcriptional activation ( 7 ). Phosphorylationof Ser392 was upregulated in MMCs exposed to 25 mM glucose (Fig. 8 D ), implicating p53 in the alterations of Bax and Bcl-2expression ( 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 mMglucose, oxidative stress promotes directional shifts in the expressionand phosphorylation status of the Bcl-2 family of proteins that favorsprogression of the apoptotic process.' b) y& d/ N1 V/ n
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Fig. 8. Quantitative immunoblot analyses of Bax, Bcl-2, Bad, andp53 expression. MMCs were maintained in C, CN, H, and HN for 16 h. A : ratio of Bax and Bcl-2 protein expression in the abovegroups. B and C : ratios of phospho-Bad(Ser112)/Bad and phospho-Bad (Ser136)/Bad, respectively. D :ratio of phospho-p53/p53 protein expression in the same groups. Thedata shown are representative immunoblots of 3-5 independentanalyses. * P 0.001, C vs. H, HN, or CN. #P 0.01, H vs. HN or CN." B6 ^! _# s8 o" b) A3 y

Cytochrome c release and caspase activation. To determine whether perturbations in the Bcl-2 family of proteins arecoupled with the release of cytochrome c from themitochondrial compartment and caspase activation, immunoblots wereperformed on cytosolic- and mitochondria-enriched fractions of MMCs. As shown in Fig. 9 A, top, 25 mM glucose (H) increased the release of cytochrome c from mitochondria compared with MMCs maintained at 5 mMglucose (C). To control for possible contamination of cytosol bymitochondrial proteins, immunoblots were probed with an antibodyagainst COX IV. To control for variations in loading conditions,Coomassie blue-stained gels (Fig. 9 B ) are shown that document equal loading conditions in cytosolic and mitochondrial fractions. As shown in Fig. 9 A, bottom, COX IVwas not detected in the cytosol of MMCs maintained at 5 or 25 mMglucose. The inclusion of NAC in MMC cultures maintained at 25 mMglucose markedly attenuated cytochrome c release (Figs. 9, A and C ). Immunoblot analysis of the cytochrome c enriched mitochondrial fraction did not detect changes incytochrome c content among the different groups.
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Fig. 9. Immunoblot analysis of cytochrome c insubcellular fractions. MMCs were maintained in C, H, or N for 16 h. A : levels of cytochrome c and COX IV incytosolic and mitochondrial fractions. B : Coomassie bluestaining of the protein fractions used for immunoblotting. C : densitometric analysis of cytochrome c levelsin cytosolic fraction. MW, molecular weight markers. Values aremeans ± SE and represent 6 independent observations. **C vs. H( P 0.01). ##H vs. N ( P 0.01).
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Figure 10 shows an upregulationof cleaved caspase-3 expression in MMCs maintained at 25 mM glucose.The antibody [cleaved caspase-3 (Asp175) antibody; Cell SignalingTechnology] used in this analysis, recognizes cleaved caspase-3 at 17 and 19 kDa and does not detect full-length caspase-3 at 32-35 kDa.Equal loading conditions for cell lysates were confirmed by Coomassieblue staining (data not shown). The increment in cleaved caspase-3expression was inhibited by NAC and DPI, implicating ROS orROS-dependent pathways in the regulation of this proteolytic enzyme.Taken together with the above findings, perturbations in the expressionand phosphorylation status of Bcl-2 proteins activate the death programin mitochondria, resulting in the release of cytochrome c and caspase activation.4 n1 T4 l) f* t* ^
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Fig. 10. Effect of glucose-induced ROS generation on cleavedcaspase-3 expression. MMCs were maintained under one of the followingconditions for 16 h: C, CN, H, HN, or HD. Values are means ± SE from 3-9 independent analyses. *C vs. HD ( P 0.05). ***C vs. H or HN ( P 0.001). ###H vs. HN, HD,or CN ( P 0.001).

