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285:1138-1148, 2003. doi:10.1152/ajprenal.00397.2002 Am J Physiol Renal PhysiolGreene Durazo-Arvizu, Sally E. Self, Martin Kuhlmann, John R. Raymond and Eddie L. Milos N. Budisavljevic, LeAnn Hodge, Kelli Barber, John R. Fulmer, Ramon A.

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Oxidative stress in the pathogenesis of experimentalmesangial proliferative glomerulonephritis

Milos N. Budisavljevic,1,2 LeAnn Hodge,1 Kelli Barber,1

John R. Fulmer,1 Ramon A. Durazo-Arvizu,3 Sally E. Self,4

Martin Kuhlmann,5 John R. Raymond,1,2 and Eddie L. Greene61Division of Nephrology, 3Department of Biometry, and 4Department of Pathology and Laboratory Medicine,Medical University of South Carolina, and 2Medical and Research Services, Ralph H. JohnsonVeterans Affairs Medical Center, Charleston, South Carolina 29425; 5Department of InternalMedicine, Universitatskliniken des Saarlandes, 66421 Homburg/Saar, Germany; and 6Division ofNephrology, Department of Medicine, Mayo Clinic, Rochester, Minnesota 55905

Submitted 7 November 2002; accepted in final form 11 August 2003

Budisavljevic, Milos N., LeAnn Hodge, Kelli Barber,John R. Fulmer, Ramon A. Durazo-Arvizu, Sally E.Self, Martin Kuhlmann, John R. Raymond, and EddieL. Greene. Oxidative stress in the pathogenesis of experi-mental mesangial proliferative glomerulonephritis. Am JPhysiol Renal Physiol 285: F1138F1148, 2003; 10.1152/ajprenal.00397.2002.Reactive oxygen species (ROS) are in-creasingly believed to be important intracellular signalingmolecules in mitogenic pathways involved in the pathogene-sis of glomerulonephritis (GN). We explored the effects of theantioxidants -lipoic acid and N-acetyl-L-cysteine on ERKactivation in cultured mesangial cells and the role of ERKactivation in the severity of glomerular injury in a rat modelof anti-Thy 1 GN. In cultured mesangial cells, growth factorsstimulated ERK phosphorylation by 150450%. Antioxidantsreduced this increase by 5060%. Induction of anti-Thy 1nephritis in rats led to a 210% increase in glomerular ERKphosphorylation. This increase in phosphorylated ERK wasreduced by 50% in animals treated with -lipoic acid. Treat-ment with -lipoic acid resulted in significant improvementof glomerular injury. Cellular proliferation was reduced by100%, and the number of proliferating cell nuclear antigen-positive cells was reduced by 64%. The increased expressionof glomerular transforming growth factor-1 protein andmRNA in rats with anti-Thy 1 nephritis was significantlyattenuated and mesangial cell transformation into myofibro-blasts was completely prevented by treatment with -lipoicacid. The effects of -lipoic acid were at least partially due toinhibition of oxidative stress. In rats with anti-Thy 1 nephritis,ROS production was increased 400500%, and this increasewas inhibited by 55% by treatment with -lipoic acid. Wesuggest that ROS may mediate glomerular injury by inducingERK phosphorylation. -Lipoic acid should be considered apotential therapeutic agent in certain types of human GN.

-lipoic acid; anti-Thy 1 nephritis; extracellular signal-regu-lated kinase; -smooth muscle actin; transforming growthfactor-1

EARLY CELLULAR PROLIFERATION followed by subsequentfibrosis is a prominent hallmark of proliferative glo-

merulonephritis (GN), and it may ultimately lead toend-stage renal disease (17). The involvement of extra-cellular stimuli, such as growth factors, cytokines, ac-tivated complement, and immune complexes, in thepathogenesis of experimental and human GN has beenknown for many years. However, the intracellular me-diators that transduce signals from noxious extracel-lular stimuli to unfettered cellular proliferation andaccompanying excess extracellular matrix depositionare only recently being unraveled (35). Precise delin-eation of these intracellular mediators is essential fordevelopment of strategies that will ameliorate the pro-gression of disease and prevent the inexorable loss ofrenal function. Experiments performed initially in cul-tured glomerular cells and, more recently, in certainmodels of experimental GN implicate the activation ofERK in glomerular cellular proliferation (6). ERK, amitogen-activated protein kinase, is a member of thefamily of serine/threonine kinases that regulates theexpression of many genes important for cellulargrowth, mainly by phosphorylating transcription fac-tors, including c-myc, c-Jun, the STAT proteins,NFIL6, ATF2, ETS1, and ELK1. Binding of extracel-lular ligands to G protein- or tyrosine kinase-coupledreceptors leads to a series of protein-protein interac-tions that culminate in phosphorylation of the ERKkinase (MEK) that is a specific activator of ERK. Theactivated (phosphorylated) ERK can then translocateto the nucleus, where it targets specific transcriptionfactors, which, when activated, result in increased cel-lular proliferation and multiple other effects related tocell function (10, 34, 40).

Enhanced generation of reactive oxygen species(ROS) has also been demonstrated in human and ex-perimental GN (14, 33, 36). However, many of theprevious reports evaluating the effects of ROS haveconcentrated on the direct damaging effects of ROS onrenal structural integrity, particularly those caused by

Address for reprint requests and other correspondence: M. N.Budisavljevic, Nephrology Div., Medical University of South Caro-lina, 171 Ashley Ave., Charleston, SC 29425 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

Am J Physiol Renal Physiol 285: F1138F1148, 2003;10.1152/ajprenal.00397.2002.

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formation of lipid peroxidation products. Because ofmore recent evidence implicating ROS as signalingmolecules (37), we explored the roles of ROS as regu-lators of intracellular signaling in mitogenic pathwaysinvolved in the injury produced in an in vivo model ofGN. In the present study, we extended our previousfindings of ROS as major participants in ERK phos-phorylation in cultured mesangial cells (12). We sug-gest that oxidative stress can cause glomerular injury,not only by inflicting direct damage on the glomerularstructures but also by inducing ERK activation andcellular proliferation. Furthermore, suppression of ox-idative stress results in inhibition of ERK activation,reduction of transforming growth factor-1 (TGF-1)mRNA transcription, and amelioration of glomerularinjury.

