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American Journal of Padtology, Vol. 140, No. 5, May 1992Copyright C American Association ofPatbologts

A Histologic Study of the ExtracellularMatrix During the Development ofGlomerulosclerosis in Murine ChronicGraft-versus-Host Disease

E. C. Bergijk, C. Munaut, J. J. Baelde, F. Prins,J. M. Foidart, P. J. Hoedemaeker,and J. A. BruijnFrom the Departmnmt ofPathology, University ofLeiden,Leiden, The Netherlands, and the Department ofBiology,University of Liege, Liege, Belgium

The development of glomerulosclerosis was studiedin murine chronic graft-versus-host disease (GvHD),which is a model for human systemic lupus erythe-matosus. The authors investigated the distributionpatterns of six components of the extracellular ma-trix (ECM), ie., laminin, fibronectin, collagen typesI,III, IV, and VI during the course of the disease. All ofthese ECM components except collagen type I werefound in the glomeruli of normal mice, where all ofthem were intrinsic constituents of the mesangium.Laminin, fibronectin, and collagen type IV were alsofound in the glomerular capillary walls. Starting 6weeks after the induction ofGvHD and continuing atweek 8, the onset of an expansion of the mesangialmatrix was observed At the same time, the amountsof laminin, fibronectin, and collagen typesIV and VIincreased Ten weeks after the onset of the disease,glomerulosclerosis developed. Traces of the intersti-tial collagen typef werefound in sclerotic glomeruliThe levels offourECM components, i.e., collagens III,IV, VI, and laminin were markedly decreased in thesclerotic glomeruli as compared with week 8. In con-trast, the amount offibronectin in the sclerotic glo-meruli increased dramatically. Immunoelectron mi-croscopic examination showed fibronectin in thesclerotic lesions, in contrast to laminin, collagentype I, and collagen type IV It is concluded that thesclerotic lesions in murine chronic GvHD containfibronectin The small amounts of the ECM compo-nents laminin, as well as collagens III, IV, and VI inthe sclerotic glomeruli in GvHD, might representremnants of mesangial material and collapsed cap-illary walls These components areprobably replacedby increasedproduction and/or accumulation ofcol-

lagen type I and fibronectin.140:1147-1156)

(Am J Pathol 1992,

Progressive glomerular sclerosis is a severe complica-tion of many forms of renal disease. In the course of thisprocess, structures in the glomerulus are replaced byfibroblasts and matrix.1 This in turn leads to impairment ofthe filtration function of the kidney. Not much is knownabout the underlying mechanisms. Hemodynamic andgenetic factors may play a role.2'3 Interleukin 1 and trans-forming growth factor-p are known to stimulate extracel-lular matrix (ECM) production by glomerular cells andhave been postulated to play a central role in the patho-genesis of glomerulosclerosis.4 A better understandingof the development of glomerulosclerosis will improve di-agnosis and treatment in patients. Research in this fieldhas focused on experimental models.

Murine graft-versus-host disease (GvHD) can beused as a model for systemic lupus erythematosus inhumans.7 GvHD is induced by transfer of DBA/2 lympho-cytes into (C57BU1 O*DBA/2)F1 hybrids. This leads to analloimmune reaction of parental T lymphocytes activatedby H-2-incompatible structures of the recipient mice.8'9According to Via and Shearer,10 the disease is the resultof a deficient cytotoxic T-cell response in the DBAI2-derived inoculate. This leads to uncontrolled proliferationof autoantibody-producing B cells in the host.11 The re-sult is an SLE-like syndrome in which severe immune-complex glomerulonephritis (ICGN) is one of the majorsymptoms. In this model, ICGN is characterized by de-position of autoantibodies in the mesangium and alongthe GBM, resulting in thickening of the GBM, the forma-tion of spikes and expansion of the mesangial matrix.From week 10 on, the development of glomerulosclerosiscan be observed by light microscopic examination and

Supported by a grant from the Dutch Kidney Foundation (C89-847).Accepted for publication December 26,1991.Address reprint requests to Dr. E. C. Bergijk, Department of Pathol-

ogy, University of Leiden, P.O. Box 9603, 2300 RC Leiden, The Nether-lands.

