The Immunohistopathology of GlomerularAntigens THE GLOMERULAR BASEMENTMEMBRANE, COLLAGEN, AND ACTOMYOSINANTIGENS IN NORMAL AND DISEASEDKIDNEYS

Jon I. Scheinman, … , Alfred J. Fish, Alfred F. Michael

J Clin Invest. 1974;54(5):1144-1154. https://doi.org/10.1172/JCI107858.

The immunofluorescent localization of antisera to human glomerular basement membrane(GBM), collagen, and smooth muscle actomyosin was examined in 15 specimens of normalrenal tissue and 98 specimens from patients with renal disease. The anti-GBM andanticollagen antisera normally localize to GBM, while antiactomyosin localizes to themesangium. Diabetic nephropathy revealed a striking expansion of mesangial materialreacting with antiactomyosin. In contrast, the expanded mesangium inmembranoproliferative glomerulonephritis did not react with antiactomyosin, and the GBMlocalization of anti-GBM and anticollagen sera was similarly lost. The thickened GBM indiabetes mellitus and membranous nephropathy reacted with anti-GBM and anticollagen,but with accentuation of staining on the inner aspect of the GBM. In proliferativeglomerulonephritis there was a moderate increase in the distribution of actomyosin.Glomerular sclerosis and hyalinization in all diseases studied was accompanied by a lossof immunofluorescent staining for all glomerular antigens, including collagen.

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The Immunohistopathology of Glomerular Antigens

THE GLOMERULARBASEMENTMEMBRANE,COLLAGEN,AND

ACTOMYOSINANTIGENS IN NORMALANDDISEASED KIDNEYS

JON I. SCHEINMAN,ALFREDJ. FISH, and ALFREDF. MICHAEL

From the Department of Pediatrics, University of Minnesota,Minneapolis, Minnesota 55455

A B S T R A C T The immunofluorescent localization ofantisera to human glomerular basement membrane(GBM), collagen, and smooth muscle actomyosin wasexamined in 15 specimens of normal renal tissue and98 specimens from patients with renal disease. The anti-GBM and anticollagen antisera normally localize toGBM, while antiactomyosin localizes to the mesangium.Diabetic nephropathy revealed a striking expansion ofmesangial material reacting with antiactomyosin. Incontrast, the expanded mesangium in membranopro-liferative glomerulonephritis did not react with antiacto-myosin, and the GBM localization of anti-GBM andanticollagen sera was similarly lost. The thickenedGBMin diabetes mellitus and membranous nephropathyreacted with anti-GBM and anticollagen, but with ac-centuation of staining on the inner aspect of the GBM.In proliferative glomerulonephritis there was a moder-ate increase in the distribution of actomyosin. Glomeru-lar sclerosis and hyalinization in all diseases studied was

accompanied by a loss of immunofluorescent stainingfor all glomerular antigens, including collagen.

INTRODUCTIONImmunopathologic studies of the human kidney are

generally utilized to demonstrate deposition of serum

proteins or foreign antigens. Normal structural glomer-ular antigens have also been demonstrated by immuno-fluorescence: antigenic components of the glomerularbasement membrane (GBM) ' react with heterologous

Presented in part at the American Society of Nephrology,20 November 1973, Washington, D. C.

Dr. Fish is an Established Investigator of The AmericanHeart Association.

Received for publication 10 May 1974 and in revised formn22 July 1974.

'Abbreviations used in this paper: FITC, fluorescein iso-thiocyanate; GBM, glomerular basement membrane; u,calculated ionic strength; SDS, sodium dodecyl sulfate;TBM, tubular basement membrane.

nephrotoxic anti-GBM serum, anti-GBM antibodies ofGoodpasture's syndrome (1), and antibody to solu-blized skin collagen (2). The glomerular mesangiumhas been shown to react with antibody to smoothmuscle actomyosin (3).

In the present studies the distribution and amountsof glomerular antigens in the diseased kidney areevaluated by immunopathologic methods using specificantisera to GBM, collagen, and actomyosin. Specialattention has been directed to those diseases in whichspecific alterations in glomerular architecture are usu-ally found, but where the relationship of these altera-tions to the normal glomerular components is un-known: the GBMthickening of diabetes mellitus andmembranoproliferative glomerulonephritis, the replace-ment of normal glomerular architecture in focal sclero-sis (segmental hyalinization), and end-stage glomerularobsolescence (diffuse hyalinization).

