Kidney International, Vol. 41(1992), pp. 590—594

Platelet-derived growth factor: A potentially importantcytokine in glomerular disease

RICHARD JOHNSON, HIROYUKI IIDA, AsHlo YosHIMu1, JURGEN FLOEGE,and DANIEL F. BOWEN-POPE

Division of Nephrology, Department of Medicine and Department of Pathology, University of Washington Medical Center, Seattle,Washington, USA

Many glomerular diseases, including IgA nephropathy, mem-branoproliferative glomerulonephritis (GN), variants of idio-pathic focal sclerosis, and lupus nephritis are characterized bymesangial cell proliferation. There is increasing evidence thatmesangial cell proliferation in GN may not be a benign process,but may be associated with activation of the mesangial cell, asreflected by the acquisition of a smooth muscle cell-like pheno-type [1] and by the synthesis and secretion of increasedamounts of normal and abnormal mesangial matrix components[21 and proteinases (David Lovett, manuscript submitted).Thus, an understanding of the mechanisms that initiate andmaintain the proliferative response in active GN may provideuseful insights into future therapeutic strategies of these dis-eases.

Mediators of mesangial cell proliferation in GN

Mesangial cells in culture will proliferate in response tonumerous growth factors (Table 1) [3—101. However, littleinformation is available as to the type, relative importance, andcellular source of the growth factors involved in mesangial cellproliferation in vivo in glomerular diseases.

The most likely cellular candidates for releasing growthfactors within the glomerulus would include infiltrating plateletsand monocyte-macrophages, and endogenous glomerular cells(especially the glomerular endothelial and mesangial cell) (Ta-ble 2). At the present time, very little information is available asto the growth factors produced by glomerular epithelial cells.

PlateletsThe mitogenic activity of serum is largely derived from

factors released by platelets during clotting. Platelets containnumerous growth factors, including platelet-derived growthfactor (PDGF), an EGF/TGF-cx like factor, TGF-/3, IGF-l, ahepatocyte growth factor (HGF), and a platelet-derived endo-thelial cell growth factor (PD-ECGF) [10—14]. Activated plate-lets also express interleukin-l (IL-l) on their surface [15]. Thus,it is not surprising that platelet releasates and lysates canstimulate proliferation of rat and rabbit mesangial cells inculture [16, 171.

There is also increasing evidence to support a role for

© 1992 by the International Society of Nephrology

platelets in mediating glomerular cell proliferation in vivo.Many experimentally-induced glomerular diseases that result inglomerular cell proliferation are characterized by an initialinjury to the glomerular endothelium or mesangium [18—23]. Inmany of these diseases, an influx of platelets into glomeruli canbe demonstrated at the time of the cellular injury [18—20, 23,241. For example, in the rat model of glomerular injury inducedwith oxidants generated by neutrophil myeloperoxidase and itssubstrates, hydrogen peroxide and halides, a pronounced en-dothelial cell injury with massive intraglomerular platelet accu-mulation can be demonstrated within minutes, and is followedby an impressive proliferation of glomerular cells [24].

An influx of platelets into glomeruli can also be demonstratedin a rat model of subendothelial immune complex nephritis(induced by the renal artery perfusion of concanavalin A andanti-concavalin A antibody) that histologically resembles dif-fuse proliferative lupus nephritis [25]. By electron microscopy,the platelets can be shown to be activated, and many aredegranulated and adherent to areas of the glomerular basementmembrane where the endothelium has been denuded (Fig. 1). Amarked proliferation of glomerular endothelial and mesangialcells occurs in the initial days following the platelet influx (Fig.2), eventually resulting in a markedly hypercellular lesion.Consistent with these observations, Clark et al have shown thatan increased renal uptake of platelets occurs in diffuse prolifer-ative lupus nephritis in humans [26].

The first evidence that the association of intraglomerularplatelet accumulation with subsequent glomerular cell prolifer-ation represented a cause and effect relationship was providedby Cattell in the experimental model of glomerular injuryinduced with Habu snake venom [27]. In this model, theintravenous injection of Habu snake venom results in injury toonly a minority of glomeruli, and is characterized by an acutedissolution of the mesangium with loss of mesangial cells("mesangiolysis") followed by mesangial cell proliferation [20].Platelets and platelet aggregates are frequent in the areas ofmesangiolysis [20]. When rats were rendered thrombocytopenicwith an anti-platelet serum prior to injection of venom, the ratsstill developed the acute mesangiolysis, but the subsequentmesangial cell hypercellularity was largely prevented [27].Treatment of rats with the anti-platelet agents, dipyridamole[281 or sulindac [29], have also been reported to reduce themesangial cell hypercellularity in this model, whereas aspirin

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Johnson ci at: PDGF and glomerulonephritis 591

