Role of Oxidative Stress in Advanced Glycation End Product-Induced Mesangial Cell Activation
Role of Oxidative Stress in Advanced Glycation End Product-Induced Mesangial Cell Activation
Role of Oxidative Stress in Advanced Glycation End Product-Induced Mesangial Cell Activation
20062014
Role of oxidative stress in advanced glycation end product- velopment of kidney disease in animal models of diabe-
induced mesangial cell activation. tes [2, 3] and experimental evidence obtained from in
Background. Levels of advanced glycation end products
vitro studies shows that prevention of ROS generation
(AGE) are elevated in individuals with advancing age, renal
failure, and diabetes, and accumulation of these molecules may defends against the damaging effects of a hyperglycemic
contribute to disease progression. The mechanism by which milieu on mesangial cell function [4, 5]. In both models,
AGE proteins alter glomerular mesangial cell function, how- the therapeutic effect of antioxidants appears directly
ever, is not completely understood. The present study assessed related to their ability to normalize key early intracellu-
the involvement of oxidative stress in AGE-dependent mesan- lar signaling events linked to the development and pro-
gial cell signaling events.
Methods. Primary cultures of rat renal mesangial cells were gression of diabetic nephropathy.
exposed to in vitro AGE-BSA and H2O2. Nuclear factor-B Advanced glycation end products (AGEs) also play a
(NF-B) and protein kinase C (PKC) isoform activation were significant role in the pathogenesis of vascular and renal
studied using confocal microscopy and Western blotting. Quan- complications associated with diabetes [68]. These toxic
titative polymerase chain reaction (PCR) was used to measure molecules represent a heterogeneous group whose level
transforming growth factor-1 (TGF-1) levels. The involve-
ment of oxidative stress was assessed by supplementing or
in plasma and tissue is determined by glycemic control,
compromising cellular antioxidant capacity. turnover rate of proteins, and renal function [9, 10]. AGEs
Results. NF-B was dose-dependently activated by AGE. accumulate in glomerular regions of the kidney [8, 11,
PKC activation was not involved in this response, but analysis 12] and infusion of AGE-modified proteins into experi-
of PKC-1 activation showed a stimulatory effect of AGE mental animals leads to renal complications similar to
proteins on this isoform. Transcription of TGF-1 was stimu-
those observed in diabetic nephropathy [13, 14]. Direct
lated by AGE and was prevented by PKC inhibition. Challenge
with H2O2 had similar downstream effects on mesangial cell cellular challenge with AGE has been shown to cause
signaling. Antioxidants, vitamin E and nitecapone, prevented oxidative stress in various cell types [1517] including
AGE-dependent NF-B activation and normalized PKC activ- mesangial cells [18], but the specific involvement of ROS
ity and associated TGF-1 transcription. Depletion of the intra- in AGE-dependent mesangial cell signaling has not been
cellular antioxidant, glutathione, effectively lowered the AGE examined previously.
concentration needed for mesangial cell activation of NF-B
and PKC-1. Treatment with a suboptimal AGE dose, under The current study was therefore designed to identify
glutathione-depleted conditions, revealed a synergistic effect the extent to which ROS are involved in AGE-induced
on both parameters. alterations in mesangial cell function. To probe this re-
Conclusion. The results support a central role for oxidative lationship, we examined the regulation of nuclear fac-
stress in AGE-dependent mesangial cell signaling and empha- tor-B (NF-B), protein kinase C (PKC), and trans-
size the importance of ROS in determining cell responsiveness.
forming growth factor-1 (TGF-1), since these factors
occupy key positions in the cellular events associated
with diabetes. We demonstrate here that AGE cause
Several lines of evidence demonstrate that oxidative
increased mesangial cell oxidative stress and that this
stress or reactive oxygen species (ROS) are involved in
early event is implicated in the independent activation
the pathogenesis of diabetes [1]. We and others have
of NF-B and the PKC pathway. Additionally, we show
shown that antioxidant therapy protects against the de-
that ROS play a key role in determining the magnitude
of AGE-induced activation of PKC-1 and NF-B.
Key words: AGE, diabetes, oxidative stress, NF-B, protein kinase C,
TGF-1.