METHODS

Unless otherwise specified, all drugs, chemicals, reagents,and antibodies were obtained from Sigma (St. Louis, MO).The studies were approved by the Institutional Animal Careand Use Committee of the Medical University of South Caro-lina. All in vivo animal experiments were conducted in ahumane manner in accordance with the National Institutesof Health Guide for the Care and Use of Laboratory Animals.

Mesangial Cell Culture

Glomerular isolation and establishment of primary mes-angial cell cultures were performed as described previously(31). Cells from passages 612 were used for all in vitroexperiments. For experiments investigating ERK phosphor-ylation, cell quiescence was induced by placing cell culturesthat were 6070% confluent in RPMI 1640 medium contain-ing 0.5% BSA, 100 U/ml penicillin, and 100 g/ml streptomy-cin for 48 h before stimulation with growth factors. Equalnumbers of quiescent cells (2 105 cells) in 12-well cultureplates were treated with epidermal growth factor (EGF, 10ng/ml), basic fibroblast growth factor (bFGF, 10 ng/ml), plate-let-derived growth factor (PDGF, 10 ng/ml), thrombin (1 M),or vehicle for 10 min in the presence or absence of 100 and250 M -lipoic acid or 30 mM N-acetyl-L-cysteine (NAC).

Similarly, in [3H]thymidine experiments designed to mea-sure cellular proliferation, cell quiescence was induced whencells were 50% confluent by treatment of the cells with RPMI1640 medium containing 0.1% fetal calf serum (FCS) andpenicillin-streptomycin for 48 h. Cells were then stimulatedwith 10% FCS or PDGF (10 ng/ml) in the presence or absenceof the MEK inhibitor PD-98059 (2 and 10 M) or -lipoic acid(100 M) or vehicle (Tris buffer). At 18 h after stimulation, asecond dose of -lipoic acid or vehicle was given, and [3H]thy-midine was added to all wells for 56 h. Cells were thencollected into vials containing scintillation fluid and countedon a scintillation counter (model LS-6500, Beckman). Incellular proliferation assays, a lower concentration of -lipoicacid was used, because we observed mild cytotoxicity at thehigher dose during prolonged incubation periods.

Induction of Anti-Thy 1 Nephritis

Anti-Thy 1 nephritis was induced in 200-g male Wistarrats by a single injection (1 mg/kg iv) of the monoclonalantibody OX-7 (ECACC, Salisbury, UK). For each experi-ment, the animals were divided into two groups. The treatedgroup (n 12) received daily injections of -lipoic acid (100mg/kg ip; Thioctacid 600 T, Asta Medica) starting 1 day

before the induction of GN. The untreated group (n 12)received intraperitoneal injections of vehicle according to thesame protocol. Animals were killed 4 and 7 days after induc-tion of GN. In each experiment, kidneys from normal ani-mals, i.e., those without anti-Thy 1 nephritis, served ascontrols. The lower pole of one kidney from each animal wasplaced in Carnoys solution and processed for histology andimmunohistochemistry. The remaining kidney tissue wasused for isolation of glomeruli by selective sieving, as de-scribed previously (31). The purity of glomerular isolates(90%) was routinely established by light microscopy. Iso-lated glomeruli were used for analysis of ERK activation byWestern blot and TGF-1 message induction by relativequantitative PCR (see below).

Western Blot Analysis for Total and Phosphorylated ERK

Cultured mesangial cells. This assay was performed asdescribed previously (13). Briefly, 20 l of cell lysates wereloaded onto 420% Tris-glycine gradient miniature precastgels (Novex, San Diego, CA) for electrophoresis. Proteinswere transferred onto Immobilon-P (Millipore, Bedford, MA)membranes and probed with a phosphorylation state-specificERK1/2 antibody or a total ERK1/2 antibody that recognizesphosphorylated and nonphosphorylated ERK (New EnglandBiolabs, Beverly, MA). After incubation with goat anti-rabbitalkaline phosphatase-conjugated secondary antibody, bandswere visualized using a CDP Star kit (New England Biolabs).Band intensity was determined by densitometry.

Glomeruli from animals with anti-Thy 1 nephritis in vivo.Soluble lysates from isolated glomeruli were prepared aspreviously described with minor modifications (3). Briefly,isolated glomeruli were placed immediately in 500 l of lysisbuffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 0.1mM sodium orthovanadate, and a proteinase inhibitor cock-tail containing 150 nM aprotinin, 1 M leupeptin, 500 M4-(2-aminoethyl)benzenesulfonyl fluoride HCl, 1 M E-64,and 0.5 mM EDTA (Calbiochem, San Diego, CA) at 4C. Afterincubation for 5 min, the lysates were sonicated for 1 min andthen centrifuged for 15 min at 10,000 g at 4C. A smallaliquot of the supernatant was set aside for determination ofprotein concentration using the bicinchoninic acid proteinassay (Pierce, Rockford, IL), and the remainder was stored at20C until the day of assay. To detect total and phosphor-ylated ERK, the supernatant (20 g of protein) was mixed 1:1with 2 Laemmli buffer, boiled for 5 min at 95C, and loadedonto 420% polyacrylamide gradient gels (Novex). After elec-trophoresis, proteins were processed for immunoblotting asdescribed above.

TGF-1 mRNA Expression

Total RNA was prepared from glomeruli isolated fromcontrol rats and rats with anti-Thy 1 nephritis. Briefly,glomeruli were washed in PBS, centrifuged at 400 g, andresuspended in TRIzol (GIBCO BRL, Gaithersburg, MD; 1ml/100 mg of glomeruli). Subsequent steps were carried outas recommended by the manufacturer. The isolated RNA wasstored at 70C until the day of the assay.