1147

1148 Bergijk et alAJP May 1992, Vol. 140, No. 5

leads to severe renal failure and finally to the death of theanimal.

Molecular dissection of the ECM in humans and lab-oratory animals, suggested that the sclerotic process ischaracterized by an abnormal production of ECM com-ponents.'2-14 To obtain more insight into the pathogen-esis of glomerulosclerosis in our model of experimentallupus nephritis, we successively investigated the depo-sition patterns of the ECM components laminin, fibronec-tin, and collagen types 1, 111, IV, and VI. For this purpose,we used the techniques of immunofluorescence, immu-noelectron microscopic examination, and the recentlydeveloped technique of reflection-contrast microscopywith peroxidase staining. This new technique providesenhanced detection sensitivity in immunostaining15 andhas not been previously applied for the study of glomer-ulosclerosis.

Methods

Animals

C57BU10 (H-2b/b) and DBA/2 (H-2d/d) mice were pur-chased from Olac 1976 Ltd. (Bicester, Oxfordshire OX6OTP, UK). (C57BU1 0*DBA/2)F1 hybrid mice were bredin our animal facilities. Female DBA/2 mice aged 7 to 8weeks served as donors. Female Fl hybrids aged 8 to 10weeks served as recipients. For the present study, weused 25 experimental Fl mice and 5 age- and sex-matched control mice.

Induction of GvHD

Spleens, lymph-nodes (mesenteric, cervical, and in-guinal), and thymi from DBA/2 donors were removed un-der aseptic conditions. Single-cell suspensions were pre-pared by mincing the tissue in sterile RPMI 1640 andgently pressing the fragments through a steel sieve (150,um pore diameter). The suspensions were filteredthrough a nylon sieve (71 ,um pore diameter), after whichthe cells were passed through a sterile Pasteur pipetteloosely packed with cotten wool. The suspensions werewashed twice in RPMI 1640 medium and centrifuged for10 minutes at 250g. The pellets were resuspended inRPMI 1640. The total number of cells and the proportionof vital cells were determined by Trypan blue exclusionand phase-contrast microscopic examination. The sus-pensions were washed again, and the pellets were re-suspended in Hanks' balanced salt solution (HBSS). Thesuspensions were mixed. On days 0, 3, 7, and 10, the Flrecipients were injected intravenously with 50*106 viableDBA/2 cells in 0.25 ml HBSS. The dose was composed of

approximately 60% spleen cells, 30% thymocytes, and10% lymph-node cells. Control mice were injected simul-taneously with 0.25 ml phosphate-buffered saline (PBS).

Follow-up of Fl Mice

The albumin content of the urine of the GvHD Fl andcontrol mice was determined 1 week before and 2 weeksafter the first injection of parental cells and then at two-weekly intervals. The animals had been kept in urine-collection cages for 18 hours with free access to waterand food. The albumin level was assessed by rocketelectrophoresis against rabbit anti-mouse albumin, whichhad been produced by immunization of a rabbit with pu-rified mouse serum albumin (Sigma Chemical Corpora-tion, St. Louis, MO). This mouse albumin was also usedas standard.16 After urine collection, the mice were anes-thetized with diethylether and blood was collected fromthe orbital plexus. Serum samples were prepared andtested to detect the presence of autoantibodies.9 Thedirect Coomb's test was used to assess the in vivo fixa-tion of immunoglobulins by erythrocytes.9 Groups of fivemice each were killed every other week starting at week4 and ending at week 12. After perfusion with Dulbecco'sphosphate-buffered saline (PBS), the kidneys were re-moved. Small pieces of kidney tissue were embedded inLowicryl for electron and reflection microscopic examina-tion. Some of the tissue was fixed in 10% buffered forma-lin, dehydrated, and then embedded in Paraplasts (Am-stelstad, Amsterdam, The Netherlands) for light micro-scopic examination. The remaining kidney tissue wasfrozen in CO2 ice-cooled isopentane and stored at- 70°C until use for the immunofluorescence studies. Forelectron and reflection microscopic examination, the kid-neys were perfusion-fixed with paraformaldehyde and0.25% glutaraldehyde in 0.1 mol/I PBS, and embeddedin Lowicryl.