METHODS
Received for publication August 23, 2001
and in revised form January 23, 2002 Materials
Accepted for publication January 24, 2002 Nitecapone [3-(3-dihydroxy-5-nitrophenyl)methylene-2,
2002 by the International Society of Nephrology 4-pentadione] was kindly provided by Orion Pharma (Es-
2006
Lal et al: AGE-induced mesangial cell activation 2007
poo, Finland). Rabbit polyclonal PKC isoform-specific to treatment with AGE or other agents according to the
and NF-B p65 antibodies were purchased from Santa various treatment conditions. Following stimulation, cells
Cruz (Santa Cruz, CA, USA). Texasred-conjugated anti- were fixed with 3.5% formaldehyde/PBS for 10 minutes,
rabbit secondary antibody was from Molecular Probes permeabilized with 0.1% Triton X-100 for 15 minutes,
(Leiden, The Netherlands). Moloney-murine leukemia and blocked with 1% goat serum, 0.1% BSA for one
virus (M-MLV)-reverse transcriptase, RNasin, and Oligo hour. Cells were then stained with NF-B (1:200) or
dT were obtained from Promega (Madison, WI, USA). PKC-1 (1:200) antibodies for one hour at room temper-
Taq polymerase and dNTPs were from Perkin Elmer ature or overnight at 4C, followed by washing and subse-
(Foster City, CA, USA). All other chemicals were pur- quent recognition with Texas-red-conjugated secondary
chased from Sigma Chemical Co. (St. Louis, MO, USA). antibody (1:200). The cells were washed again and
mounted in Prolong antifade. Fluorescence images were
Isolation and primary culture of mesangial cells recorded using a Zeiss LSM410 inverted confocal scan-
Kidney cortex, isolated from male Sprague-Dawley ning laser microscope with excitation at 543 nm and long-
rats, was chopped on ice and passed through a series of pass detection at 570 nm (Carl Zeiss Instruments, Zurich,
nylon meshes of graded pore sizes (150, 105, and 77 Switzerland). Optical sections through the center of the
mol/L) by applying gentle pressure and repeated wash- cells were used for localization of the immunofluorescent
ing with phosphate-buffered saline (PBS; pH 7.4) supple- signal. For the NF-B studies, cell staining was semi-
mented with antibiotics (100 U/mL of penicillin and 100 quantitatively estimated by measuring the redistribution
g/mL of streptomycin). Glomeruli collected from the of immunofluorescent signal between the nucleus and
final 77 mol/L collection sieve were rinsed and collagen- cytoplasm. Two persons blind to the protocol performed
ase (100 mg/100 mL) digested for 15 minutes prior to the calculations using Scion Image software. Nucleus
plating in Dulbeccos modified Eagles medium (DMEM; localization was confirmed with Sytox Green nuclear
Life Technologies) containing 20% fetal bovine serum stain (Molecular Probes). Cells revealed no auto-fluo-
(FBS; Life Technologies), antibiotics, 24 mmol/L NaHCO3, rescence and control sections incubated without primary
and 20 mmol/L HEPES. Mesangial cells obtained from antibody and with secondary antibody displayed no sig-
glomerular outgrowths were used from passages 3 to 10. nificant background signal. Negative controls for PKC-1
Mesangial cells had a predominantly fusiform or stellate immunofluorescence performed with primary antibodies
appearance on phase-contrast microscopy. Positive im- preabsorbed to its immunizing peptide showed very weak
munofluorescent staining for smooth muscle -actin and staining. Three to five individual experiments were per-
negative staining for von Willebrand factor and cytoker- formed and 9 to 25 cells were analyzed for each experi-
atin confirmed the purity of mesangial cell cultures and mental condition.
absence of contaminating endothelial and epithelial cell
types. Under all experimental treatment conditions, mes- Determination of thiobarbituric
angial cells were serum-starved over night in DMEM acid-reactive substances
containing 0.5% FBS prior to treatment. Analysis of mesangial cell lipid peroxide content was
determined by measuring the abundance of thiobarbi-
AGE-BSA preparation turic acid-reactive substances (TBARS) [19]. Briefly,
Glycated bovine serum albumin (BSA) with AGE for- mesangial cells, treated for 72 hours with 100 g/mL
mation was prepared in vitro by incubating 50 mg/mL AGE in the presence or absence of antioxidants, were
BSA with 0.15 mol/L ribose in sterile phosphate buffer scraped off 60 mm plates and sonicated in 1.15% KCl
(pH 7.4) containing antibiotics at 37C for 14 days. Con- solution followed by solubilization with an equal volume
trol non-glycated BSA (50 mg/mL) was prepared in the of 8% sodium dodecyl sulfide (SDS). To this mixture,
same manner without ribose supplementation. AGE- 20% acetic acid and 0.8% thiobarbituric acid were added.