Total RNA was used to synthesize first-strand cDNA usinga SuperScript first-strand synthesis system for RT-PCR(GIBCO BRL) according to the manufacturers directions.Briefly, 1 g of total RNA was incubated with a 10 mM dNTPmixture and 1 l of random hexamers in nuclease-free waterfor 5 min at 65C and then chilled on ice for 1 min. Areaction mixture containing 10 RT buffer, 25 mM MgCl2,0.1 M DTT, and an RNase inhibitor was added, and the

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samples were incubated for 2 min at room temperature. RT(50 U) was added, and the samples were incubated for 10 minat room temperature and then for 50 min at 44C. Fortermination of the reaction, the sample was heated to 70Cfor 15 min, placed on ice for 1 min, and treated with RNase Hfor 20 min at 37C. Samples were stored at 20C until theywere used for PCR.

Relative semiquantitative PCR was done using a thermalcycler (model 9700, Perkin-Elmer) and a TGF-1 gene-spe-cific relative RT-PCR kit (Ambion, Austin, TX) according tothe manufacturers directions. Briefly, the kit uses a multi-plex reaction to coamplify a TGF-1 target and an 18S ribo-somal target, which is used as an internal standard. Sampleswere amplified using a hot start and 28 cycles of 94C for 30 s,57C for 30 s, and 72C for 45 s. PCR products were visual-ized on 1% agarose gels with ethidium bromide staining.Band intensities were measured using a UMAX 4.3 scanner,Adobe Photoshop 4.0, and Scananalysis 2.5 software (Biosoft1995, Ferguson, MO).

Chemiluminescent Detection of O2 Production

in Glomerular Cells

The procedure of Ohara et al. (27) was used to measure O2

production in glomerular fractions of rat kidney. Briefly, ratswere killed 24 h after induction of anti-Thy 1 nephritis, andglomeruli were collected in a Krebs-HEPES (K-H) bufferconsisting of (in mM) 99.01 NaCl, 4.69 KCl, 1.87 CaCl, 1.20MgSO4, 1.03 K2HPO4, 25.0 NaHCO3, 20.0 Na-HEPES, and11.1 glucose. The glomerular fractions were centrifuged at3,000 rpm for 7 min at 4C, and the pellets were resuspendedin 400 l of K-H buffer. Assays were performed using 100 lof this sample suspension in 2 ml of K-H buffer. After thesesamples were heated at 37C for 30 min, lucigenin was addedto the tubes for a final concentration of 0.25 mM. The tubeswere left at room temperature for 15 min, and then chemi-luminescence of each sample was measured over a period of3060 min using a luminometer (Femtomaster FB12, Zylux).A blank containing all components except sample suspensionwas read before each series of sample readings and wassubtracted from the sample readings.

Immunohistochemistry of Proliferating CellNuclear Antigen

Slides with 3-m sections of kidney tissue were preparedby heating at 60C for 30 min. Sections were deparaffinizedin three changes of xylene, gradually hydrated through mul-tiple changes of ethanol, and rinsed in 1 TBSA-BSAT (150mM NaCl, 0.1% BSA, 5 mM Tris, 0.1% Triton X-100, and0.05% sodium azide, pH 7.4). Endogenous peroxidase activitywas quenched with 3% H2O2 in methanol for 10 min. Theslides were then denatured in 4 N HCl for 20 min andstabilized in 100 mM sodium tetraborate for 5 min. Nonspe-cific staining was blocked using 10 TBSA-BSAT for 30 minat room temperature. Sections were then incubated over-night at 4C with a monoclonal anti-proliferating cell nuclearantigen (PCNA) antibody (Calbiochem), rinsed, incubated for2 h at room temperature in a horseradish peroxidase-labeledgoat anti-mouse IgG (Kirkegard and Perry, Gaithersburg,MD), developed using 3,3-diaminobenzidine, and counter-stained in eosin.

In initial experiments, before the rats were killed, theywere injected with bromodeoxyuridine, and slides werestained with antibromodeoxyuridine antibodies in additionto anti-PCNA antibodies. We obtained nearly identical re-

sults. Therefore, we presented only data with anti-PCNAstaining.

Immunofluorescent Studies

Staining for -smooth muscle actin. Slides were deparaf-finized and hydrated as described above, rinsed in PBS, andimmersed in 10 mM sodium citrate solution (pH 6.0) for 5min at 95C. The solution was changed, and the process wasrepeated once. Slides were allowed to cool in the same solu-tion for 20 min and rinsed in PBS. Nonspecific staining wasblocked with PBS 1% BSA 10% normal goat serum for1 h at room temperature. Slides were incubated overnight at4C in monoclonal anti-human smooth muscle actin (-SMA)antibody (Dako, Carpinteria, CA). Antibodies were visualizedusing a goat anti-mouse FITC-labeled secondary antibody.

Staining for TGF-1 protein. Slides were prepared as de-scribed for -SMA and incubated with goat anti-TGF-1antibody (Santa Cruz Biotechnology, Santa Cruz, CA) anti-body. Staining was visualized using a donkey anti-goatFITC-labeled secondary antibody.

Complement 3 staining. Untreated or -lipoic acid-treatedanimals were killed 1 h after induction of anti-Thy 1 nephri-tis. Kidneys were placed in liquid nitrogen and prepared forcryostat sections. Sections were rinsed three times in PBS

1% BSA and incubated overnight at 4C with sheep anti-ratcomplement 3 antibody (Biogenesis, Poole, UK). Antibodieswere visualized using a donkey anti-sheep FITC-conjugatedsecondary antibody.

Scoring of Glomerular Injury

Kidney sections were stained with hematoxylin and eosinor periodic acid-Schiff and scored for cellularity and numberof PCNA-positive cells, mitoses, and microaneurysms, aspreviously described (16). To assess cellular proliferation andthe number of PCNA-positive cells, the cell count was deter-mined in 30 glomeruli per kidney section, and the resultswere averaged. To assess cellularity, glomeruli were scoredand normalized to control values. PCNA is reported as thenumber of positive cells per glomerulus. The numbers ofmitoses and microaneurysms were also counted in 30 glomer-uli. All glomerular scoring was done by a pathologist (S. E.Self) who was unaware of the experimental design.