Antibodies

We used rabbit-anti-mouse IgG obtained from Janssen(Olen, Belgium), affinity-purified goat-anti-type 1, anti-type111, and anti-type IV collagen antisera from Sera-lab (Sus-sex, England), rabbit-anti-EHS laminin from E-Y laborato-ries (San Mateo, CA), polyvalent rabbit serum againsttype VI collagen from Heyl (Berlin, Germany), and poly-clonal antiserum to human fibronectin from Sigma (StLouis, MO). As second antibodies in the immunofluores-cence studies, we used FITC-labeled goat-anti-rabbitIgG and rabbit-anti-goat IgG from Nordic (Tilburg, TheNetherlands). For reflection contrast microscopic exami-nation, we used peroxidase-labeled swine anti-rabbit

ECM and Glomerulosclerosis in Murine Chronic GvHD 1149AJP May 1992, Vol. 140, No. 5

and peroxidase-labeled swine anti-goat antibodies(DAKO, Denmark) and gold-labeled goat-anti-rabbit-G15 as second-step antibodies (Amersham). In the im-munoelectron microscopic studies, rabbit-anti-goat IgGwas used as second antibody. Gold-labeled goat-anti-rabbit-G15 was used as third antibody, and as secondantibody in laminin staining. Specificities were deter-mined using indirect and direct ELISA, indirect immuno-fluorescent staining, indirect immunocytochemical anal-ysis, and dot blotting techniques.

Histologic Techniques

The kidney tissue was processed for light microscopicand immunofluorescence studies as described else-where.17 The light-microscopic slides were stained withperiodic acid-Schiff (PAS) or hematoxylin-eosin (H&E).The immunofluorescence slides were scored double-blind by two observers independently of each other. Im-munoelectron microscopic examination was completedaccording to Abrahamson et al.18 On the grids the sec-tions were poststained with uranyl acetate and Reynold'slead citrate, and examined in a Philips CM10 electronmicroscope, operating at 60 kV. Reflection-contrast mi-croscopic examination and postembedding histochemi-cal analysis were done according to Cornelese-ten Veldeet al15 with a few modifications. Briefly, the kidneys wereperfusion-fixed with 1% paraformaldehyde and 0.25%glutaraldehyde in 0.1 mol/l PBS (pH 7.4). Dehydrationand embedding were performed according to Altman etal.19 Ultrathin sections (±80 nm) were caught on ami-nocylane-coated slides. The slides were then preincu-bated in 1% bovine serum albumin (BSA) in PBS, andreincubated overnight at 40C with the first antibody. Thesecond antibody was incubated for 2 hours. When a per-oxidase-labeled second antibody had been used, thesections Were incubated 10 minutes in a solution of 50mg/i 00 ml diaminobenzidine and 0.01% hydrogen per-oxide in 0.05 mol/I TRIS-HCI (pH 7.6). The slides werewashed in PBS and in double-distilled water, before be-ing examined. The slides were not counterstained or cov-ered with a coverslip.

Results

Development of GvHD

The albumin excretion of the diseased animals rosesharply and reached the highest levels in week 12(30,000 ± 2,140 ,ug/18 hr vs 40 ± 2 ,ug/18 hr for thecontrol mice). Ascites and edema developed in the ma-jority of the treated animals. Antinuclear antibodies devel-

oped in all treated animals. In the course of the disease,92% of the animal sera became positive in the directCoomb's test, indicating the development of autoimmu-nity. Light and electron microscopic examinationsshowed a lupus type of nephritis with glomerular prolifer-ation as well as membranous nephritis, complicated byglomerular sclerosis starting 10 weeks after the inductionof GvHD. At this stage, Bowman's capsule was markedlythickened, especially at the hilus. Furthermore adhesionswere seen between the visceral and parietal glomerularepithelia.