BSA and control BSA were dialyzed against PBS over- The complete mixture was heated to 95C for 60 minutes,
night to remove ribose, sterile-filtered through 0.2 m after which the chromogenic substrate was extracted into
filters (Sartorius), aliquoted and stored at 80C until the organic phase with butanol/pyridine (15:1). Absor-
needed. Estimation of AGE content by spectrofluorom- bance of the organic layer was measured with a spectro-
etry with excitation wavelength of 390 nm and emission fluorometer with excitation wavelength of 515 nm and
wavelength of 450 nm revealed a 10.7-fold increase in emission wavelength of 553 nm. Absorbance was mea-
fluorescence for AGE-BSA compared to control BSA sured relative to a standard using 1,1,3,3-tetramethoxy-
at a concentration of 200 g/mL. propane.
with 200 g/mL AGE for 60 minutes in the presence or protein loading between lanes within individual blots
absence of antioxidants. Following treatment, cells were was confirmed with amido black dye staining.
rinsed with ice-cold PBS and resuspended in buffer A
containing 50 mmol/L Tris-HCl (pH 7.4), 120 mmol/L Semiquantitative PCR of TGF-1
NaCl, and Complete protease inhibitor cocktail (Roche, Confluent cultures of mesangial cells were treated for
Mannheim, Germany) followed by brief sonication (3 72 hours with 100 g/mL AGE in the presence or ab-
5 seconds at setting 2 using a Branson Sonifier). The cell sence of antioxidants. Total RNA was extracted using a
sonicate was centrifuged at 100,000 g for 60 minutes commercially available kit (RNeasy KIT; Kiagen, Hil-
at 4C. The supernatant was kept as the cytosolic fraction den, Germany) and 1 g was reverse transcribed in a
and the resultant pellet was resuspended in buffer A standard reaction mixture [2]. PCR analysis was carried
supplemented with 1% (vol/vol) Triton X-100, sonicated out for 29 cycles according to the following sequence:
and incubated on ice for 30 minutes. Centrifugation at denaturation (95C for 30 seconds), primer annealing
100,000 g for 60 minutes at 4C produced a supernatant (57C for 45 seconds), and extension (72C for 60 sec-
(membrane) fraction containing detergent-soluble pro- onds). TGF-1 primers: 5-AAG CAC CCG GGT TGT
teins. Protein concentration was determined using the GTT GGT T-3, 5-GTC AGA CAT TCG GGA AGC
Bio-Rad detergent-compatible protein assay with BSA AGT G-3. -actin primers: 5-TCC TGA CCC TGA
as a standard. AGT ACC CCA TTG-3, 5-GTC GCC TTC ACC GTT
For assessing NF-B nuclear translocation after treat- CCA GTT-3. PCR products were separated by poly-
ment, mesangial cells were rinsed with PBS and resus- acrylamide gel electrophoresis (PAGE) and were quanti-
pended in ice-cold hypotonic buffer containing 10 mmol/L fied using a phosphor imager system and Biorad Quantity
HEPES, pH 7.9, 10 mmol/L KCl, 1.5 mmol/L MgCl2, and One software.
Complete protease inhibitor cocktail. After 15 minutes, Statistical analysis
NP-40 was added to 0.1% (vol/vol) and lysates were
centrifuged at 500 g for 5 minutes. The resulting super- Data are expressed as means SE. Differences be-
natant was collected and used as the cytosolic fraction. tween means were evaluated by analysis of variance
Crude nuclear pellets were rinsed twice with hypotonic (ANOVA) and the Student t test. Statistical significance
buffer and resuspended in hypertonic buffer containing was accepted at P 0.05.
