Kidney International, Vol. 59 (2001), pp. 1324–1332

PDGF signal transduction inhibition ameliorates experimentalmesangial proliferative glomerulonephritis1

RICHARD E. GILBERT, DARREN J. KELLY, TARA MCKAY, STEVEN CHADBAN,PRUDENCE A. HILL, MARK E. COOPER, ROBERT C. ATKINS, and DAVID J. NIKOLIC-PATERSON

Department of Medicine, University of Melbourne, St. Vincent’s Hospital; Department of Nephrology, Monash Medical Centerand Department of Medicine, Monash University; Department of Anatomy and Cell Biology, University of Melbourne;Department of Medicine, University of Melbourne, Austin and Repatriation Medical Center, Melbourne, Victoria, Australia

PDGF signal transduction inhibition ameliorates experimental merular diseases in humans, a major cause of end-stagemesangial proliferative glomerulonephritis. renal failure throughout much of the world. The mecha-

Background. Platelet-derived growth factor (PDGF) has nisms underlying these changes are incompletely under-been consistently implicated in the cell proliferation and extra-stood, although studies conducted over the past 15 yearscellular matrix accumulation, which characterize progressivesuggest a key role for growth factors in the pathogenesisglomerular disease. In the present study, the effects of a potent

and selective inhibitor of PDGF receptor tyrosine kinase, STI of renal injury [1]. While many act in a pleiotropic fash-571, were examined in vitro and in vivo. ion, increasing evidence suggests that the effects of spe-

Methods. Cultured mesangial cells were incubated with PDGF cific growth factors predominate in certain physiological(50 ng/mL) and fibroblast growth factor-2 (FGF-2; 50 ng/mL)and pathological situations [2–5].and treated with STI 571 (0.13 to 2.0 mmol/L). Experimental

Platelet-derived growth factor (PDGF) has been widelymesangial proliferative glomerulonephritis was induced inmale Wistar rats with monoclonal OX-7, anti-rat Thy-1.1 anti- implicated in the pathogenesis of progressive renal injurybody with rats randomized to receive either STI 571 (50 mg/kg in both human disease and experimental models [6].intraperitoneally daily) or vehicle. Animals were examined six PDGF is synthesized by resident renal cells [7, 8] anddays later.

by infiltrating macrophages [8] that are frequently associ-Results. In vitro, both PDGF and FGF-2 induced a threefoldincrease in mesangial cell 3H-thymidine incorporation. STI 571 ated with progressive renal injury [9]. The actions ofreduced PDGF but not FGF-2–stimulated mesangial cell prolif- PDGF include stimulation of mesangial cell proliferationeration in a dose-dependent manner, with complete abolition [10], increased extracellular matrix (ECM) synthesis [11],at 0.4 mmol/L. In animals with Thy-1.1 glomerulonephritis,

and increased expression of the prosclerotic cytokine,PDGF receptor tyrosine kinase blockade was associated withtransforming growth factor-b (TGF-b) [12]. Further-significant reductions in mesangial cell proliferation (P ,

0.001), the number of activated (a-smooth muscle positive) more, PDGF levels are also increased in response to amesangial cells, and glomerular type IV collagen deposition variety of factors that have been implicated in renal(P , 0.001). disease, including angiotensin II [13], endothelin [14],Conclusion. The amelioration of the pathological findings

inflammatory cytokines [15], and advanced glycation endof experimental mesangial proliferative glomerulonephritis byblockade of PDGF receptor activity suggests the potential clini- products [11, 12].cal utility of this approach as a therapeutic strategy in glomeru- Specific inhibition of PDGF action is therefore a majorlar disease. target for therapy in glomerular disease. Recently, selec-

tive, receptor tyrosine kinase (RTK) inhibitors with in vivoactivity have been synthesized. Of this class of 2-phenyl-

Cellular proliferation and extracellular matrix accu-aminopyridines, signal transduction inhibitor (STI) 571

mulation are characteristic features of progressive glo-is a potent and selective inhibitor of PDGF RTK andv-Abl kinase (with which it shares substantial homology)

1 See Editorial by Floege and Ostendorf, p. 1592 [16]. Such agents may therefore provide therapeutic po-tential in the treatment of disorders characterized byKey words: platelet-derived growth factor, extracellular matrix, cell

proliferation, progressive renal disease, STI 571. overexpression of PDGF or v-Abl such as glomerulardisease and chronic myeloid leukemia (CML).Received for publication August 8, 2000

The present study sought first to examine the effectsand in revised form October 26, 2000Accepted for publication November 6, 2000 of PDGF-RTK inhibition with STI 571 on mesangial cell

proliferation in vitro, and also to determine the efficacy 2001 by the International Society of Nephrology

1324

Gilbert et al: PDGF signal transduction inhibition 1325

of this approach on the pathological hallmarks of experi- counting was performed with a b counter (Wallace Rack-mental mesangial proliferative nephritis, mesangial pro- beta, Wallac Oy, Turku, Finland). Replicates of six wellsliferation, and matrix accumulation. were used.