DISCUSSION& B5 x8 r7 V/ l$ T

In the present study, we demonstrate that in vitro exposure ofMMCs and NHMCs to 25 mM glucose activates the genetic program forapoptosis. Glucose-induced apoptosis was independent ofmechanical strain and osmolar forces and triggered by oxidant stress.Direct evidence is also provided for perturbations in the pro- andantiapoptotic members of the Bcl-2 family, culminating in therelease of cytochrome c from mitochondria and caspaseactivation. Finally, we demonstrated that inhibition of theredox-sensitive transcription factor NF- B prevents glucose-inducedMMC apoptosis.) @& r% Q' y: i+ ]4 N2 Z8 {# X
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High glucose promotes mesangial cell apoptosis. Hyperglycemia dominates the pathophysiology and clinical course of type1 and type 2 diabetes. Compelling evidence from the Diabetes Controland Complications Trial indicates that rigorous control of bloodglucose reduces the risk of long-term microvascular complications (6a).In the present study, we demonstrate for the first time that highambient glucose concentration activates the genetic program for MMC andNHMC apoptosis. The cytotoxic property of high glucose wasindependent of osmolar forces, because the percentage of apoptoticMMCs with an osmolar equivalent glucose-mannitol media did not differfrom control values. The latter property may serve to protect mesangialcells from transient elevations in osmolarity or reflect the uniqueability of high glucose to trigger activation of intracellularsignaling molecules involved in the expression of the death program.Hyperglycemia has recently been reported to induce apoptosis incardiac myocytes ( 7, 8 ), whereas in vascular smooth musclecells, hyperglycemia was found to protect against apoptotic celldeath ( 14 ). These observations suggest that the cytotoxicproperties of glucose vary across cell lines. Several factors mayoperate to limit detection of this form of cell death under in vivoconditions. First, cell loss is not a prominent characteristic duringthe early phases of diabetic glomerulopathy ( 38 ), in whichthickening of the basement membrane and glomerular hypertrophy are themost characteristic deviations from normal ( 25 ). Second,during the late phases of this disorder, expansion of the mesangialmatrix dominates the histological picture ( 28 ) along withsclerosis and occlusion of glomeruli ( 4, 29 ). Third, celldeath by apoptosis does not result in sclerosis or residualscarring ( 16 ) and, in the absence of an immunocytochemical analysis, cannot be detected morphologically ( 7 ). Takentogether, our finding that high glucose, independent of hemodynamic orphysical forces, promotes mesangial cell apoptosis raisesadditional questions concerning the fate of mesangial cells in diabetic nephropathy.7 P7 I% D8 G* ~5 W
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High glucose, ROS, and mesangial cell apoptosis. Multiple lines of evidence have established a role for ROS as importantmediators of cell biology ( 9, 21, 34 ).O 2 − · is the first product of the univalent reductionof oxygen. O 2 − · is converted toH 2 O 2 and oxygen by SOD ( 6 ). BothO 2 − · and H 2 O 2 have beenreported as capable of activating death programs ( 42 ).Recent work has emphasized the importance of mitochondrial generationof ROS, in response to high-ambient-glucose concentration, as thetrigger for hyperglycemia-related metabolic events, including thecovalent modification of proteins by advanced glycation end products( 27 ). In the present study, we demonstrate that high glucose also promotes the generation of ROS in MMCs and NHMCs, implicating ROS as potential mediators of glucose-induced mesangial cell apoptosis. Two main sites for the generation of ROS have been identified at the inner mitochondrial membrane, the NADH dehydrogenase at complex I, and the interface between ubiquinone andcomplex III ( 27 ). Although ROS are not classically thought of as signaling molecules, alterations in the redox status of cells hasbeen shown to modulate the activation of transcription factors( 9 ) and ionic channels ( 13 ). Previous workhas documented that H 2 O 2 activates the deathprogram in mesangial cells ( 17 ); however, this is thefirst report demonstrating that high ambient glucose concentrationpromotes mesangial cell apoptosis by an oxidant-dependentmechanism. Although hyperglycemia is known to be a potent stimulus forthe activation of PKC isozymes, which modulate a myriad of biologicalfunctions, the PKC inhibitor chelerythrine did not attenuate orincrease MMC or NHMC apoptosis. Conversely, the antioxidantsNAC, DPI, and ascorbic acid suppressed transmission of the death signalin both cell lines. Our findings are consistent with the growing bodyof work implicating ROS in the pathogenesis of diabetic complications( 3, 12, 41 ). In this regard, the balance of evidencepoints toward the O 2 − · as the mediator of cellinjury ( 6, 27 ); however, evidence also supports a role forthe cytotoxic effects of H 2 O 2 ( 17, 42 ). Taken together, the present study provides evidence that glycooxidative stress is an activating signal for the apoptosis gene program in mesangial cells.