Table 1. Peptide growth factors capable of inducing mesangial cellproliferation in vitro

Platelet derived growth factor (PDGF)Interleukins I and 6 (IL-I, IL-6)Tumor necrosis factor alpha (TNF-a)Epidermal growth factor (EGF)EndothelinInsulin-like growth factor I (IGF-l)Basic fibroblast growth factor (bFGF)Transforming growth factor alpha (TGF-a)InsulinArginine-vasopressinTransforming growth factor beta (TGF,3)a

At low concentrations (0.01 to 0.1 ng/ml) TGF-J3 potentiates theproliferative response of human mesangial cells to EGF or TGF-a [8].TGF-/3 also induces proliferation in mouse mesangial cells at confluence[91.

Table 2. Growth factors present in various cell populations that mayhave a role in glomerulonephritis

Infiltrating cells Glomerular cells

Mesangial EndothelialaPlatelets Macrophages

PDGF PDGF PDGF PDGFEGF/TGF-a IL-l IL-I bFGFPD-ECGF TNF lL-6 IL-6TGF-/3 IL-6 TNF IL-IHGF TGF-J3 TGF-13 EndothelinIL-i IGF-lIGF-l Endothelin

CSF-Ia With the exception of PDGF, the growth factors listed are based on

studies of enbdothelial cells from other sites.

was not protective [28]. Unfortunately, studies utilizing aspirinare complicated by the fact that this agent does not blockplatelet adherence to collagen or its subsequent release reaction[30, 31].

We have investigated the role of platelets in another model ofmesangial proliferative nephritis which is induced with anantibody to the Thy 1.1 antigen that is present on the ratmesangial cell membrane [32]. This model is also associatedwith an acute mesangiolysis and platelet influx followed bymesangial cell proliferation [23]. As in the Habu snake venommodel, depletion of platelets with an anti-platelet antibodysignificantly reduces glomerular cell proliferation without anydemonstrable effect on mesangiolysis [32]. In contrast, comple-ment depletion (with cobra venom factor) prevents both themesangiolysis (and platelet influx) and the secondary prolifera-tive response [23, 33].

MacrophagesMonocyte-macrophages can be demonstrated in glomeruli in

many types of GN characterized by glomerular cell prolifera-tion, including diffuse proliferative nephritis, IgA nephropathy,lupus nephritis, and membranoproliferative GN [reviewed in34]. In several experimental models of GN, monocyte-macro-phage depletion with an anti-macrophage serum [35,361 or withirradiation [371 resulted in a reduction in glomerular hypercel-lularity, but it is unclear in these studies whether the decreasein glomerular cellularity can be attributed to the reduction ininfiltrating monocytes, or if it is associated with a significant

Fig. 1. Involvement of platelets in subendothelial immune complexnephritis. Ten minutes after induction of nephritis by renal arteryperfusion of concanavalin A (con A) and anti-con A antibody, numer-ous platelets can be demonstrated in glomerular capillaries, often inclose association with the endothelium and basement membrane. Evi-dence for platelet activation includes the centralization of granuleswithin platelets and the presence of filopodia. Key: C, capillary lumen;p, platelet (x7IOO).

Fig. 2. A marked cell proliferation can be documented in glomeruli ofrats with subendothelial immune complex nephritis induced by theperfusion of con A and anti-con A antibody. Shown is a glomerulusfrom a rat two days after induction of GN, in which proliferating cellscan be identified by immunostaining for the proliferating cell nuclearantigen (PCNA). Positive cells have a dark nuclear stain. (Immunoper-oxidase with methyl green counterstain, x400).

decrease in endogenous glomerular cell number. However,Matsumoto and Hatano have recently reported that macro-phage depletion reduces the glomerular hypercellularity in a ratmodel of anti-GBM nephritis, and that this treatment signifi-cantly reduces the number of mesangial cells that can beobtained from isolated glomeruli of these animals in culture[38].

The potential mechanisms by which the macrophage medi-ates glomerular cell proliferation could involve release by themacrophage of growth factors [reviewed in 39] (Table 2), orcould involve stimulation by the macrophage of other cell types

592 Johnson et a!: PDGF and glomerulonephritis

to release growth factors within the glomerulus. It is clear thatmany growth factors present in macrophages, such as IL-I,IL-6, PDGF, and TNF-a, mediate mesangial cell proliferationin vitro (Table 1).