Rat anti–Thy-1 nephritisMETHODS

Male Wistar rats (150 to 170 g) were obtained fromDrug preparation Monash Animal Services (Melbourne, Australia). Anti–

STI 571, the properties of which have been previously Thy-1 nephritis was induced in two groups of eight ratsdescribed in detail elsewhere [16], was obtained from by an intravenous injection of 5 mg/kg OX-7 IgG, asNovartis Pharmaceuticals (generous gift of Dr. E. Buch- previously described [20]. Starting one day after OX-7dunger, Novartis, Basel, Switzerland). For in vitro studies, IgG administration, animals received daily intraperito-STI 571 was diluted to 10 mmol/L in dimethyl sulfoxide neal injections with either STI 571 (50 mg/kg) or vehicle(DMSO). This stock solution was then diluted in cell cul- control (10% DMSO in saline) until killed on day 6, theture medium to a final concentration of 0.013 to 2.0 mmol/L. peak of mesangial proliferation in this model [20]. TheFor in vivo studies, fresh solution was prepared daily by dose of 50 mg/kg/day was selected on the basis of previ-dissolving the compound in DMSO (200 mg/mL) and ous studies on the inhibition of tumor growth [16]. Thediluting this stock solution 1:10 in normal saline. administration of STI 571 was commenced 24 hours after

OX-7 administration as mesangiolysis is complete by thisAntibodiestime [20]. Three hours prior to sacrifice, all rats were

Monoclonal OX-7, anti-rat Thy-1.1 antibody was used given an intraperitoneal injection of 50 mg/kg bromo-for the induction of mesangial proliferative glomerulone- deoxyuridine (BrdU) in order to label cells in the DNAphritis [17]; macrophages were detected using ED1, anti- synthetic (S) phase of the cell cycle. A group of eightrat CD68 [18]. Myofibrobalsts were identified by labeling normal rats was also injected with BrdU three hourswith 1A4, anti-human a-smooth muscle actin (Sigma before sacrifice.Immunochemicals, St. Louis, MO, USA), and M744, anti-BrdU (Dako, Glostrup, Denmark) was used to identify Proteinuria and renal function assessmentproliferating cells. A polyclonal goat anti-bovine/anti- Twenty-four hour urine collections and blood sampleshuman type IV collagen antibody (Southern Biotechnol- were taken on day 23 (prior to experiment) and dayogy, Birmingham, AL, USA) was also used to examine

6. Urinary protein concentration was measured by theextracellular matrix.

benzethonium chloride method [21]. Serum and urinecreatinine levels were measured using the Jaffe rate reac-Mesangial proliferationtion [22].A well-characterized cloned mesangial cell line (1097)

isolated from Sprague-Dawley rats [19] was used be- Histologytween passages 20 and 30. For these experiments, cells

Tissues were fixed in 10% neutral-buffered formalinwere cultured in RPMI 1640 Medium (GIBCO, Grandand were embedded in paraffin. Kidney sections (4 mm)Island, NY, USA) with heat-inactivated fetal calf serumwere stained with periodic acid-Schiff’s reagent (PAS).(FCS), 100 U/mL penicillin, and 100 mg/mL streptomycinQuantitation of nuclei was performed by examining 50in humidified 5% CO2 atmosphere at 378C. Mesangialhilar glomeruli per animal.cells were plated out at low density in 96-well flat-bot-

tomed microtiter plates in RPMI/10% FCS and allowedImmunohistochemistry

to adhere overnight. The subconfluent cells were thenType IV collagen. Immunostaining for type IV colla-starved for three days in RPMI/0.5% FCS. A 48-hour