Statistical Analysis

Values are results of at least two separate experiments.The results between experiments were consistent, and datafrom typical experiments are presented in Figs. 13, 5, 6, 8,10, and 11. Results from treated and untreated animals inwhich anti-Thy 1 nephritis was induced were normalized toresults from healthy control animals. A two-sample two-sided t-test was used to evaluate treatment, i.e., with -lipoicacid, for significance against the untreated group. Analyseswere confirmed using nonparametric statistics, the tradi-tional Mann-Whitney test, and approximated permutationtest. Experiments involving repeated measures were ana-lyzed using a generalized estimating equations approach.The generalized estimating equations approach providesmodel-based regression methods applicable for analysis ofthe correlated data that result from the repeated-measuresexperiments. Differences between treatments were consid-ered statistically significant for P 0.05. Values aremeans SE. Analyses were performed using the statisticalsoftware STATA.

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RESULTS

Role of Antioxidants in Growth Factor-Induced ERKPhosphorylation in Cultured Rat Mesangial Cells

Quiescent cultured mesangial cells were stimulatedwith EGF, bFGF, PDGF, thrombin, or vehicle, andERK phosphorylation was determined (Fig. 1). EGF,bFGF, PDGF, and thrombin induced 350, 250, 150,and 250% increases in phosphorylated ERK, respec-tively, compared with treatment with vehicle alone.Conversely, when cells were treated with these growthfactors in the presence of the thiol-derived antioxidants-lipoic acid (250 M) and NAC (30 mM), ERK phos-phorylation was reduced significantly by 5060% foreach growth factor tested. Treatment with 100 M-lipoic acid modestly decreased phosphorylated ERKby 2030%. Growth factors had no effect on total ERK,and neither -lipoic acid nor NAC, when used alone,led to a significant change in ERK phosphorylationcompared with vehicle (data not shown). These datademonstrate the ability of two chemically distinct an-tioxidants to significantly reduce growth factor-in-duced ERK phosphorylation in cultured mesangialcells. Our data suggest that ERK phosphorylation in-duced by growth factors is at least partly mediatedthrough the generation of ROS.

ERK Phosphorylation Is Involved in Growth Factor-Stimulated Mesangial Cellular Proliferation

We, as well others, previously showed that an MEKinhibitor, PD-98059, attenuates increased ERK phos-phorylation in agonist-stimulated mesangial cells. Wewanted to determine whether this inhibition has ef-fects on cellular proliferation. Cultured rat mesangialcells were stimulated with PDGF (10 ng/ml) alone or inthe presence of PD-98059 or vehicle. PDGF stimulationresulted in a twofold increase in [3H]thymidine incor-poration that was regularly attenuated by 515% in

the presence of 2 M PD-98059 (Fig. 2). However,coincubation of mesangial cells with 10 M PD-98059resulted in a significant 60% reduction in [3H]thymi-dine incorporation. PD-98059 or vehicle alone did nothave effects on cellular proliferation (not shown). Thisexperiment demonstrated that ERK phosphorylationhas important roles in growth factor-induced mesan-gial cellular proliferation.

-Lipoic Acid Attenuates Proliferation of GrowthFactor-Stimulated Cultured Rat Mesangial Cells

The effects of -lipoic acid on cellular proliferationwere tested in primary cultures of rat mesangial cells.Cells were stimulated with PDGF or 10% FCS, andcellular proliferation was assessed by [3H]thymidineincorporation. PDGF at 10 ng/ml consistently inducedat least a twofold increase in thymidine incorporation(Fig. 3). Coincubation of PDGF-stimulated mesangialcells with 100 M -lipoic acid resulted in a significant28% reduction in [3H]thymidine incorporation. The ve-hicle in which -lipoic acid was dissolved had no effecton PDGF-induced cellular proliferation. Similarly, 100M -lipoic acid caused a significant decrease in thy-

Fig. 1. Inhibition of growth factor-stimulated ERK phosphorylationby the antioxidants -lipoic acid and N-acetyl-L-cysteine (NAC) incultured mesangial cells. Cell stimulation with growth factors [epi-dermal growth factor (EGF, 10 ng/ml), basic fibroblast growth factor(bFGF, 10 ng/ml), platelet-derived growth factor (PDGF, 10 ng/ml),and thrombin (1 M)] induced a 2- to 4-fold increase in phosphory-lated ERK above stimulation with vehicle. Coincubation of growthfactor-stimulated cells with 250 M -lipoic acid or 30 mM NACresulted in a statistically significant 5060% reduction in phosphor-ylated ERK. *P 0.05 vs. growth factor alone.

Fig. 2. Inhibition of ERK kinase attenuates PDGF-induced mesan-gial cellular proliferation. In PDGF (10 ng/ml)-stimulated cells,[3H]thymidine incorporation was increased 2-fold compared withunstimulated cells. Coincubation of cells with PD-98059 (PD) re-sulted in a 60% reduction in the increase in [3H]thymidine incorpo-ration. Coincubation of cells with vehicle had no effect (not shown).Values [counts per minute (cpm)] are means SE of an experimentperformed in quadruplicate. *P 0.01 vs. PDGF.

Fig. 3. Effects of -lipoic acid (LA) on [3H]thymidine incorporationby PDGF-stimulated rat mesangial cells. Incubation of culturedmesangial cells with 100 M -lipoic acid resulted in 28% inhibitionof PDGF-stimulated [3H]thymidine incorporation. Values aremeans SE of an experiment performed in quadruplicate. *P 0.05vs. PDGF alone or PDGF vehicle.





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midine incorporation in mesangial cells stimulatedwith FCS (data not shown). Incubation of mesangialcells with -lipoic acid in the absence of growth factorsinduced a small, insignificant inhibition of cellularproliferation (data not shown). These studies suggestthat treatment of mesangial cells with -lipoic acid canattenuate growth factor-induced cellular proliferation.