The glomerular deposition patterns of the ECM mole-cules were determined semiquantitatively by immunoflu-orescence techniques. The results are summarized inTable 1. We also studied the localization patterns of lam-inin, fibronectin, collagen type 1, and type IV, and the IgGdeposits at the ultrastructural level, using reflection-contrast and immunoelectron microscopic examinations.Four sequential ultrathin sections of material representingGvHD week 10 material were cut, immediately followedby a section with a thickness of 0.5 n.m. The ultrathinslides were stained with antibodies against IgG, laminin,collagen IV, and fibronectin. The thicker section wasgiven a PAS-staining. The results are shown in Figure 2.On this basis conclusions could be drawn concerningthe distribution of these ECM components in relation tothe IgG-containing deposits and the sclerotic lesions. Thesignals of the other ECM components studied were tooweak for detection by these methods.

IgG Deposits

The immunofluorescence and reflection-contrast micro-scopic studies showed IgG in a mainly linear patternalong the glomerular capillary wall, starting 2 weeks afterthe induction of GvHD. The distribution of IgG changed toa more granular pattern after 6 to 8 weeks. At this stage,the kidneys of diseased animals showed electron-densedeposits in the mesangium and subepithelially along theglomerular capillary wall at the ultrastructural level. Incu-bation with anti-IgG antibodies led to staining of the elec-tron-dense deposits (Figure 1). The GBM showed irreg-ular thickening with formation of spikes. With the devel-opment of glomerular sclerosis, the presence of IgG atthe sites of the sclerotic lesions decreased (Figure 2b).Immunoelectron microscopic examination showed fewdeposits of IgG in the sclerotic lesions as compared withthe presence of IgG in the electron-dense deposits in theremnants of the mesangial matrix and GBM (Figure 3a).

Laminin was localized in the mesangium and the glo-merular capillary wall in normal mice (Figure 2e). Sixweeks after the induction of GvHD, the amount of lamininin the mesangium as well as that in the glomerular cap-

1150 Bergijk et alAJP May 1992, Vol. 140, No. 5

Table 1. Deposition ofExtracellular Matrix Molecules in Kidneys ofMice Sufferingfrom GvHD, as DeterminedSemiquantitatively by Immunofluorescence Studies

lam col col IlIl col IV col VI fn

nFl mes ++ - + ++ + ++GCW + + +BC + + + ++ + +TBM + + + + + + + +int + + ++ +

wk4 mes ++ - + ++ + ++GCW + - _ + _ +BC + + + + + + +TBM + + + + + + + +int + + ++ - - +

wk6 mes +++- + +++ +/++ +++GCW +/+ + + ++BC ++ ± + ++ + +TBM + + + + + + +int + ++ + - +

wk8 mes +++- + +++ +/++ +++GCW +/++ _ ++BC ++ + + ++ + +TBM + + - + + + + +int + ++ + - +

wk 10/12 gsl - - - - -+ + + +mes + + + + + + + +GCW +/++ + _ ++BC + +++ + ++TBM + + + + + + + + +int + + ++ +

mes = mesangium, GCW = glomerular capillary wall, BC = Bowman's capsule, TBM = tubular basement membrane, int = interstitium,gsl = glomerular sclerotic lesion, lam = laminin, col = collagen type 1, fn = fibronectin.

illary wall increased (Figure 2f). Two weeks later, the lo-calization of laminin had not changed. At week 10, how-ever, the distribution of laminin changed radically at thesites where glomerulosclerosis developed. In the mesan-gial areas that were not yet sclerotic, the laminin distribu-tion was the same as at week 8. However, in the scleroticregions the presence of laminin was dramatically de-creased relative to the occurrence at week 8 (Figure 2g).

Immunoelectron microscopic examination at weeks 6and 8 showed laminin around the deposits, and concen-trated mainly at the top of the spikes (Figure 3b). In scle-rotic glomeruli at week 10, laminin was found in mesan-gial and capillary material around the sclerotic lesions(Figure 3c).

Collagen was found in small amounts in the intersti-tium, the tubular basement membrane (TBM), Bowman's

IS i.i.:-r fi> AI. !t

Figure 1. Electronmicrograph of the glomerular basement membrane of a mouse 8 weeks after induction of GvHD. The electron-densedeposits react with anti-IgG antibodies (x23000). US = urinay space; CL = capillwy lumen.