gen was performed as previously described [23]. In brief,proliferation assay was then performed. STI 571 0.013kidney sections were rehydrated and treated with 1%to 2.0 mmol/L or drug diluent was then added to cells inH2O2/methanol followed by incubation in Protein Block-RPMI/0.5% FCS followed in 30 minutes by the additioning Agent (Lipshaw-Immunon, Pittsburgh, PA, USA) forof either recombinant PDGF-BB 50 ng/mL (Boehringer-20 minutes at room temperature. Sections were thenMannheim, Mannheim, Germany) or recombinant fi-incubated with type IV collagen antibody for 60 minutesbroblast growth factor-2 (FGF-2; Calbiochem, La Jolla,at room temperature, washed in PBS, and incubated withCA, USA) 50 ng/mL. During the last six hours of culture,biotinylated goat anti-rabbit immunoglobulin (Dako, Car-0.5 mCi/well 3H-thymidine was added. Cells were thenpinteria, CA, USA) followed by incubation with avidin-harvested by removing the medium, washing twice inbiotin peroxidase complex (ABC; Vector, Burlingham,warm phosphate-buffered saline (PBS), and dissolvingCA, USA). Peroxidase conjugates were subsequently lo-in 100 mL 0.2 mol/L sodium hydroxide. This solution was

then neutralized with hydrochloric acid, and scintillation calized using diaminobenzidine tetrahydrochloride (DAB)

Fig. 2 Fig. 4

Gilbert et al: PDGF signal transduction inhibition 1327

b

Fig. 2. Photomicrograph of PAS-stained kidney section from controlanimals (A), rats with experimental mesangial proliferative glomerulo-nephritis receiving either vehicle (B) or STI 571 (C). Magnification3360. Compared with control animals, untreated rats show markedglomerular hypercellularity and mesangial matrix expansion. STI 571–treated rats show near normal glomerular histology. Reproduction ofthis figure in color was made possible by a grant from Novartis, Aus-tralia.

b

Fig. 4. Effects of STI 571 on mesangial cell proliferation as assessedby double immunostaining with BrdU (blue color) and ED1 (browncolor). Photomicrograph showing macrophages (ED 11, brown), prolif-erating macrophages (BrdU1/ED 11 cells, blue and brown) and prolif-erating mesangial cells (BrdU1/ED 12 cells, blue) in control animals Fig. 1. Effects of STI 571 on platelet-derived growth factor (PDGF)-(A) and rats with experimental mesangial proliferative glomerulone- stimulated DNA synthesis. Data are expressed as mean 6 SD. *P ,phritis receiving either vehicle (B) or STI 571 (C) treatment was associ- 0.001 vs. PDGF (50 ng/mL) without STI 571. †P , 0.001 vs. medium.ated with reduced mesangial cell proliferation. Magnification 3360.Reproduction of this figure in color was made possible by a grant fromNovartis, Australia.

as a chromogen. Sections were then counterstained withMayer’s hematoxylin.

a-Smooth muscle actin. Immunostaining for a-smoothmuscle actin was performed in formalin fixed tissue sec-tions using a microwave-based technique to prevent anti-body cross-reactivity, as previously described [24]. Inbrief, sections were microwave treated for 10 minutes in0.01 mol/L sodium citrate buffer, pH 6.0, and then la-beled with a-smooth muscle actin antibody using a three-layer peroxidase-antiperoxidase method and developedwith 3,3-DAB (Sigma) to produce a brown color.

ED 1 and BrdU: Double staining. Double immunohis-tochemical staining was performed in formalin-fixed tis-sue sections using a microwave-based technique to pre-vent antibody cross-reactivity, as previously described[24]. In brief, sections were labeled with ED 1 as de- Fig. 3. Glomerular cellularity as assessed by the number of nuclei

(mean 6 SD) per glomerular cross-section (gcs) in 50 hilar glomeruliscribed above for a-smooth muscle actin. Sections wereper animal. Glomerular hypercellularity was significantly attenuated by

then microwave treated for a second time, labeled at STI 571. *P , 0.05 vs. control; †P , 0.05 vs. untreated.48C with M744 anti-BrdU antibody using the three-layerphosphatase-antiphosphatase method, and developedwith fast blue BB salt (Ajax Chemicals, Melbourne, Aus-

were scored under high power (3400), and the differenttralia) to produce a blue color. Sections had a weakpopulations were expressed as the mean 6 SD per glo-counterstain with PAS and mounted in aqueous medium.merular cross-section. As podocytes do not proliferateNegative controls. Sections incubated with proteinand glomerular endothelial cells account for ,3% ofblocking agent instead of primary antisera served as neg-proliferating cells in this OX-7–induced disease modelative controls. Tissues were also incubated with irrele-[24], proliferating cells were identified as either macro-vant isotype control antibodies as previously describedphages (ED 11BrdU1) or mesangial cells (ED 12[24]. Tissues treated in this manner showed no positiveBrdU1) as previously described [24]. All scoring wasstaining.performed with the observer masked to the study group.