To exclude the possibility that inhibition of thymi-dine incorporation was the result of cell death, wetested the viability of mesangial cells after exposure to-lipoic acid, PD-98059, or vehicle. Mesangial cellsfrom some plates were not recovered for counting afterincubation with the inhibitors and [3H]thymidine butwere left to grow after the medium was changed. Wefound no difference in cell appearance and, in particu-lar, nuclear morphology between cells that were ex-posed to -lipoic acid and control cells that were neverexposed to -lipoic acid and [3H]thymidine. Finally, westained cells exposed to -lipoic acid or PD-98059 withtrypan blue and found no difference in mortality com-pared with cells exposed to vehicle.

Effects of -Lipoic Acid on ERK Phosphorylation andPathological Changes in Anti-Thy 1 Nephritis

After demonstrating that -lipoic acid could decreaseERK phosphorylation and cellular proliferation in cul-tured mesangial cells, we wanted to explore the rele-vance of this finding in vivo. For in vivo experiments,we employed a rat model of mesangial proliferativeGN, i.e., anti-Thy 1 nephritis. We first established thattreatment of animals with -lipoic acid does not inter-fere with the induction of nephritis. Complement bind-ing to the glomerular mesangium, a crucial step in the

development of anti-Thy 1 nephritis, was not impededby treatment with -lipoic acid (Fig. 4). This findingallowed us to proceed with experiments in which ef-fects of -lipoic acid on the pathogenesis and severity ofglomerular injury in anti-Thy 1 nephritis could betested.

Attenuation of ERK phosphorylation in glomeruli ofrats with anti-Thy 1 nephritis by -lipoic acid. To studyERK phosphorylation, anti-Thy 1 nephritis was in-duced in male Wistar rats (Fig. 5). The treated groupreceived daily intraperitoneal injections of -lipoicacid, while the untreated group received vehicle ac-cording to the protocol described in METHODS. Total andphosphorylated ERK were measured in isolated glo-meruli from each animal. Four days after induction ofGN, phosphorylated ERK was increased by 210% inglomeruli of untreated animals compared with healthycontrol animals in which anti-Thy 1 nephritis was notinduced. We observed a significant, 50% reduction inphosphorylated ERK in rats treated with -lipoic acidcompared with anti-Thy 1 nephritis animals treatedwith vehicle alone. The levels of total ERK did notchange during the course of nephritis and were notinfluenced by treatment with -lipoic acid. Thus treat-ment with the antioxidant -lipoic acid blunts ERK

Fig. 4. Complement binding to glomerular mesangium in rats withanti-Thy 1 nephritis treated with -lipoic acid. Kidney tissue wassnap-frozen in liquid nitrogen, and the cryostat section was incu-bated with sheep anti-rat complement 3 antibody. Binding wasvisualized using a donkey anti-sheep FITC-conjugated secondaryantibody.

Fig. 5. A: representative samples of total and phosphorylated ERKin glomeruli of control rats and rats with anti-Thy 1 nephritis withand without -lipoic acid. Amount of total ERK was not differentbetween control rats and rats with anti-Thy 1 nephritis with orwithout -lipoic acid. Each lane represents a sample obtained froman individual rat. B: densitometric analysis of phosphorylated ERK.ERK phosphorylation was increased 200% 4 days after induction ofanti-Thy 1 nephritis. Rats treated with -lipoic acid demonstrated asignificant 50% decrease in phosphorylated ERK. Intensity of bandsin phosphorylated ERK is corrected to amount of total ERK. Valuesare means SE from 6 rats in each experimental group. *P 0.05vs. untreated.





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activation in this model of GN. These experimentsdemonstrate that ERK is activated during the prolif-erative phase of this model of experimental GN. Theyfurther support our data obtained in cultured mesan-gial cells that ROS are involved in ERK activation.

Amelioration of glomerular injury with -lipoic acidin rats with anti-Thy 1 nephritis. The effects of -lipoicacid on the severity of glomerular injury were evalu-ated by assessing glomerular damage in the course ofanti-Thy 1 nephritis. We evaluated cellular prolifera-tion and the number of mitoses and PCNA-positivecells in the glomeruli of control animals and animalswith anti-Thy 1 nephritis with and without -lipoicacid treatment. At 4 days after induction of anti-Thy 1nephritis, untreated animals developed the typical fea-tures of mesangial proliferative GN, with an increase

in glomerular cellularity (Fig. 6A) compared with con-trol animals as well as a ninefold increase in thenumber of PCNA-positive cells (Fig. 6B). Treatmentwith -lipoic acid significantly reduced cellular prolif-eration and the number of PCNA-positive cells by 100and 64%, respectively. Mitoses were very rarely seen innormal glomeruli. At 4 days after induction of anti-Thy1 nephritis, we observed 1 mitosis per 60 screenedglomeruli in the untreated group. Although the differ-ence did not reach statistical significance, treatmentwith -lipoic acid decreased the incidence of mitoses by50% to 1 in 120 screened glomeruli (data not shown).From the data presented in Figs. 5 and 6, we cansuggest that glomerular cellular proliferation corre-lated with ERK activation and that -lipoic acid treat-ment not only blunted ERK activation but also signif-icantly reduced glomerular cellular proliferation.

We did not investigate the cellular origins of ROS,but this issue was studied by Nakamura et al. (24)using cell-specific monoclonal antibodies. Their find-ings suggest that, in the early phase of anti-Thy 1nephritis, ROS are mainly generated by infiltratingmacrophages, but by 4 days after induction of anti-Thy1 nephritis, mesangial cells appear to produce a sub-stantial amount of ROS.

Antioxidant -Lipoic Acid Prevents PhenotypicTransformation of Glomerular Mesangial Cellsin the Course of Anti-Thy 1 Nephritis

In the course of experimental and human GN, pro-liferating mesangial cells undergo phenotypic changeswhile they acquire the characteristics of myofibroblasts(1, 17). This is typically demonstrated by positive stain-ing of kidney sections with antibodies against -SMA.Normal glomeruli did not stain positive for -SMA(Figs. 7A and 8). Staining was detected only in themedia of blood vessels. Four days after GN induction,-SMA staining became positive in 75% of glomeruli(Figs. 7B and 8). In animals treated with -lipoic acid,3% of glomeruli expressed positive staining for-SMA (Figs. 7C and 8). These experiments demon-strate that preventing the production of ROS and/orremoving ROS with -lipoic acid prevents the pheno-typic changes in proliferating resident mesangial cells.