METHODS

AntigensActomyosin was isolated from a human uterus weighing

160 g obtained at autopsy from a 27-yr-old woman 16 hafter accidental death by asphyxiation. The patient hadaborted 2 wk previously in the 20th wk of pregnancy. Theisolation was performed at 4°C after the methods of Becker(3) and Finck (4). Initially, the washed and homogenizedmyometrium was extracted for 18 h in 400 ml of a buffer(0.3 M KCl, 0.17 M K2HPO4, 0.001 M EDTA, pH 8.5)of calculated ionic strength (,t) =0.8, to which 0.08%ATP' was added. The brei was filtered through Mira-cloth3 and the protein precipitated by dilution to I = 0.03,collected by centrifugation at 3,300 g for 15 min, andwashed twice. The precipitate was solubilized in 200 mlof a solution of ,u = 0.57 (0.13 M KCl, 0.095 M KH2PO4,0.075 M K2HPO4, 0.001 M EDTA, pH 6.5) by dispersionwith a Pasteur pipette and slow stirring for 2 h. This andsubsequent solutions were clarified by centrifugation at37,000 g for 90 min. The protein was again precipitated by

2Sigma Chemical Co., St. Louis, Mo.'Calbiochem, San Diego, Calif.

The Journal of Clinical Investigation Volume 54 November 1974*1144-11541144

dilution to u = 0.03, solubilized in A = 1.15 buffer (0.81 MKCl, 0.11 M KH2PO4, 0.1 M K2HPO4, 0.001 M EDTA,pH 6.5), and precipitated at A = 0.3. Solubilization in 90 mlof the A = 1.15 buffer and precipitation at A = 0.3 were re-peated. Next, the protein was twice solubilized at ,uk = 1.15and precipitated by dilution to , = 0.1. The protein wasdissolved in 40 ml of 0.02 M Tris-HCl buffer containing0.5 M KCl, pH 7.4, stabilized by the addition of 40 ml of5.0 M LiCl, pH 7.0, and precipitated at 4°C with (NH4)2-S04 at 39% saturation (vol/vol). After the precipitate waswashed, solubilized in 10 ml of the 0.5 M KCl solution, anddialyzed, 3 ml (approximately 10 mg by OD at 280 nm [4]was applied by reverse flow to a 1.5 X 100-cm closedjacketed column of Sephadex G-200 Superfine 4 packed onsiliconized 6-mm glass beads (5), running at 7 ml/h at4°C. The column was supported on a Teflon mesh ' toavoid precipitation of the protein during loading. The pro-tein emerged at the void volume in a single peak. It waspreserved in 50%o glycerol and used for immunization ofrabbits and immunodiffusion tests. Portions of the unchro-matographed protein were used for ATPase assay (6) andreleased 3.6 ,ug P/mg protein/30 min. Polyacrylamide gel(7.5%o) chromatography in sodium dodecyl sulfate (SDS)(7) was performed by Ms. Juille Johnson in the labora-tories of Drs. M. Han and E. Benson (Departments ofLaboratory Medicine and Pathology, University of Minne-sota Medical School). The gel electrophoresis pattern ofour actomysin preparation (in SDS) was compared withpurified samples of rabbit skeletal muscle actin and myosinand revealed multiple bands similar to those of actin, as wellas two bands in the region of myosin.

Collagen was isolated from skin samples from neonatalautopsies. Acid-soluble collagen was extracted at 4° C indilute acetic acid (pH 3.2) and isolated by neutral precipi-tation and acid solubilization three times, as described byRothbard and Watson (2). The collagen was lyophilizedand dissolved in 5 M guanidine-HCl and dialyzed against8 M urea for immunization.

Human GBMwas isolated from two normal kidneys atautopsy within 5 h of death as described previously in thislaboratory (8).

Immunologic techniques

Fluorescein isothiocyanate' (FITC) labeling of the IgGfraction of antisera was performed and the optimallylabeled fractions (fluorescein: protein ratio 6-10 X 10')were obtained by DEAE6 chromatography by using themethod of Cebra and Goldstein (9). Direct and indirectimmunofluorescent staining was performed as describedpreviously in this laboratory (10).