20 mmol/L HEPES, pH 7.9, 420 mmol/L NaCl, 1.5 mmol/L
MgCl2, 25% glycerol and Complete protease inhibitor RESULTS
cocktail. The suspension was rotated for 30 minutes at The current series of experiments tested whether
4C and then centrifuged at 10,000 g for 30 minutes AGE-BSA was an adequate stimulus for activation of
at 4C to yield a supernatant containing extracted nuclear the nuclear transcription factor, NF-B, since regulation
proteins. Protein concentration was determined as de- of this protein has previously been shown to be sensitive
scribed above. to oxidant stress [20]. In its inactive state, NF-B is main-
tained as a latent form present in the cytoplasm where
Immunoblotting of PKC-1 and NF-B
it is bound to a protein complex that masks the nuclear
Equal amounts of protein (cytosolic and membrane localization signal. Exposure of serum-starved mesangial
or nuclear) were loaded onto 8% SDS-PAGE gels, elec- cells to 200 g/mL AGE, under normal glycemic condi-
trophoresed, and transferred to polyvinylidine difluoride tions (5.5 mmol/L glucose) for 30 minutes resulted in
(PVDF) membranes (Amersham Pharmacia Biotech). NF-B activation, as depicted by its enhanced transloca-
Membranes were blocked in 5% milk/PBS for one hour tion to the nucleus (Fig. 1A). Neither non-glycated BSA
at room temperature or overnight at 4C. Membranes nor AGE-BSA prepared in the presence of the nonenzy-
were subsequently rinsed with 0.1% Tween 20 PBS matic glycation inhibitor, 200 mmol/L aminoguanidine
(TPBS) and incubated with PKC-1 (1:2000) or NF-B hemisulfate (AG), had a significant effect on NF-B
(1:5000) antibody for one hour. Following primary anti- nuclear translocation (Fig. 1 A, B). Semiquantification
body incubation, membranes were rinsed and exposed of AGE-mediated NF-B activation revealed a time- and
to goat anti-rabbit secondary antibody (1:5000) for one dose-dependent pattern of nuclear translocation (Fig. 1C).
hour. Immunosignals were visualized using Amersham NF-B nuclear translocation remained elevated by two-
Pharmacia Biotechs ECL Plus reagents according to the fold after an extended AGE exposure of 48 to 72 hours.
manufacturers specifications. Single bands for PKC-1 Western blotting of total cell protein with anti-NF-B
and NF-B were quantified using BioRad Quantity One antibody revealed a consistent level of NF-B expression
software. Specificity of the PKC-1 signal was confirmed following short- and long-term AGE treatment. Cell frac-
by the disappearance of the putative signal by preincuba- tionation of cytosolic and nuclear proteins followed by
tion of the antibody with its competing peptide. Accurate Western blotting confirmed NF-B translocation to the
Lal et al: AGE-induced mesangial cell activation 2009
Fig. 1. Effect of advanced glycation end products (AGE)-bovine serum albumin (BSA) on mesangial cell nuclear factor-B (NF-B) immunolocali-
zation. (A) Representative confocal micrographs of NF-B staining in mesangial cells treated with 200 g/mL non-glycated BSA, 200 g/mL AGE,
and aminoguanidine (AG)-BSA for 30 minutes. Enhanced nuclear luminosity can be clearly identified in AGE treated cells. (B, C ) Cumulative
semiquantitative data depicting the increase in NF-B nuclear staining obtained from confocal images of cells treated under the various conditions.
Summarized data are means SEM of 4 to 5 different experiments and are representative of data collected from 45 to 125 cells. Symbols in C
are: () 120 minutes; () 60 minutes; () 30 minutes; () 15 minutes; (X| ) BSA. * P 0.005 compared to AGE-treated cells.
of oxidatively modified cellular components [26, 29, 30] gions within the cell is important because translocation
provide evidence for increased oxidative stress in diabe- of PKC is indicative of kinase activation and modification
tes. The current study shows, to our knowledge for the of PKC signaling processes. The significance of PKC-
first time in mesangial cells, that oxidative stress is a cen- activation, and lack of PKC- activation, by AGE-BSA
tral mediator of AGE-dependent activation of NF-B indicates a certain degree of isoform selectivity [18, 25].
and PKC/TGF-1 pathways, and highlights the poten- While the nature of the AGE-dependent activation of
tial importance of this signaling system in diabetic ne- PKC-1 remains undefined, preliminary data obtained
phropathy. using sodium borohydride-reduced AGE suggests that
The transcription factor NF-B is a pleiotropic regula- the observed response is specific to AGE compounds,
tor of the inducible expression of many genes and it is and not to Amadori products present in our AGE prepa-
activated by a wide variety of stimuli associated with ration [34]. The mechanism and importance of isoform-
stress or injury [20]. It is a dimer of two proteins, (one specific PKC activation in the face of AGE challenge
of which is p65), that reside in the cytoplasm of unstimu- and the significance of similar in vitro findings to diabetic
lated cells, and can be rapidly induced to enter the nu- nephropathy are currently under examination.