ECM and Glomerulosclerosis in Murine Chronic GvHD 1151AJP May 1992, Vol. 140, No. 5

capsule, and more abundantly in vessel walls, but not inthe glomerular capillary network of normal Fl mice. Up to8 weeks after the onset of the disease, a slight increase inthe interstitium, the TBM, and Bowman's capsule oc-curred. Starting at week 10, traces of collagen wereseen in the glomeruli. At that time there was a dramaticincrease of this type of collagen in the interstitium, theTBM, and Bowman's capsule (Figure 4). Immunoelectronmicroscopic examination at week 10 showed smallamounts of collagen type in the mesangial matrix, butnot in the glomerular sclerotic lesions (Figure 3d).

Collagen III was found in the mesangium of glomeruliof normal mice. There was no staining of the glomerularcapillary wall. In the course of the disease, staining of themesangium showed no to a slight increase. Sclerotic glo-meruli at week 10 showed a decrease of collagen Ill (Fig-ure 5). The sclerotic regions did not stain for collagentype 111. Compared with normal glomeruli, the amount ofcollagen Ill increased, especially interstitially, after GvHDinduction.

Collagen IV was present in the mesangium and theglomerular capillary wall of normal mice. Bowman's cap-sule was also strongly positive for collagen type IV. Start-ing at week 6 the amount of collagen IV in the mesangiumincreased. At weeks 10 and 12, in the sclerotic lesions,the amount of collagen type IV was smaller that it hadbeen in week 8. Parts of the glomeruli that had not yetbecome sclerotic, showed a distribution pattern similar tothat seen in week 8 (Figure 2c).

Immunoelectron microscopic examination showedcollagen type IV in the mesangial matrix and in the glo-merular capillary wall around the electron-dense depos-its at week 6 and 8. In sclerotic glomeruli, collagen type IVwas present in the mesangial matrix and the remaindersof the GBM, but not in the sclerotic lesions (Figure 3e).

Collagen VI was found in the mesangium, Bowman'scapsule, the TBM, and around the vessel walls in normalmice. Six weeks after the induction of the disease, expan-sion of the mesangium began and an increase of colla-gen type VI occurred. At weeks 10 and 12, a markedincrease had occurred in the interstitium, especiallyaround the vessel walls. In the glomeruli, collagen VI hadalmost totally disappeared. Nonsclerotic parts of glomer-uli showed mesangial staining resembling that seen atweek 8 (Figure 6).

Fibronectin was found both in the mesangium and theglomerular capillary wall of control mice. Bowman's cap-sule and the TBM were positive. Six weeks after the onsetof GvHD, fibronectin increased in the mesangium, butremained unchanged in the capillary wall. This picturepersisted in week 8. The sclerotic glomeruli appearing atweek 10 showed a large amount of fibronectin (Figure2d).

Immunoelectron microscopic examination showed adiffuse distribution of fibronectin in the sclerotic lesions.

Within the lesions, differences in the degree of electrondensity occurred. The more electron-dense material inthe sclerotic lesions stained more intensely for fibronectinthan the less electron-dense parts did. Fibronectin wasalso present in the mesangial matrix and in the remnantsof GBM around the sclerotic lesions (Figure 3f).

Discussion

In the present study, we investigated glomerular sclerosisas a complication of immunologically mediated glomer-ulonephritis. Improvement of the diagnosis and treatmentof patients requires a better understanding of the mech-anisms underlying the development of glomerulosclero-sis. To this end, we studied the distribution patterns of anumber of extracellular matrix components, i.e., laminin,fibronectin, and collagen types 1, 111, IV, and VI in theexperimental murine GvHD model, making use of immu-nofluorescence, immunoelectron microscopy, and therecently developed technique of reflection-contrast mi-croscopy with peroxidase staining. The reflection-contrast microscopic technique offers a useful supple-ment to the well-known technique of immunofluores-cence. The detection level of the former method is slightlylower, because less antigen is present in the ultrathinsections.15 However, the staining is much more precise.The minimal thickness of these sections makes it possi-ble to define a sequential range of serial sections througha single glomerulus. The morphology shown by theseultrathin sections is comparable, which means that thedistribution patterns of the various ECM components canbe compared in relation to each other and to immuno-globulins present.