Tissues were placed on moistened Onkosponge no. 17and snap frozen in isopentane prechilled in liquid N2 andstored at - 70°C until sectioning at 2-4 lm on a Lipshaw1500 Cryotome cryostat (Lipshaw Mfg. Co., Detroit, Mich.)or at 1-2 jum on a Harris MS-100 Microtome cryostat(Harris Mfg. Co., Cambridge, Mass.). No deteriorationor changes in the fluorescent staining reactions reportedhere were noted when restaining freshly cut tissue storedup to 20 mo in isopentane at - 70°C. Tissues were ex-amined with a Zeiss fluorescent or Ultraphot III microscope

4Pharmacia Fine Chemicals, Inc., Piscataway, N. J.5BBL, BioQuest Div., Becton, Dickinson & Co., Cockeys-

ville, Md.6Bio-Rad Laboratories, Richmond, Calif.7Histamed, Patterson, N. J.

with a Zeiss FITC interference filter and an OG-4 barrierfilter (Carl Zeiss, Inc., New York).

Immunodiffusion was performed as previously reported(10). Actomyosin was studied at 4VC on 1% Noble agarin 0.5 M KCl, 0.02 M Tris-HCl, pH 7.4; antigen wasplaced in wells 5 mmin diameter and the antiactomyosinantisera in 1.3-mm wells and allowed to diffuse for 3-7days at 4VC. Immunodiffusion of acid-soluble collagen wasattempted in acetic acid, guanidine, urea (2), and collagen-ase solutions, on agarose and Noble agars, and in glycineand Veronal buffers.

Other studiesCollagenase' digestion of normal human kidney frozen

tissue sections was described by Rothbard and Watson (11)before immunofluorescent staining. Thioflavine T stain foramyloid was performed on frozen sections after formalinfixation (12). Tissue targets for immunofluorescence in-cluded the following: frozen sections of a biopsy of gravidhuman term uterus; normal skin from an 11-yr-old boywith ulcerative colitis and purpura; a normal bowel biopsy;normal human kidney obtained at autopsy within 4 h ofdeath in an accident victim. The buffy coat of human bloodsmears was aid dried, fixed in absolute alcohol-ether (1: 1),and stained for immunofluorescence.

AntiseraAntiactomyosin (antiserum to actomyosin). Actomyosin,

1 mg in 50% glycerol-0.5 M KCl solution in completeFreund's adjuvant, was injected subcutaneously into eachrabbit weekly for 5 wk. These rabbits were then boostedevery 2 wk and antisera harvested for up to 8 more wk.The strongest pool of antiserum was absorbed with nor-mal human serum on a Sepharose 4B immunoabsorbentcolumn (13). This antiserum showed two precipitin linesby immunodiffusion against the actomyosin used for im-munization. These lines, at differing dilutions, formed linesof identity with those formed by antiactomyosin antiserumprovided by Dr. C. G. Becker (N. Y. Hospital, CornellMedical Center). The immunodiffusion reactions were abol-ished by two absorptions of our antiserum with precipitateduterine actomyosin (approximately 5 mg/ml of undilutedantiserum). There was no reaction between this antiserumand normal human serum at dilutions of both up to 1: 512.

Antiactomyosin stained frozen sections of a gravid (term)human uterus strongly, as well as bowel wall muscularisand smears of platelets, segmented neutrophils, and somemononuclear cells. Glomerular mesangial staining in thenormal human kidney and staining of gravid uterine musclewere completely inhibited on indirect immunofluorescenceafter two absorptions with precipitated actomyosin. Mini-mal reduction of mesangial staining was produced by ex-haustive absorption of the antiserum with lyophilized GBM(25 mg/ml undiluted antiserum). No inhibition was seenafter two consecutive absorptions with collagen (12.5 mg/mlundiluted antiserum, performed twice).