cleus by appropriate signaling events, including ROS Exposure of mesangial cells to AGE proteins has been
challenge, as observed here. A study by Scivittaro et previously shown to activate PKC-2 [18] and to increase
al demonstrates that AGE cause ROS generation and TGF-1 expression [35]. The results presented here sug-
oxidative stress in mesangial cells, but the role of these gest a central role for ROS in precipitating both AGE-
modified proteins in NF-B activation was not described induced PKC activation and TGF-1 transcription. As
[18]. Based on the current observations, the largely simi- supported by our results obtained after AGE or H2O2
lar inhibitory effects of nitecapone and vitamin E on both treatment, mesangial cell PKC-1 activation is sensitive
H2O2 and AGE-induced NF-B translocation suggests a to, and at least partially determined by, cellular oxidative
common mechanism of activation involving ROS. AGE- stress levels [36]. In diabetic animals and mesangial cells
induced NF-B activation has been described in other exposed to hyperglycemic media, antioxidants are thought
cell types [15, 17, 31], but to our knowledge this is the to prevent TGF-1 induction through their inhibitory ac-
first study to address the response in mesangial cells. tion on upstream PKC [25]. The ability of both anti-
The oxidative stress-sensitive steps involved in NF-B oxidants and PKC inhibitors to reduce AGE-induced
activation remain unexamined, but our preliminary find- TGF-1 levels, as seen here, indicates a similar mode of
ings indicate that IB, the inhibitory subunit that main- action. Accordingly, it appears that AGE proteins induce
tains NF-B in an inactive state, is degraded upon short- an oxidative stress that impinges on a pathway leading to
term AGE treatment. This suggests that ROS likely act PKC activation and associated TGF-1 expression. We
at a site located upstream of NF-B. Another important speculate the following about the central role of cellular
issue regarding the signaling cascade evoked by AGE antioxidant homeostasis in determining mesangial cell re-
treatment is the mechanism by which it causes oxidative sponse sensitivity.
stress. AGE may interact with any one of a number of Cells actively maintain a proper level of intracellular
heterogeneous AGE-binding proteins expressed in the ROS through defense systems including antioxidant en-
plasma membrane of various cell types [7]. Previous stud- zymes and low molecular weight antioxidants. In diabe-
ies indicate that AGE proteins prepared in vitro possess tes the antioxidant capacity is compromised [2, 26, 28,
similar cross-reactive AGE epitopes that are common 29], and this may be important in determining the extent
to proteins modified by AGE in vivo [31], and that inter- of AGE-mediated cell signaling events. Glutathione is
action of these molecules with the cell-associated recep- the major intracellular antioxidant and incubation of cells
tor for AGE (RAGE) is associated with ROS generation with DEM lowers glutathione levels and correspondingly
and NF-B activation [16, 17]. The mechanism of NF-B exposes cells to an increased sensitivity to oxidative stress
specific activation under conditions of oxidative stress [37, 38]. Here, we found that DEM sensitized mesangial
remains elusive, but a recent study suggests that NADPH cell AGE-responsiveness and lead to enhanced NF-B
oxidase plays an important role in AGE-RAGE depen- and PKC-1 activation. Based on these results, we pro-
dent ROS generation and gene activation [32]. Mesan- pose that depletion of cellular glutathione reduces the
gial cells express RAGE and additional experiments are antioxidant capacity of the cell and increases sensitivity
needed to confirm whether this receptor is a relevant to oxidative stress-dependent AGE signaling. We think
mediator of the responses observed here. it likely that depletion of GSH antioxidant capacity by
Protein kinase C activation is a well-recognized, early DEM is the major contributing factor since we found
step associated with the initiation and progression of that inhibition of the glutathione-synthesizing enzyme,
cellular dysfunction accompanying diabetic microvas- -glutamylcysteine synthetase, with 0.1 mmol/L buthio-
cular disease [24, 33]. The observed redistribution of cell- nine sulfoximine, a strategy that also depletes cells of
ular PKC-1 to perinuclear and detergent-soluble re- glutathione, also increased cellular sensitivity to sub-
Lal et al: AGE-induced mesangial cell activation 2013
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