Quantitation of immunohistochemistry The magnitude of immunostaining for a-smooth mus-cle actin or type IV collagen was quantitated using com-Sections double stained with BrdU and ED 1 antibodyputer-assisted image analysis as previously describedwere used to quantitate for ED 11BrdU1, ED 11

BrdU2, and ED 12BrdU1 cells. Fifty hilar glomeruli [25, 26]. In brief, for each tissue section, images from

Gilbert et al: PDGF signal transduction inhibition1328

three nonoverlapping, randomly selected fields were ex-amined by light microscopy (Olympus BX-50; OlympusOptical, Tokyo, Japan) and digitized using a high-resolu-tion camera (Fujix HC-2000; Fujifilm, Tokyo, Japan).All images were obtained using a 320 objective lens.Digitized images were then captured on a Power Macin-tosh G3 computer (Apple Computer Inc., Cupertino,CA, USA) equipped with an in-built graphic board andopened using analytical software (Analytical ImagingSoftware, Ontario, Canada).

The area of brown on an immunoperoxidase-stainedsection was selected for its color range, and the propor-tional area of tissue with this range of color was thenquantitated on 50 hilar glomeruli per animal such thatthe magnitude of immunolabeling was expressed as theproportional area of the tissue section that stained brown.

StatisticsFig. 5. Mesangial cell proliferation as assessed by the number of

All data are shown as mean 6 SE unless otherwise BrdU1/ED 12 cells (mean 6 SEM) per glomerular cross-section (gcs)in 50 hilar glomeruli per animal. Mesangial cell proliferation was signifi-specified. Data were analyzed by analysis of variancecantly attenuated by STI 571. *P , 0.001 vs. control; †P , 0.001 vs.(ANOVA) using the StatView IV program (Brainpower,untreated.

Calabasas, CA, USA) on a Macintosh G3. Comparisonsbetween group means were performed by Fisher’s leastsignificant difference method. A P value of less than 0.05was considered statistically significant. Glomerular cellularity and mesangial cell proliferation

in rats with anti–Thy-1 nephritis

Animals with anti–Thy-1 nephritis displayed moderateRESULTSglomerular hypercellularity when compared with control

PDGF-induced mesangial cell proliferation in vitroanimals, as assessed by the nuclear counting; however,

The ability of PDGF to stimulate proliferation in se- glomerular hypercellularity was significantly reduced inrum-starved mesangial cells was inhibited by pretreat- rats treated with STI 571 (Fig. 3). Mesangial cellment of cells with STI 571 in a dose-dependent fashion (BrdU1ED 12) proliferation was increased 16-fold com-(Fig. 1). Complete inhibition of PDGF-induced mesan- pared with control rats and significantly reduced by treat-gial 3H-thymidine incorporation was seen at 2 mmol/L. ment with STI 571 (Figs. 4 and 5). Similarly, the propor-In contrast, mesangial proliferation induced by FGF-2 tional area of glomeruli immunostained for a-smoothwas unaffected by STI 571 (data not shown). Mesangial muscle actin indicating activated mesangial cells was alsocells remained viable, as evidenced by trypan blue exclu- increased in untreated rats with anti–Thy-1 nephritis andsion and the maintenance of normal mesangial cell ap- significantly reduced by the administration of STI 571pearance and, in particular, nuclear morphology. (Figs. 6 and 7). In contrast, STI 571 had no effect on

either glomerular macrophage numbers (Fig. 8A) orClinical characteristics their proliferative activity (Fig. 8B).