Anticollagen (antiserum to collagen). Collagen, 15 mg,dissolved in 8 M urea, was used for each immunization incomplete Freund's adjuvant. Seven rabbits and one goatwere immunized weekly in multiple subcutaneous sites for4 wk and then biweekly for 16 wk with weekly test bleeds.Antiserum localizing by indirect immunofluorescence to

8Collagenase, CLSPA, Worthington Biochemical Corp.,Freehold, N. J.

9Pharmacia Fine Chemicals, Inc.

Immunohistopathology of Glomerular Antigens 1145

human GBMappeared in two of seven rabbits; of the tworabbits developing antibody, the stronger was used for sub-sequent studies. The IgG fraction (10 mg/ml) of this anti-serum did not react on immunodiffusion against solubilizedcollagen nor with normal human serum. None of the re-maining rabbits developed detectable antibody after 4 mo ofimmunization. The goat developed weak antibody, stainingthe glomerulus diffusely without specific GBMlocalization.

Anticollagen localized on sections of human dermis tobundles of fibrillar material which extended into the retepegs, as well as to the mesangium of the glomerulus. Allfluorescent reactions were inhibited by prior absorption ofthe antiserum with lyophilized collagen (12.5 mg/ml ofundiluted antiserum, performed twice), but not by similarabsorption with GBMor actomyosin.

A sample of rabbit antiserum to 0.5 M acetic acid-soluble NaCl-precipitable human collagen (14) was alsokindly supplied by Dr. Dov Michaeli (Department of Bio-chemistry, University of California, San Francisco). Byindirect immunofluorescence this antiserum stained the glo-merulus more diffusely than did ours, without apparentlyspecific GBM localization. However, other tissue stainingreactions by indirect immunofluorescence were similarthough somewhat stronger than ours and were abolished bytwo absorptions with 0.7 mg of our lyophilized collagenpreparation per ml of a 1: 64 dilution of the antiserum.

Anti-GBM (antiserum to GBM). 1 mg of lyophilizedGBMwas injected in complete Freund's adjuvant into arabbit by using multiple subcutaneous sites; animals were

boosted 5 wk and 8 mo later and bled 1 wk later. Thisantiserum localized to human GBMand tubular basementmembranes (TBM) by indirect and direct immunofluores-cence. Its absorption with lyophilized GBM (12.5 mg/mlof undiluted antiserum, performed twice) completely in-hibited its localization to basement membranes by indirectimmunofluorescence. Similar absorption of unlabeled anti-serum with lyophilized collagen, actomyosin, or normalhuman serum did not alter the fluorescent staining reaction.Two absorptions of the FITC-labeled antiserum with acto-myosin did not inhibit its staining of the GBM. The anti-serum did not react on immunodiffusion against normalhuman serum.

Human (Goodpasture's) anti-GBM antibody was elutedfrom the nephrectomy specimen of a patient with pulmonaryhemorrhage, rapidly progressive nephritis, and linear IgGdeposition along the GBMseen by immunofluorescence (1).The Goodpasture's antibody localized only to GBMby in-direct immunofluorescence.

Patient material113 percutaneous and open kidney biopsies and nephrec-

tomy specimens from 109 patients admitted to the Pediatric,Medical and Surgical services of the University of Minne-sota Hospitals were examined in this study: normals in-cluded biopsies from 11 kidney transplant donors and fourautopsy specimens, obtained within 4 h of accidental death.

Tissues from the following diseases were studied (TableI): membranous nephropathy, 16 patients (including six

TABLE IAlterations in Glomerular Antigens in Renal Disease

GBMfluorescence using antibodies Mesangial fluorescence using antibodyto GBMand collagen to actomyosin

No. of Absent Absent Expandedtissues or or

Disease examined Normal trace Thickened* Normal trace Mildly Markedly

Normal 15 1S 1S

Membranoproliferative glomerulonephritis 19 19 17

Membranous nephropathy 16 2 14 8 7

Diabetes mellitusOnset 1 1 1Early 2 2 2Late 16 1 10 9End-stage 5 4 1 5

Proliferative nephritis 11 11 11

Nephrotic syndrome 11 11 8 3

Glomerular hypertrophy 2 1 1 1 1

Amyloid nephropathy 3 3 2

End-stage kidney 12

Residual glomeruli 8 8Hyalinized glomeruli 12 12

Predominant findings for fluorescent localization of antisera in renal diseases. GBMstaining absent or trace indicates very thin or absent linear staining ofcapillary loops. Thickened GBMis judged at its narrowest appearance among different planes of focus. Mesangial staining absent or trace indicates absenceof, or only small residual patches of, mesangial staining. Mildly expanded mesangial staining with antiactomyosin indicates loss of the limited mesangialdistribution and extension to the periphery and often around glomerular loops. Markedly expanded mesangial staining with antiactomyasin indicates thatthe widened mesangium is stained to the full extent of that observed by light microscopy in these tissues.