In normal (C57BL110*DBA/2)F1 hybrid mice, wefound the components laminin, fibronectin, collagentypes 111, IV, and VI, in the glomerular mesangium. Lam-inin, fibronectin, and in small amounts, collagen type IV,were also found in the glomerular capillary wall.

The finding of collagen type III in the glomeruli of nor-mal mice is in itself remarkable. Most other authors didnot find collagen type III in normal glomeruli of hu-mans122021 or rats 13,22,23 On the other hand, Downer etal24 reported finding trace amounts of interstitial collagenin some glomeruli of control New Zealand White rabbits.Thus, differences in collagen Ill distribution might existbetween species or even between strains.

The amounts of all the ECM components studied in-creased during the course of the disease. Changes be-gan to occur 6 weeks after the induction of GVHD. At thisstage, the mesangium started to expand. Irregular thick-ening of the GBM and the formation of spikes were alsoobserved. IgG-containing electron-dense deposits werepresent both subendothelially and subepithelially alongthe GBM.

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ECM and Glomeruloscierosis in Murine Chronic GvHD 1153AJP May 1992, Vol. 140, No. 5

Figure 2. a: Glomerulus 10 weeks after the induction of GvHD, stained according to the PAS technique. b: IgG distribution in glomerulusat week 10. c: Collagen type IVat week 10. d: Fibronectin at week 10. e: Laminin distribution in a glomerulus ofa normal Fl hybrid mouse.f: Laminin distribution 6 weeks after induction ofGvHD. g: Laminin distribution at week 10; (a-d) and (g) show sequential sections ofoneglomerulus; (b-g) the peroxidase reflection contrast microscopical technique was used (x630).

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Figure 3. Immunoelectron micrographs of (a) IgG distribution in a glomerulus 10 weeks after the induction ofGvHD, arrows indicate IgGcontaining electron-dense deposits; (b) glomerular basement membrane showing localization oflaminin at week 8; (c) Laminin ditributionat week 10; (d) collagen I (arrows) at week 10; (e) collagen IVat week 10; and (f) fibronectin at week 10. Magnification a, c-f, x 7000;b, X28000. GSL = glomerular sclerotic lesion, MM = mesangial matrix, CL = capillary lumen.

1154 Bergijk et alAJP May 1992, Vol. 140, No. 5

Figure 4. Immunofluorescence micrographshowing the distibution pattern of collagentypeI 10 weeks after GvHD induction (x630).

Starting 10 weeks after induction of the disease, glo-merular sclerosis developed. This was associated withadhesion to and thickening of Bowman's capsule. Thefirst striking finding was the appearance of traces of theinterstitial collagen type in the glomerulus at this time-point. Our finding is in agreement with the observationsmade by Foellmer et al,16 who suggested an associationbetween the presence of collagen and interruption ofBowman's capsule in rats with anti-GBM nephritis. A ge-notype switch of glomerular cells leading to local produc-tion of collagen type has also been suggested as an

explanation for the presence of interstitial collagens in aglomerulus.25 Our finding of collagen type in the mes-

angial matrix and not in the glomerular sclerotic lesionsmight be in agreement with this hypothesis. In addition,diminished collagenase activity in sclerotic glomerulicannot be ruled out as an explanation for the presence ofcollagen type in sclerotic glomeruli. This could be due todamage of the cells producing this enzyme.

Second, we found striking differences between thedistribution patterns of the ECM components in the scle-rotic glomeruli. Fibronectin was present in the scleroticlesions. The other components we investigated showeda sudden decrease relative to the level at week 8. We hadthe impression that these components were replaced bythe sclerotic lesions. The traces of ECM componentsfound around the lesions are believed to have been rem-nants of mesangial material and/or of collapsed capillarywalls. The finding of differences in the occurrence of in-dividual ECM molecules in sclerotic glomeruli is in itselfinteresting. Recently, Ikeda et al26 found differences inthe localization of various ECM components in diabeticglomerular lesions. They found that collagen IV occurredin the core portion of the nodular lesion, whereas colla-gen VI was seen in the surrounding laminated acellulararea. These authors believed that the formation of thelesions in diabetic microaneurysms might be based onthe increase of type VI collagen.