The administration of OX-7 IgG both with and with-Glomerular matrix accumulationout STI 571 was well tolerated by all experimental ani-

mals with no weight loss or abnormalities in hemoglobin, Marked accumulation of immunostainable type IVleukocyte, or platelet counts. Proteinuria was mildly collagen was present in untreated rats with anti–Thy-1increased in animals that received OX-7 IgG compared nephritis and was significantly reduced by the adminis-with control rats and was unaffected by STI 571 treat- tration of STI 571 (Figs. 6 and 7).ment (normal control, 2.1 6 0.2 mg/24 hours; vehicle-treated Thy-1 nephritis, 13.4 6 7.2 mg/24 hours; STI 571-

DISCUSSIONtreated Thy-1 nephritis, 16.4 6 7.0 mg/24 hours). InPAS-stained sections, mesangial hypercellularity and in- In the present study, blockade of PDGF receptor activ-creased mesangial matrix were noted in glomeruli of ity in experimental mesangial proliferative glomerulone-untreated rats. These pathological changes were not seen phritis was associated with a reduction in the pathological

hallmarks of this disease: hypercellularity and extracellu-in rats receiving STI 571 (Fig. 2).

Gilbert et al: PDGF signal transduction inhibition 1329

Fig. 6. Immunostaining for a-smooth muscle actin (left panel) and type IV collagen (right panel) in photomicrograph of kidney section fromuntreated (A), and STI 571-treated rats (B) with experimental mesangial proliferative glomerulonephritis. Magnification 3360.

lar matrix accumulation. Additionally, the antiprolifera- sion may be particularly relevant to more chronic glo-merular diseases in which mesangial matrix correlatestive effects of PDGF receptor blockade on mesangial

cells were confirmed in vitro. These findings attest to the closely with declining glomerular filtration rate [27–29].In the present study, the reduction in extracellular matrixcentral role of PDGF in glomerular disease and provide

a new therapeutic strategy for its treatment. resulting from STI 571 administration may reflect changesin both direct and indirect PDGF related processes. ForThe major findings of the present study were the ame-

lioration of mesangial hypercellularity and matrix accu- instance, while PDGF may directly stimulate extracellu-lar matrix synthesis [30], it is also possible that the ob-mulation by treatment with STI 571. Not only was

PDGF-induced mesangial proliferation abrogated in vitro, served changes may have been a consequence of thefewer activated, synthetic-type, a-smooth muscle actin-but also in vivo, as shown by a reduction in cell nuclei

and BrdU1ED 12 cells. In addition to these effects on positive mesangial cells [31]. In addition, the ability ofPDGF to stimulate expression of the prosclerotic cyto-cell proliferation, mesangial matrix expansion was also

reduced by PDGF-RTK inhibition as evidenced in both kine TGF-b in mesangial cells [12] suggests that suchinteractions may also contribute to the reduction in ex-PAS-stained sections and in tissues immunostained for

the major glomerular extracellular matrix protein, type tracellular matrix observed in the present study.The cellular actions of PDGF follow the binding ofIV collagen. Such effects on extracellular matrix expan-

Gilbert et al: PDGF signal transduction inhibition1330

Fig. 7. Activated mesangial cells (A) and type IV collagen deposition(B) as assessed by the proportional area immunostained with a-smoothmuscle actin and type IV collagen antibodies, respectively. Data areexpressed as (mean 6 SEM) per glomerular cross-section (gcs) in 50hilar glomeruli per animal from untreated and STI 571–treated rats.*P , 0.05.

ligand to the extracellular part of its receptor. This inturn leads to its dimerization and autophosphorylation

Fig. 8. Quantitation of macrophage numbers (ED 11, A) and macro-ultimately leading to protein kinase C activation and con-phage proliferation (B), as assessed by the number of ED 11/BrdU1sequent cell responses [6, 32]. STI 571 is a potent and cells (mean 6 SEM) per glomerular cross-section (gcs) in 50 hilar

selective inhibitor of the PDGF receptor (PDGF-R) and glomeruli per animal. *P , 0.01 vs. control.Abl protein tyrosine kinases [33]. While STI 571 is apotential treatment for Bcr-Abl–positive leukemias, itsinhibitory effects on PDGFR activation suggests a poten-

no effect on FGF-2–induced mesangial 3H-thymidine in-tial role in nonmalignant diseases in which PDGF hascorporation. By selectively targeting PDGF signaling,been strongly implicated, such as glomerulonephritis. InSTI 571 administration in the in vivo setting resulted incontrast to its effects on the PDGF receptor, ligand-cell-specific inhibition of mesangial cell, but not macro-induced autophosphorylation of the receptors for epider-phage proliferation within the glomerulus.mal growth factor, insulin-like growth factor I, and FGF

Numerous studies have reported PDGF overexpres-are insensitive to STI 571, with IC50 .200-fold greatersion in a variety of human [34–38] and experimentalthan for PDGF RTK [33]. Indeed, in the present study,glomerular diseases [39–41]. However, while there is sub-STI 571 specifically inhibited PDGF-induced mesangial

cell proliferation in a dose-dependent manner, but had stantial evidence suggesting that this overexpression of