The number of tissues examined exceeds the number for which results are given, due to inadequate tissue quality or autofluorescence precluding satis-factory evaluation. More than three glomeruli were evaluated for each result given, except that two glomeruli were present in two sections from membrano-proliferative glomerulonephritis, one section from nephrotic syndrome, and one section from poststreptococcal glomerulonephritis.* The GBMhad a laminated appearance, with the inner layer more densely stained in nine of 14 with membranous nephropathy, six of 10 with late dia-betes, and one with end-stage diabetes.

1146 J. 1. Scheinman, A. J. Fish, and A. F. Michael

mMM-FIGURlE 1 Immunofluorescent localization of glomerular antigens in the normal glomerulus.(A) Anti-GBM localization to the GBMand Bowman's capsule. The mesangium is notstained (anticollagen staining is similar, not illustrated). (B) Antiactomyosin staining of themesangium. Sections 2 /m, all photographs 1,000 X.

with systemic lupus erythematosus); membranoprolifera-tive glomerulonephritis, 19 specimens from 16 patients;diabetes mellitus, 24 patients; nephrotic syndrome, includingthree nil-lesion, one steroid responsive with focal sclerosis,five steroid resistant with focal sclerosis, and two congenital(Finnish type); proliferative nephritis including three post-streptococcal, three anaphylactoid purpura nephritis, fourfocal mesangial proliferative nephritis, and one rapidly pro-gressive nephritis; glomerular hypertrophy, one patient withcyanotic congenital heart disease and proteinuria (15) andone child with renal failure due to bilateral renal hypo-plasia with oligonephronia (16); amyloid nephropathy,three specimens from two patients. End-stage renal diseaseincluded two obstructive pyelonephritis, one congenital dys-plasia, three medullary cystic disease, one cystinosis, andfive unknown causes.

Immunopathologic analysesTissues were evaluated by the senior author (J. I. S.), and

all significant deviations from normal patterns were readblindly by at least one of the other authors. More thanthree glomeruli were present per section except where in-dicated in Table I. A complete set of stained sections fromthe same normal tissue accompanied each group of stainsfor comparison. The intensity of glomerular staining wasjudged relative to nonglomerular structures: anti-GBMstaining of GBMwas compared to TBM (approximatelyequal in normal kidney); anticollagen staining of GBMwas compared to interstitial staining (slightly less intensein normal kidney); antiactomyosin staining of the mesan-gium was compared to peritubular capillary staining (ap-proximately equal in normal kidney). However, because ofthe difficulty in judging intensity of fluorescence, abnormali-

ties were primarily judged by distribution (as defined inthe legend to Table I): GBMstaining was described astrace or absent, normal or thickened; mesangial stainingwas described as trace or absent, normal, or expanded(mildly or markedly).

Autofluorescence of GBMand mesangial areas, found es-pecially in several diabetic tissues (17), precluded satisfac-tory evaluation and necessitated elimination of these tissuesfrom the results.

RESULTS

Glomerular antigens in normal renal tissue

Normal immunochemical reactions of FITC-labeledantisera on normal renal tissue are illustrated in Fig. 1and diagramatically presented in Fig. 2 and Table II.Within the glomerulus, anti-GBM (Fig. 1A and Fig.2) and anticollagen antisera stain the GBM almostexclusively. Antiactomyosin (Fig. 1B and Fig. 2)fixes to the mesangium.

Collagenase digestion of normal tissue, as describedby Rothbard and Watson (11), abolished fluorescentstaining of the GBMby anticollagen and anti-GBM,whereas antiactomyosin staining of the mesangiumremained intact.