Figure 5. Immunofluorescence micrographshowing the distibution pattern of collagentype III 10 weeks after GvHD induction

,, , ~~~(X630).

ECM and Glomerulosclerosis in Murine Chronic GvHD 1155AJP May 1992, Vol. 140, No. 5

Figure 6. Distrbution of type VI collagen ina sclerotic glomerulu-s 12 weeks after the in-

pK~~~T.~~duction of GvHD. Tyvpe W7 collagen had al-disappeared in the sclerotic areas (ar-

rowheads). Immunofluorescence micrograph(x630).

Last year, Funabiki et al27 reported fluorescence stud-ies in patients with glomerulonephritis and diabetic ne-phropathy. In the advanced stages of glomerulonephritis,they found a marked increase of types IV and VI colla-gen, laminin, and fibronectin in the mesangium. The dis-tribution extended along the glomerular capillary walls aswell. During the progression of glomerulosclerosis, theyfound a significant decrease in the amount of type IVcollagen, laminin, and fibronectin, whereas the occur-rence of interstitial collagen types and Ill was observedfocally in the glomerular lesions.

The observed influx of collagen type and the de-creased amounts of laminin and collagen IV in our modelin the sclerotic stage is comparable with Funabiki's find-ings in the aforementioned renal diseases. Markedly di-vergent, however, is our finding of fibronectin in the scle-rotic lesions. Unfortunately, our model does not allow in-vestigation of the composition of sclerotic lesions duringthe progression of glomerulosclerosis, because the micedo not live long enough.

The glomerular sclerotic lesions appeared to consistof material of variable electron density in immunoelectronmicroscopic examination as well as in routine electronmicroscopic examination. The more electron-dense partsof the lesions contained more fibronectin than the lesselectron-dense parts did. The sclerotic lesions in GvHDprobably contain a variety of molecules. Albumin, trans-ferrin, and fatty acids were not among them (unpublishedresults).

There are two possible explanations for the increasedpresence of fibronectin in the glomeruli of GvHD mice.First, there might be an accelerated increase of the syn-thesis and excretion of fibronectin or decreased break-down during the development of glomerulosclerosis. Thesecond hypothesis is that there is an accumulation of

exogenous fibronectin from the circulation. During thecourse of GvHD, a dramatic change in glomerular mor-phology was seen, including thickening of the glomerularcapillary walls and narrowing of the capillary loops. Thismight lead to trapping of exogenous fibronectin and othercomponents from the circulation into the glomerulus. Theother ECM components might be pushed aside or cov-ered by the accumulated material. The accumulation offibronectin in the sclerotic lesions might be facilitated byfibronectin-binding receptors. Recently, a pilot studyshowed increased expression of adhesion molecules ofthe integrin family. Furthermore histologic staining for al-bumin, transferrin, and fatty acids showed that the devel-opment of glomerulosclerosis in murine chronic GvHD isnot a process of nonspecific trapping of proteins from thecirculation. However, it is accompanied by bloodclottingas shown by fibrin and fibrinogen staining (unpublishedresults).

Further investigations, including mRNA studies, areneeded to establish which mechanisms underlie the de-velopment of glomerulosclerosis in murine chronic GvHDand the abnormal composition of the ECM in these le-sions. We recently speculated about the nature of pro-gression in experimental lupus nephritis.28 Investigationsin this field can contribute to a better understanding of thedevelopment of glomerulosclerosis, and thus to improve-ment of the diagnosis and treatment of patients with glo-merulonephritis.

Acknowledgments

The authors thank J. van der Ploeg for technical assistance andDr. E. de Heer for stimulating discussions.

1156 BergijketalAJP May 1992, Vol. 140, No. 5

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