Endogenous glomerular cellsAlthough studies with glomerular endothelial cells have been

sparse due to the difficulty in establishing these cells in culture,both endothelial cells from other sites and glomerular mesangialcells secrete numerous growth factors [reviewed in 40, 41](Table 2). Many of the growth factors released by mesangialcells, including PDGF, IL-I, IL-6, IGF-l, and TGF-f3, bind toreceptors present on the mesangial cell membrane, thus func-tioning as autocrine growth factors [3, 6, 42, 43]. Consistentwith the hypothesis that mesangial cells may produce autocrinegrowth factors in GN has been the observation that severalproliferative glomerular diseases in animals and humans areassociated with increased growth factor gene or protein expres-sion in renal cortex or glomeruli [6, 44—47]. However, in manyof these studies, the possibility that the macrophage wasresponsible for the increased expression of these factors wasnot definitively excluded.

Platelet-derived growth factorPDGF is a 30 kd molecular weight cationic protein consisting

of two disulfide-bonded chains existing as a heterodimer(PDGF-AB) or as a homodimer (PDGF-AA or PDGF-BB)[reviewed in 10]. The B-chain is almost identical to the trans-forming protein of the Simian Sarcoma virus (p28sis), and iscoded by the c-sis proto-oncogene [10]. In platelets, PDGF isstored in the alpha granules, from where it is released into theextracellular environment following activation of the platelet bynumerous substances, including platelet activating factor,thrombin, collagen, and immune complexes [reviewed in 48].PDGF is also produced by many other cell types, includingrenal microvascular endothelial cells, mesangial cells, macro-phages, and smooth muscle cells [3, 10, 49].

PDGF may have an important role in mesangial cell prolifer-ation. PDGF is a potent mitogen for mesenchymal cells inculture [10], including mesangial cells [3]. Although somestudies suggest that PDGF may be a "complete" growth factorfor mesangial cells [3], others have reported that PDGF is a"competence" factor, inducing cells to enter the cell cycle, asopposed to "progression" factors which allow the cell tocomplete the cell cycle once it has been initiated. Thus, PDGFhas been reported to make mesangial cells "competent" so thatthey can further respond to IL-I [4] and IGF-l [50].

Recent studies also suggest that PDGF may have an addi-tional role as an intermediary in mediating the effects of othergrowth factors. A variety of growth factors, including EGF,TNF-a, and bFGF will induce mesangial cells in culture toproduce PDGF and PDGF mRNA [51]. PDGF itself will alsoinduce mesangial cells to produce PDGF mRNA [51]. Mostimportantly, the mitogenic effects of EGF on mesangial cells[51] and of IL-I on fibroblasts [52] can be inhibited withanti-PDGF antibody. This suggests that PDGF may have acritical role as an autocrine growth factor for a variety of celltypes, including mesangial cells.

In contrast to mesangial cells, PDGF appears to have minimalor no mitogenic effect for glomerular endothelial cells or epi-

thelial cells in culture [9], However, PDGF may have additionaleffects besides being a mesangial cell mitogen. PDGF is a potentchemotactic factor for smooth muscle cells [53] and mesangialcells [54]. Recent studies suggest that the chemotactic proper-ties of PDGF may play an important role in the smooth musclecell migration into the neointima that occurs in the rat carotidfollowing balloon angioplasty (Note added in proof, A).

Binding of PDGF to the cell membrane PDGF receptor(PDGF-R) is dependent on the PDGF isoform. The PDGFreceptor consists of two subunits (a, j3) in which the a-subunitcan bind the A- or B-chain but the $-subunit only binds theB-chain [55]. Thus, PDGF-R aa binds all forms of PDGF,receptor aJ3 binds either PDGF-AB or PDGF-BB, and receptorf3f3 binds only PDGF-BB [55]. Shortly after binding, the recep-tor-ligand complex is endocytosed with loss of receptor expres-sion on the cell surface [10]. Activation of tyrosine kinase leadsto autophosphorylation of the receptor as well as phosphoryla-tion of cytoplasmic proteins [10]. Activation of phospholipase Cleads to generation of inositol triphosphate (1P3) which mobi-lizes intracellular Ca2 and diacyiglycerol which activates pro-tein kinase C, and this has been demonstrated in mesangial cells[56, 57]. Within minutes to hours activation of the proto-oncogenes c-myc and c-fos occurs [10], and additional poorly-defined intracellular signaling eventually culminates in newDNA synthesis.

Role of PDGF in GN

The observation that PDGF may be an important growthfactor for mesangial cells suggests that it may be involved inglomerular diseases associated with mesangial cell prolifera-tion, including lupus nephritis, IgA nephropathy, and membra-noproliferative GN. Indeed, PDGF has been identified byimmunostaining in the glomerular capillary walls of some pa-tients with diffuse proliferative lupus GN [58]. Recently, Gesu-aldo et al have also reported that PDGF A- and B-chaintranscripts are increased in whole kidney RNA obtained frommice with mesangial proliferative GN [461.