Glomerular Expression of TGF-1 Protein and mRNAin the Course of Anti-Thy 1 Nephritis

Proliferating mesangial cells upregulate genes im-portant in production of extracellular matrix compo-nents (7). The generation of ROS has previously beenshown to upregulate TGF-1 mRNA and activity incultured mesangial cells (13). TGF-1 message induc-tion contributes to the increase in mesangial matrix ina number of experimental and human glomerulone-phritides. To explore the effects of -lipoic acid onglomerular TGF-1 expression, we used immunohisto-chemical methods to assess the glomerular expressionof TGF-1 in the course of anti-Thy 1 nephritis. Innormal glomeruli, we did not observe any significant

Fig. 6. Severity of glomerular injury in rats with anti-Thy 1 nephri-tis with or without -lipoic acid compared with control rats (withoutanti-Thy 1 nephritis). A: 4 days after induction of anti-Thy 1 nephri-tis, rats developed increased glomerular cellularity compared withcontrol animals. Treatment with -lipoic acid resulted in 100%attenuation of glomerular hypercellularity. B: 9-fold increase innumber of proliferating cell nuclear antigen (PCNA)-positive cells issignificantly reduced 65% by treatment with -lipoic acid. *P 0.05vs. untreated.





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staining with antibodies against TGF-1 (Fig. 9A). Aminimal quantity of scattered staining could be seen insome glomeruli early (by 4 days after induction ofnephritis) in the course of glomerular injury (Fig. 9B).However, by 7 days after induction of anti-Thy 1 ne-phritis, virtually all glomeruli demonstrated pro-nounced immunoreactivity for TGF-1 (Fig. 9C). Inglomeruli from -lipoic acid-treated rats with anti-Thy1 nephritis, TGF-1 expression was greatly attenuated7 days after induction of nephritis (Fig. 9D). To assessglomerular TGF-1 expression in the course of anti-Thy 1 nephritis and the effects of -lipoic acid on thisexpression in more quantitative terms, we analyzedTGF-1 mRNA expression. Total RNA was isolatedfrom glomeruli of individual animals, and a relativequantitative RT-PCR was performed. Figure 10 dem-onstrates the relative abundance of TGF-1 mRNAsin the glomeruli of control rats and in untreated or-lipoic acid-treated anti-Thy 1 nephritis rats. Four daysafter induction of anti-Thy 1 nephritis, a small increasein TGF-1 mRNA expression was noted in anti-Thy 1compared with control animals without anti-Thy 1 nephri-tis. In -lipoic acid-treated rats with anti-Thy 1 nephritis, asmall decrease in glomerular TGF-1 mRNA expressionwas demonstrated. At 7 days after induction of nephri-tis, TGF-1 mRNA expression was increased 10-fold.Treatment of anti-Thy 1 rats with -lipoic acid resultedin a significant 68% attenuation of TGF-1 mRNAexpression. These findings suggest that treatment withthe antioxidant -lipoic acid can significantly attenuate theincrease in glomerular TGF-1 mRNA expression in theanti-Thy 1 model of GN.

Fig. 7. Immunofluorescent staining of kidney tissue for -smoothmuscle actin (-SMA) in control rats and untreated and -lipoicacid-treated anti-Thy 1 nephritis rats. A: in control rats, i.e., withoutanti-Thy 1 nephritis, positive staining for -SMA was seen only inmedia of blood vessels, while glomeruli are uniformly negative. B: inrats with anti-Thy 1 nephritis, positive staining for -SMA wasobserved within glomeruli. C: in -lipoic acid-treated anti-Thy 1nephritis rats, glomerular staining became negative.

Fig. 8. Percentage of glomerular staining for -SMA. At 4 days afterinduction of anti-Thy 1 nephritis, 75% of glomeruli expressedpositive staining for -SMA. Treatment with -lipoic acid reducedstaining to 3% of glomeruli. *P 0.01 vs. untreated.





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Effects of -Lipoic Acid on ROS Expressionin Anti-Thy 1 Nephritis

To determine whether the effects of -lipoic acidwere attributable to the attenuation of oxidative stress,we assayed glomerular ROS expression in control ani-mals and in untreated and -lipoic acid-treated ani-mals with anti-Thy 1 nephritis. Glomeruli were iso-lated in K-H buffer, and glomerular ROS productionwas determined by measuring lucigenin-inducedchemiluminescence. Development of anti-Thy 1 ne-phritis was associated with a fivefold increase in glo-merular ROS production compared with control ani-mals without anti-Thy 1 nephritis (Fig. 11). In -lipoicacid-treated animals with anti-Thy 1 nephritis, therewas a significant 55% decrease in glomerular ROSproduction. These experiments suggest that the effectsof -lipoic acid on mesangial cellular proliferation, invitro and in vivo ERK phosphorylation, glomerularTGF-1 expression, and mesangial cell phenotypictransformation are mediated through attenuation ofROS generation.

DISCUSSION

The role of oxidative stress as a major participant inthe pathogenesis of human and experimental GN hasbeen increasingly investigated (14, 18, 25, 26, 32, 33,36). Our previous work in cultured rat mesangial cellsdemonstrated that ROS generation is involved in thesignal transduction pathway linking activated cell sur-face receptors to ERK phosphorylation and that thiseffect was attenuated by coincubation of cells with-lipoic acid (12). In addition, we have shown thatreceptor stimulation produces measurable amounts ofH2O2 and O2

in a time scale similar to that of ERKphosphorylation and that three structurally differentoxidants applied to mesangial cells were able to phos-phorylate ERK to a similar degree. That work mapped

ROS activity downstream from the PKC and proximalto the MEK in the ERK cascade. In the present studies,we suggest a role(s) for ROS in intracellular signaltransduction pathways that lead to ERK activation inthe anti-Thy 1 model of GN.