Oxidant-dependent NF- B activation and mesangial cellapoptosis. NF- B comprises an inducible family of transcriptional factors thatare important regulators of host immune and inflammatory responses. Inaddition, NF- B-dependent signaling pathways have been implicated incell survival ( 19, 20, 22, 32, 39, 44, 46 ). Stimulation ofthe NF- B signaling pathway occurs by phosphorylation and degradationof the NF- B inhibitory protein I B, with subsequent translocationof NF- B to the nucleus ( 5, 19 ). Our results indicate adifferential effect of 25 mM glucose on NF- B binding activity, whichis ROS dependent. The upregulation in p65/c-Rel activity was markedlysuppressed in the presence of NAC or DPI, implying that ROSpreferentially 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 studiesin other cell lines ( 27, 46 ) have documented anassociation between glucose-induced ROS generation and NF- Bactivity, suggesting this transcription factor may modulate cellularresponses to a high-glucose environment. The observation that NAC andDPI not only blocks the recruitment of NF- B but also prevents MMCapoptosis is consistent with such a hypothesis. The cumulativeresults strongly suggest that hyperglycemia recruits NF- B viaROS-dependent signals and implicates oxidant-dependent activationof NF- B in the signaling cascade of glucose-induced MMC apoptosis.
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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 wasviewed as prosurvival and antiapoptotic ( 22, 44 ). Itis now recognized that NF- B may also assume a proapoptoticfunction through the regulation of apoptosis genes. The nucleartranscription factor p53 regulates proapoptotic gene programs( 20, 32 ), and NF- B has been reported to be aprerequisite 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 inassociation with p65/c-Rel inhibition is consistent with such ahypothesis. Alternatively, we demonstrate phosphorylation of p53 atSer392, a modification of the p53 protein linked with transcriptional activation ( 7 ). Our results also indicate that MMCapoptosis was accompanied by an increase in the Bax/Bcl-2ratio, an alteration that favors progression of apoptosis( 16 ). High-glucose concentration attenuatedphosphorylation of Bad at Ser112 and Ser136. The latter findingis consistent with the proapoptosis function of Bad ( 30 ). Of note, the antioxidant NAC restored phosphorylation of Bad to levelsdetected at 5 mM glucose. The increase in p53 phosphorylation detectedat 25 mM glucose was prevented by the addition of NAC, as was theincrease in the Bax/Bcl-2 ratio. It seems reasonable to infer thateither oxidant stress directly activates p53 ( 33 ) orNF- B and p53 may cooperate to regulate the expression of Bax ( 20, 32, 44 ). Additional studies will be required to test this hypothesis.( @) \* x1 F) B- H8 u
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Oxidant stress and mitochondrial dysfunction. The mitochondria are key determinants of cell death and cell survival( 1, 11 ). Cytochrome c release by mitochondriaand caspase activation are critical events in triggeringoxidant-induced apoptosis ( 11 ). Anti- andproapoptotic proteins of the Bcl-2 family possess a COOH-terminaldomain, which serves to target proteins to specific cell compartments( 16 ). Several mechanisms are utilized by theantiapoptotic protein Bcl-2 to interrupt transmission of deathsignals, direct antioxidant effect, protein-protein interaction, andinhibition of cytochrome c release from mitochondria( 16 ). This latter mechanism enables Bcl-2 to directlyinterfere with cytochrome c -dependent activation ofcaspases, a key event in the execution of the death signal. Theantiapoptotic function of Bcl-2 may be neutralized by translocationof Bad and Bax from the cytosol to the mitochondria, with thesubsequent formation of heterodimers ( 16, 42 ). Cytochrome c release is tightly regulated by protein-proteininteractions among Bcl-2, Bax, and Bad ( 42 ). A growingbody of evidence suggests that release of cytochrome c frommitochondria commits a cell to die by either apoptosis ornecrosis ( 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 thecytochrome c content of mitochondrial fractions inexperimental and control groups. This may be due to the enrichedcytochrome c content of mitochondria compared with therelatively small fraction released to the cytosol. Moreover, theexpression of cleaved caspase-3 was upregulated by 25 mM glucose. Theantioxidants NAC and DPI markedly attenuated the glucose-inducedupregulation of cleaved caspase-3 expression. Taken together, our dataindicate that oxidant-induced perturbations in the Bcl-2 family ofproteins facilitate the release of cytochrome c frommitochondria, initiating the terminal cascade of the death signal.

The present study has certain limitations. First, the duration ofexposure to 25 mM glucose was brief compared with an in vivo model ofhyperglycemia. Second, although beyond the scope of this investigation,infection of mesangial cells with constitutively active mutants ofI B to establish whether NF- B is necessary and sufficient formesangial cell apoptosis was not performed. Moreover, we didnot demonstrate increased p53 DNA binding activity to the p53-dependentgene Bax. Finally, the application of an in vitrosystem to study the fate of resident cells in the diabetic glomerulusmay not mimic the in vivo condition. Future investigations directed atthe issues not addressed in the present study will be important infurther elucidating the molecular events that direct expression of thedeath program.
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In summary, the results of the present study have establishedapoptosis as a biological response to high ambient glucoseconcentration in MMCs and NHMCs. As depicted in APPENDIX A,activation of the death program is ROS dependent, and recruitment ofNF- B is an integral component of the death signaling pathway.Although the precise mechanism by which NF- B orchestrates thetransmission of the death signal remains to be defined, inhibition ofNF- B binding activity by NAC was found to prevent mesangial cellapoptosis. Perturbations in the Bcl-2 family of proteins,cytochrome c release, and caspase activation are consistentwith evolving concepts in which mitochondria are viewed as keydeterminants of cell survival and death. Future investigations mustconsider the in vivo consequences of hyperglycemia-induced oxidantstress on mesangial cell survival and the progression of diabetic nephropathy.
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APPENDIX A0 ]5 ]5 R! R% N2 M7 t  p9 K

A1. Proposed scheme for glucose-induced MMC apoptosis.& l4 P& X9 |- ?! j, O) X5 J6 J
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ACKNOWLEDGEMENTS
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We acknowledge Drs. G. P. Yangand V. Gaussin (University of Medicine and Dentistry of New Jersey) forhelp with the TUNEL assay. This work was partially supported byAmerican Heart Association Grant-in-Aid 9750856A (A. Malhotra), aresearch grant from the Foundation of University of Medicine andDentistry of New Jersey Annual Grants Program (A. Malhotra), andsupport from the Summit Area Public Foundation through the generosity of the Mrs. Elaine B. Burnett Fund (L. G. Meggs and S. Baskin)./ w8 p! v8 V. S0 [7 l( y
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