Gilbert et al: PDGF signal transduction inhibition 1331

PDGF is of major pathogenetic significance in glomeru- a direct role in the pathogenesis of this form of experi-mental glomerulopathy, although it may provide a sourcelar disease [6], in order to establish a role for a specific

cytokine in renal disease, it has been proposed that four of excessive PDGF in this disease.In summary, the strong association of both experimen-criteria need to be satisfied [2]. These criteria, an adapta-

tion of Koch’s postulates, state the following: (1) the tal and human mesangial proliferative glomerulonephri-tis with PDGF overexpression and the amelioration ofcytokine must have the relevant biological effect on tar-

get cells in vitro; (2) it should be expressed in disease the pathological findings in the present study by blockad-ing PDGF receptor activity suggest the potential clinicalstates and correlate with the proposed biological effect;

(3) its administration in vivo should reproduce the dis- utility of this approach as a therapeutic strategy in glo-merular disease.ease; and (4) blocking the cytokine should ameliorate

the disease. While there is substantial experimental datato satisfy the first three criteria for PDGF’s role in glo- ACKNOWLEDGMENTSmerular disease, only two previous studies have ad- This study was supported by the National Health and Medical Re-

search Council of Australia and the Austin Hospital Medical Researchdressed the issue of PDGF blockade. In a study by John-Foundation. The authors thank Dr. Elisabeth Buchdunger for theson et al, neutralizing anti–PDGF-B antibodies reducedgenerous gift of STI-571 and Ms. Lyn Hurst for her technical assistance.

glomerular cell proliferation and matrix accumulation in Reproduction of Figures 2 and 4 in color was made possible by a grantfrom Novartis, Australia.Thy 1.1 glomerulonephritis [42] attesting to the central

role of PDGF in this disease model. More recently, FloegeReprint requests to Richard E. Gilbert, M.D., Ph.D., University of

et al antagonized PDGF-B effects in rat anti–Thy-1.1 Melbourne, Department of Medicine, St. Vincent’s Hospital, VictoriaParade, Fitzroy, Victoria, 3065, Australia.glomerulonephritis by reducing translation of PDGF-BE-mail: gilbert@medstv.unimelb.edu.aumRNA with high-affinity nucleic acid aptamers [43]. The

current findings further support a central role for PDGFREFERENCESin pathological mesangial proliferation. However, these

previously identified means of targeting PDGF have lim- 1. Klahr S, Morrissey JJ: The role of vasoactive compounds, growthfactors and cytokines in the progression of renal disease. Kidneyited applicability to the treatment of renal disease inInt 57(Suppl 75):S7–14, 2000

humans. While neutralizing antibody administration is 2. Johnson RJ, Floege J, Couser WG, et al: Role of platelet-derivedgrowth factor in glomerular disease. J Am Soc Nephrol 4:119–128,effective in rat models of glomerulonephritis [42], the1993use of large quantities of heterologous antibodies is not

3. Border WA, Yamamoto T, Noble NA: Transforming growth fac-feasible in humans, and while the production of specific, tor-b in diabetic nephropathy. Diabetes Metab Rev 12:309–339,

1996high-affinity, monoclonal humanized antibodies is tech-4. Miller SB, Rogers SA, Estes CE, et al: Increased distal nephronnically feasible, the paucity of such antibodies in clinical

EGF content and altered distribution of peptide in compensatorypractice indicates the practical difficulties of this ap- renal hypertrophy. Am J Physiol 262:F1032–F1038, 1992proach. Aptamer therapy is similarly effective in experi- 5. Gilbert RE, Cox A, McNally PG, et al: Increased epidermal

growth factor expression in diabetes related kidney growth. Diabe-mental disease but remains an uncertain possibility fortologia 40:778–785, 1997human use. In contrast, STIs are already in clinical devel- 6. Floege J, Johnson RJ: Multiple roles for platelet-derived growth

opment [44, 45], with recent phase II studies of STI 571 factor in renal disease. Miner Electrolyte Metab 21:271–282, 19957. Shultz PJ, DiCorleto PE, Silver BJ, et al: Mesangial cells expressin CML [46]. As in experimental mesangial proliferative