We have investigated whether PDGF is expressed in theanti-Thy 1 model of mesangial proliferative nephritis in the rat[58, 59]. A marked increase in PDGF A- and B-chain mRNAcould be documented in isolated glomerular RNA obtainedthree and five days after inducing nephritis. The increasedexpression of the PDGF mRNA occurred at the time of maxi-mal mesangial cell proliferation, as documented by a 20-foldincrease in glomerular cells expressing the proliferating cellnuclear antigen (PCNA) [58]. (PCNA is a nuclear protein that isexpressed from late Gl to the G2/M phase of the cell cycle [60]).Cells expressing PDGF B-chain mRNA could also be docu-mented in glomeruli by in situ hybridization using a digoxigenin-labeled cRNA probe for the murine PDGF B-chain [59]. Inaddition, cells expressing PDGF B-chain protein were identifiedby immunostaining with the specific monoclonal antibody,PGF-007 [58, 59]. Both complement depletion and plateletdepletion significantly reduced the cell proliferation and PDGFexpression.

The identity of the cells expressing PDGF was studied. Mostof the PDGF B-chain positive cells were in mesangial locations,and by double immunolabeling >85% of the PDGF B-chainpositive cells were also positive for a-smooth muscle actin [58].We have recently demonstrated that a-smooth muscle actin is

Johnson ci a!: PDGF and glomerulonephritis 593

expressed by mesangial cells in the Thy I model [1]. In addition,macrophages and glomerular endothelial cells are negative forthis actin isoform by immunocytochemistry [lii. Therefore, thisstudy suggests that mesangial cells are the major source for theincreased PDGF expression in this model of nephritis, and thusare consistent with Abboud's hypothesis [511 that PDGF mayfunction as a mesangial cell autocrine growth factor in glomer-ular disease.

Further support for PDGF as an autocrine growth factor hasresulted from studies performed in our laboratory in the rem-nant kidney (that is, 5/6 nephrectomy) model in the rat. In thismodel, mesangial cell proliferation can be documented prior tothe development of glomerulosclerosis, and is associated withincreased glomerular mRNA and protein expression of thePDGF B-chain (Note added in proof, B).

Mesangial proliferative nephritis may also be associated withan up regulation of the PDGF-R. Recent studies have demon-strated an increase in PDGF-Rf3 protein in mesangial areas ofpatients with mesangial proliferative nephritis by immunostain-ing [61]. We have examined the involvement of the PDGF-R inthe anti-Thy 1 model [58]. An 8- to 25-fold increase in PDGF-R/3mRNA could be demonstrated in isolated glomerular RNA fromdiseased rats by Northern analysis at three and five daysfollowing induction of disease, The maximal expression of thePDGF-R 13-subunit mRNA occurred at day 5 as opposed to thatfor PDGF B-chain mRNA which occurred at day 3. By immu-nostaining, the cells expressing the PDGF-R/3 appeared to bemesangial cells. The up regulation of the PDGF-Rf3 could beblocked if proliferation was inhibited by depleting rats ofcomplement or platelets. In contrast to the PDGF-R 13-subunit,no PDGF-R a-subunit protein or mRNA could be detected innormal or diseased glomeruli in this study. This suggests thatthe mesangial cells may be interacting primarily with thePDGF-BB isoform.

In conclusion, the mechanism of mesangial cell proliferationin GN may likely involve the release of growth factors by bothexogenous (platelets and macrophages) and endogenous(mesangial and endothelial) cells. Recent evidence supports thehypothesis that PDGF may be one of the important growthfactors. In addition to being a growth factor that can be releasedby activated platelets and macrophages, PDGF may also serveas an autocrine growth factor for the mesangial cell. Studies arecurrently underway to determine if inhibition of PDGF in vivocan reduce the glomerular cell proliferation in mesangial prolif-erative GN.

Note added in proof

A. FERNS GA, RAINES EW, SPRUGEL KH, MOTANI AS, REIDY MA,Ross R: inhibition of neointimal smooth muscle accumulation afterangioplasty by an antibody to PDGF. Science 253:1129—1131, 1991

B. FLOEGE J, BURNS MW, ALPERS CF, Y05HIMURA A, PRITZEL P.GORDON K, SEIFERT R, BOWEN-POPE DF, COUSER WG, JOHNSON Ri:Glomerular cell proliferation and PDGF expression precede glomeru-losclerosis in the remnant kidney model. Kidney mt (in press)

Acknowledgments

These studies were supported by U.S. Public Health Service grantsDK-39068 and DK-43422 and by a research grant from the NorthwestKidney Foundation. The authors thank Drs. William Couser andCharles Alpers for their helpful comments.

Reprint requests to Richard Johnson, M.D., Division of Nephrology,Department of Medicine, University of Washington Medical Center,Seattle, Washington 98195, USA.

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