In our initial experiments, we examined the role ofROS in growth factor-induced ERK phosphorylation incultured rat mesangial cells. To demonstrate that therole of ROS in ERK activation is general for growthfactors, we stimulated cells with PDGF, EGF, bFGF,and thrombin. We demonstrated that two chemicallydistinct antioxidants significantly blunted growth fac-tor-induced ERK phosphorylation in cultured mesan-gial cells. We obtained consistent reductions in ERKphosphorylation with both antioxidants in the pres-ence of all growth factors tested. Previous data fromour laboratory showed that the effects of antioxidantson mesangial cell ERK phosphorylation were abolishedin the presence of buthionine sulfoximine, a potentinhibitor of glutathione synthase (12). Our previousstudy supports an intracellular location for the actionof an antioxidant on ERK, because glutathione is themost abundant and one of the most important intra-cellular antioxidants.

We next demonstrated that the MEK inhibitor PD-98059 and -lipoic acid attenuated PDGF-stimulatedmesangial cellular proliferation in vitro. To inducecellular proliferation, we employed PDGF, because thisgrowth factor has been widely implicated in the patho-genesis of the proliferation involved in glomerular in-jury in human disease and experimental models (17).Furthermore, interference with PDGF signal transduc-tion pathways and inhibition in ERK phosphorylationhave been shown to ameliorate the injury in experi-mental models of GN (5, 11). Although statisticallysignificant, the in vitro inhibition of proliferation ofPDGF-stimulated cultured mesangial cells by -lipoic

Fig. 9. Immunofluorescent staining ofkidney tissue for transforming growthfactor-1 (TGF-1) protein in controlrats and rats with anti-Thy 1 nephri-tis. A: TGF-1 protein was not detectedby immunostaining in glomeruli of con-trol rats without anti-Thy nephritis. B:some staining was seen 4 days afterinduction of anti-Thy 1 nephritis. C: by7 days after induction of nephritis, avery pronounced staining was detectedin virtually all glomeruli. D: stainingwas greatly reduced in -lipoic acid-treated anti-Thy 1 nephritis rats.





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acid was less pronounced than that by PD-98059 or-lipoic acid in the course of anti-Thy 1 nephritis.Although we do not know the exact mechanism for thisfinding, we can speculate that -lipoic acid may inter-fere with ROS production induced by other factors inthe course of experimental nephritis and that PDGFeffects on cellular proliferation may be only partiallyROS dependent. Furthermore, the concentration of-lipoic acid employed in cellular proliferation experi-ments was lower than that used in ERK phosphoryla-tion assays (see METHODS). Nevertheless, our results areconsistent with data from the literature demonstratingthat modification of oxidative stress alters renal cellu-lar proliferation and differentiation. For example,Kitamura (19) showed, in cultured rat mesangial cells,that a reducing agent (NAC) induced a cell phenotypeassociated with suppressed mitogenesis, whereas theoxidizing agents diamide and menadione had the op-posite effect. It has also been shown that ROS areinvolved in angiotensin II-induced ERK activation andsubsequent hypertrophy of renal tubular cells (15).

The potential clinical and pathophysiological rele-vance of the in vitro findings was tested in an in vivoanimal model of mesangial proliferative GN, i.e., anti-Thy 1 nephritis. First, we demonstrated that the aug-mentation in ERK phosphorylation correlates with thecellular proliferation phase of anti-Thy 1 nephritis. Apathogenic role for ERK phosphorylation in GN hasbeen previously suggested in the antiglomerular base-ment membrane model (3) and, more recently, in theanti-Thy 1 model of GN (4). Furthermore, Bokemeyeret al. (5) demonstrated that the ERK kinase inhibitorU-0126 decreased phosphorylated ERK expression andcellular proliferation in this model of experimental GN.Our results not only support the contention that ERKactivation is likely a common pathophysiological mech-anism underlying cellular proliferation in the course ofexperimental GN but also suggest that ROS play im-portant roles in its activation.

Second, we have demonstrated that a 400500%increase in oxidative stress occurs during the course ofthe anti-Thy 1 nephritis and that this increase pre-cedes measurable increases in ERK phosphorylation.Our contention of a temporal relation between ROSgeneration and ERK phosphorylation in the course ofanti-Thy 1 nephritis is supported by recent work byGaertner et al. (9) and Bokemeyer et al. (4). Theydemonstrated that increases in glomerular ROS occurwithin several hours after induction of the disease,peak at 24 h, and remain elevated for 5 days. Theassays of glomerular phosphorylated ERK detected aslight increase at 2 h, with further rises to a peak 6days after induction of anti-Thy 1 nephritis.

The present study further demonstrates that treat-ment of rats with experimental GN with an antioxi-dant (-lipoic acid) results in a significant decrease inoxidative stress and ERK phosphorylation. Thus itappears that ROS participate in ERK phosphorylationduring the course of GN in a fashion similar to thatobserved in cultured mesangial cells stimulated bygrowth factors. The present studies also demonstrate

Fig. 10. A: representative samples of glomerular expression ofTGF-1 mRNA (T) and 18S RNA (18) in control rats (C), untreatedrats 4 and 7 days after induction of anti-Thy 1 nephritis (D4 and D7,respectively), and -lipoic acid-treated rats 4 and 7 days after induc-tion of anti-Thy 1 nephritis (D4LA and D7LA, respectively). Eachlane represents a sample obtained from an individual rat. B: densi-tometric analysis of TGF-1 mRNA expression. By 4 days afterinduction of anti-Thy 1 nephritis, there was a small increase inTGF-1 mRNA expression compared with control rats without anti-Thy 1 nephritis (D4). Decrease in TGF-1 mRNA expression wasinsignificant in -lipoic acid-treated rats 4 days after induction ofanti-Thy 1 nephritis (D4LA). At 7 days after induction of anti-Thy 1nephritis (D7), glomerular TGF-1 mRNA expression was increased10-fold compared with control rats. Treatment with -lipoic acidresulted in 68% reduction in TGF-1 mRNA expression 7 days afterinduction of anti-Thy 1 nephritis (D7LA). Values are means SEfrom 6 individual rats in each experimental group. *P 0.05 vs. D7.