PDGF mRNAs and proliferate in response to PDGF. Am J Physiolglomerulonephritis, there is substantial evidence sup- 255:F674–F684, 1988porting a key pathogenetic role for PDGF in human 8. Floege J, Johnson RJ, Alpers CE, et al: Visceral glomerular

epithelial cells can proliferate in vivo and synthesize platelet-derivedglomerulonephritis characterized by mesangial prolifera-growth factor B-chain. Am J Pathol 142:637–650, 1993tion such as IgA nephropathy [6], among the most com- 9. Nikolic-Paterson DJ, Lan HY, Atkins RC: Macrophages in im-

mon causes of end-stage renal failure in the world [47]. mune renal injury, in Immunologic Renal Disease, edited by Neil-son EG, Couser WG, New York, Raven Press, 1997, pp 575–592Thus, the ability to inhibit PDGF signal transduction

10. Schultz PJ, DiCorleto PE, Siver BJ, et al: Mesangial cells expressmay offer a new therapeutic approach to renal disease PDGF mRNA and proliferate in response to PDGF. Am J Physiolin humans. 255:F674–F684, 1988

11. Doi T, Vlassara H, Kirstein M, et al: Receptor-specific increaseIn the present study, PDGF-RTK inhibition had noin extracellular matrix production in mouse mesangial cells byeffect on glomerular macrophage infiltration and prolif-advanced glycation end products is mediated via platelet-derived

eration. These findings are consistent with studies of growth factor. Proc Natl Acad Sci USA 89:2873–2877, 199212. Throckmorton DC, Brogden AP, Min B, et al: PDGF and TGF-PDGF blockade with neutralizing anti-PDGF antibody

beta mediate collagen production by mesangial cells exposed to[42] and aptamer-based PDGF antagonism [43], sug-advanced glycosylation end products. Kidney Int 48:111–117, 1995

gesting that monocyte/macrophage chemotaxis in this 13. Johnson RJ, Alpers CE, Yoshimura A, et al: Renal injury fromangiotensin II-mediated hypertension. Hypertension 19:464–474,disease model is largely PDGF independent. Further-1992more, the amelioration of mesangial cell proliferation

14. Jaffer FE, Knauss TC, Poptic E, et al: Endothelin stimulatesand matrix accumulation without altering macrophage PDGF secretion in human mesangial cells. Kidney Int 38:1193–

1198, 1990infiltration suggests that the macrophage may not have

Gilbert et al: PDGF signal transduction inhibition1332

15. Silver BJ, Jaffer FE, Abboud HE: Platelet-derived growth factor nephritis: Alpha-smooth muscle actin is a marker of mesangial cellproliferation. J Clin Invest 87:847–858, 1991synthesis in mesangial cells: Induction by multiple peptide mito-

gens. Proc Natl Acad Sci USA 86:1056–1060, 1989 32. Daniel TO, Kumjian DA: Platelet-derived growth factor in renaldevelopment and disease. Semin Nephrol 13:87–95, 199316. Druker BJ, Tamura S, Buchdunger E, et al: Effects of selective

33. Buchdunger E, Zimmerman J, Mett H, et al: Selective inhibitioninhibitor of the Abl tyrosine kinase on the growth of Bcr-ablof the platelet-derived growth factor signal transduction pathwaypositive cells. Nat Med 2:561–566, 1996by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyri-17. Mason DW, Williams AF: The kinetics of antibody binding tomidine class. Proc Natl Acad Sci USA 92:2558–2562, 1995membrane antigens in solution and at the cell surface. Biochem J

34. Frampton G, Hildreth G, Hartley B, et al: Could platelet-derived187:1–20, 1980growth factor have a role in the pathogenesis of lupus nephritis?18. Dijkstra CD, Dopp EA, Joling P, et al: The heterogeneity ofLancet 2:343, 1988mononuclear phagocytes in lymphoid organs: Distinct macrophage

35. Gesualdo L, Pinzani M, Floriano JJ, et al: Platelet-derivedsubpopulations in the rat recognized by monoclonal antibodiesgrowth factor expression in mesangial proliferative glomerulone-ED1, ED2 and ED3. Immunology 54:589–599, 1985phritis. Lab Invest 65:160–167, 199119. Kakizaki Y, Kraft N, Atkins RC: Differential control of mesan-

36. Nakajima M, Hewitson TD, Mathews DC, et al: Platelet-derivedgial cell proliferation by interferon-gamma. Clin Exp Immunolgrowth factor mesangial deposits in mesangial IgA glomerulone-85:157–163, 1991phritis. Nephrol Dial Transplant 6:11–16, 199120. Nikolic-Paterson DJ, Jun Z, Tesch GH, et al: De novo CD44

37. Fellstrom B, Klareskog L, Heldin CH, et al: Platelet-derivedexpression by proliferating mesangial cells in rat anti-Thy-1 nephri-growth factor receptors in the kidney: Upregulated expression intis. J Am Soc Nephrol 7:1006–1014, 1996inflammation. Kidney Int 36:1099–1102, 198921. Iwata J, Nishikaze O: New micro-turbidimetric method for deter-

38. Waldherr R, Noronha IL, Niemir Z, et al: Expression of cytokinesmination of protein in cerebrospinal fluid and urine. Clin Chemand growth factors in human glomerulonephritides. Pediatr25:1317–1319, 1979Nephrol 7:471–478, 199322. Larsen K: Creatinine assay by a reaction-kinetic principle. Clin

39. Yoshimura A, Gordon K, Alpers CE, et al: Demonstration ofChim Acta 41:209–217, 1972PDGF B-chain mRNA in glomeruli in mesangial proliferative ne-23. Rumble JR, Cooper ME, Soulis T, et al: Vascular hypertrophyphritis by in situ hybridization. Kidney Int 40:470–476, 1991in experimental diabetes: Role of advanced glycation end products. 40. Floege J, Burns MW, Alpers CE, et al: Glomerular cell prolifera-J Clin Invest 99:1016–1027, 1997 tion and PDGF expression precede glomerulosclerosis in the rat24. Tesch GH, Lan HY, Atkins RC, et al: Role of interleukin-1 in remnant kidney model. Kidney Int 41:297–309, 1992

mesangial cell proliferation and matrix deposition in experimental 41. Fukui M, Nakamura T, Ebihara I, et al: Low-protein diet attenu-mesangioproliferative nephritis. Am J Pathol 151:141–150, 1997 ates increased gene expression of platelet-derived growth factor

25. Lehr HA, Mankoff DA, Corwin D, et al: Application of pho- and transforming growth factor-beta in experimental glomerulartoshop-based image analysis to quantification of hormone receptor sclerosis. J Lab Clin Med 121:224–234, 1993expression in breast cancer. J Histochem Cytochem 45:1559–1565, 42. Johnson RJ, Raines EW, Floege J, et al: Inhibition of mesangial1997 cell proliferation and matrix expansion in glomerulonephritis in

26. Lehr HA, van der Loos CM, Teeling P, Gown AM: Complete the rat by antibody to platelet-derived growth factor. J Exp Medchromogen separation and analysis in double immunohistochemi- 175:1413–1416, 1992cal stains using photoshop-based image analysis. J Histochem Cyto- 43. Floege J, Ostendorf T, Janssen U, et al: Novel approach tochem 47:119–126, 1999 specific growth factor inhibition in vivo: Antagonism of platelet-

27. Klahr S, Schreiner G, Ichikawa I: The progression of renal derived growth factor in glomerulonephritis by aptamers. Am Jdisease. N Engl J Med 318:1657–1666, 1988 Pathol 154:169–179, 1999

28. Adler S, Striker LJ, Striker GE, et al: Studies of progressive 44. Gibbs JB: Anticancer drug targets: Growth factors and growthglomerular sclerosis in the rat. Am J Pathol 123:553–562, 1986 factor signaling. J Clin Invest 105:9–13, 2000

29. Mauer S, Steffes M, Ellis E, et al: Structural-functional relation- 45. Druker BJ, Lydon NB: Lessons learned from the developmentships in diabetic nephropathy. J Clin Invest 74:1143–1155, 1984 of an Abl tyrosine kinase inhibitor for chronic myelogenous leuke-

30. Floege J, Eng E, Young BA, et al: Infusion of PDGF or basic mia. J Clin Invest 105:3–7, 2000FGF induces selective glomerular mesangial cell proliferation and 46. Goldman JM: Tyrosine-kinase inhibition in treatment of chronicmatrix accumulation in rats. J Clin Invest 92:2952–2962, 1993 myeloid leukaemia. Lancet 355:1031–1032, 2000

31. Johnson RJ, Iida H, Alpers CE, et al: Expression of smooth 47. D’Amico G: The commonest glomerulonephritis in the world: IgAnephropathy. Q J Med 64:709–727, 1987muscle cell phenotype by rat mesangial cells in immune complex