Fig. 11. Effects of -lipoic acid on oxidative stress in the course ofanti-Thy 1 nephritis. One day after induction of anti-Thy 1 nephritis,glomerular lucigenin-induced chemiluminescence was increased5-fold compared with control animals without anti-Thy 1 nephritis.This increase was reduced by 55% in -lipoic acid-treated rats withanti-Thy 1 nephritis. Values are means SE from 6 individual ratsin each experimental group. *P 0.05 vs. untreated.





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that decreases in oxidative stress and ERK phosphor-ylation are associated with the amelioration of glomer-ular injury in anti-Thy 1 nephritis. We found thatexuberant cellular proliferation judged by total glomer-ular cellularity and the number of PCNA-positive cellswas significantly reduced by treatment with -lipoicacid, similar to that obtained by use of ERK kinaseinhibitor by Bokemeyer et al. (5). It thus appears thatthe effects of the antioxidant -lipoic acid parallel theeffects of ERK kinase inhibition in the course of anti-Thy 1 nephritis. However, it is obvious that ROS arenot the only factors involved in ERK phosphorylationin the course of this model of GN.

Treatment with -lipoic acid also prevented pheno-typic changes in the proliferating mesangial cells. It isinteresting that mesangial cell expression of -SMA inpatients with IgA nephropathy directly correlates withprogression to end-stage renal disease (38). Experi-mental evidence elsewhere suggests that the acquisi-tion of the expression of -SMA by cultured mesangialcell appears to be modulated by redox-sensitive signalpathways (19). Although we do not have direct proof,several lines of evidence suggest that ERK phosphor-ylation could induce phenotypic changes in the glomer-ulus and that the effect of -lipoic acid in our study wasat least partially the result of inhibition of ERKphosphorylation. For example, constitutive ERK ac-tivation has been associated with a transformed phe-notype in other cell types (8, 21, 28). Treatment ofrats with anti-Thy 1 nephritis with the ERK kinaseinhibitor U-0126 resulted in decreased stainingagainst -SMA (5).

Phenotypic transformation of mesangial cells is as-sociated with the increased production of TGF-1 (39).We previously showed that TGF-1 production by ago-nist-stimulated cultured mesangial cells was attenu-ated by ERK inhibition and antioxidants (13). Here wehave demonstrated that treatment of anti-Thy 1 ne-phritis animals with an antioxidant resulted in atten-uated TGF-1 expression in a fashion similar to thatobserved in cultured mesangial cells, although it isclear that mesangial cells may not be the only source ofglomerular TGF-1 in the course of GN (20). A cor-responding effect of -lipoic acid, decreased produc-tion of extracellular matrix material, has recentlybeen demonstrated in a rat model of diabetic ne-phropathy (23). It is interesting that a tendency for adecrease in matrix accumulation was also demon-strated in anti-Thy 1 nephritis by the ERK kinaseinhibitor U-0126 (5).

It is highly unlikely that the effects observed with-lipoic acid were the result of interference with theinduction of GN for several reasons. First, all rats withanti-Thy 1 nephritis, regardless of therapeutic inter-vention, expressed similar degrees of proteinuria whentested after induction of GN (data not shown). Further-more, staining of kidney sections of animals with anti-Thy 1 nephritis with anti-rat complement 3 antibodiesdemonstrated that treatment with -lipoic acid did notalter complement binding to the glomeruli. Comple-ment binding and resulting mesangiolysis are crucial

initial steps in the generation of anti-Thy 1 nephritis.Finally, the extent of mesangiolysis was not signifi-cantly altered by treatment with -lipoic acid (data notshown), suggesting that -lipoic acid blocked specificpathways of glomerular damage.

-Lipoic acid is a naturally occurring compound thatserves as a prosthetic group to multiple enzyme com-plexes in the mitochondria (30). Abundant experimen-tal evidence has demonstrated that -lipoic acid is alsoa potent scavenger of free radicals and an importantfactor in maintaining antioxidant status in the cell byreplenishing antioxidants such as glutathione (2, 29).It may also exert an antioxidant effect by chelation oftransition metals (30). Previous studies have shownthat treatment of rats with experimental diabetic ne-phropathy with -lipoic acid resulted in increased re-nal cortical glutathione levels and decreased malondi-aldehyde, which is an indicator of significant lipidperoxidation (23).

On the basis of the data presented here, we suggestthat the pathophysiological pathway depicted in Fig.12 may play a role in the pathogenesis of GN. Differentstimuli, such as activated complement, deposition ofimmune complexes, or local release of growth factors orcytokines, induce formation of ROS by intrinsic glo-merular cells or infiltrated leukocytes. Generated ROScan then activate the ERK cascade, culminating in asustained ERK phosphorylation. Phosphorylated ERKsubsequently translocates into the nucleus and in-duces cellular proliferation, phenotypic transforma-tion, and TGF-1 production, resulting in a clinicalpicture of GN.

In summary, we have demonstrated that ROS have aprominent role in ERK activation in a model of prolif-erative GN and that suppression of ROS productionresults in blunted ERK activation, decreased TGF-1mRNA transcription, and amelioration of glomerularinjury. On the basis of data from the present studies inan experimental model of mesangial proliferative GNand because of the low toxicity potential for -lipoicacid, we propose that -lipoic acid should be considereda potential therapeutic agent in certain types of humanproliferative glomerular injury such as IgA nephropa-thy or mesangioproliferative GN, where current ther-apeutic regimens remain suboptimal in preventingprogressive renal disease.

DISCLOSURES

This work was supported by grants from the Department ofVeterans Affairs (Merit and Research Enhancement Award ProgramAwards to J. R. Raymond), National Institutes of Health GrantsDK-52448 and HL-03710 (to J. R. Raymond and E. L. Greene), aRobert Wood Johnson Faculty Development Award (to E. L. Greene),

Fig. 12. On the basis of the present study, we suggest that reactiveoxygen species (ROS) play a role as intracellular signaling moleculesin the pathogenesis of glomerulonephritis.





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and a laboratory endowment jointly supported by the Medical Uni-versity of South Carolina Division of Nephrology and Dialysis Clin-ics, Inc. (to J. R. Raymond).

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