Glomerular antigens in renal disease (Table I)

Membranoproliferative glomerulonephritis. Therewas an almost complete loss of normal glomerular anti-

Immunohistopathology of Glomerular Antigens 1147

- Peritubular

b Capil lary

GBM

Mesangium

TABLE I IReactivity of Antisera to Renal Structures

Antisera

Anti- Anti- Antiacto-Location in normal kidney GBM collagen myosin

NonglomerularTBM 3+ 0 0Bowman's capsule 2+ tr 0Capillary basement membrane 2+ tr 0Peritubular capillary wall 0 1-2 + 2+Artery walls 0 tr 1+Interstitium 0 1-2 + tr

GlomerularGBM 3+ 1-2+ 0Mesangium 0 tr 2 +

A nt i - GBM

gen localization in membranoproliferative glomerulo-nephritis. The expanded mesangial areas did not reactwith antiactomyosin, and there was only minimal stain-ing at the periphery of the mesangial areas. The stain-ing of the GBMby anti-GBM (Fig. 3) and anticollagenwas markedly diminished or absent; even in lessseverely involved glomeruli, normal GBM antigenswere detectable only as a fine rim around capillaryloops. Certainly no splitting of the GBMwas seen, as

found on silver stains. One representative biopsy speci-men of this disease, stained by indirect immunofluores-cence with the Goodpasture's antibody, also showedtrace GBMstaining.

Membranous nephlropathy. In membranous nephrop-athy, studies with anti-GBM antibody revealed thick-ening of the basement membranes (Fig. 4). In more

severe lesions an apparent splitting of the basementmembrane was seen with an accentuation of the innerlayer. Occasionally, interruptions in staining of theouter aspect could be seen, probably corresponding toimmune deposits. Results with anticollagen were simi-lar, although the inner accentuation was less promi-

Antiac tom yosln N

FIGURE 2 A composite illustration of the fluorescent stain-ing patterns on normal kidney tissue of antisera to GBM(anti-GBM), collagen (anticollagen), and actomyosin (an-tiactomyosin). Gray shading indicates positive immuno-

fluorescence. Anti-GBM: GBM, TBM, and Bowman's cap-sule are stained, along with peritubular capillary and ar-teriolar basement membranes. No staining of the mesangiumor interstitium is found. The antibody eluted from a kidneywith anti-GBM nephritis (Goodpasture's) stains only theGBMby indirect immunofluorescence. Anticollagen: GBMis stained, but Bowman's capsule is stained only weakly.TBM are not stained and appear as negative "lines" be-tween the stained interstitium and the light autofluorescenceof tubular cytoplasm. There is only trace staining of themesangium. Peritubular capillary and arteriolar basementmembranes are weakly stained. (Anticollagen antiserumfrom Dr. Michaeli stains the glomerulus diffusely and theinterstitial staining is stronger). Antiactomyosin: GBM,TBM, and Bowman's capsule are not stained. The mesan-gium is prominently stained. In the interstitium most stain-ing is associated with capillary endothelial cells or withthe muscularis of arterioles and venules.

1148 J. 1. Scheinman, A. J. Fish, and A. F. Michael

Arter y +el /

,./t. \-\ _ \ C

Bowman'sCapsule

FIGURE 3 Membranoproliferative glomerulonephritis. (A) Marked mesangial expansion inglomerular lobules, viewed by indirect immunofluorescence, staining with FITC-labeled anti-serum to GBM. Only trace staining of the GBMis seen. (B) Phase and indirect immuno-fluorescent microscopy combined, same field as A. Three circular autofluorescent artifacts arepresent. Magnification 400 X.

nent. Staining of the mesangium by antiactomyosin wasmildly expanded in seven of 15 patients. The one pa-tient whose light microscopy (including silver methen-anine stain) was normal (but with positive immuno-fluorescent staining for C3, IgM, and IgG, typical ofmembranous nephritis) had no immunofluorescent ab-normalities with anti-GBM, anticollagen, and antiacto-myosin.

Diabetes mellitus. In tissues from nine patients withrenal failure due to diabetes mellitus, the distributionof actomyosin was markedly increased to encompass thefull extent of the expanded mesangium seen by lightmicroscopy (Fig. 5C [early] and 5D [late]). Absorp-tion of the antiactomyosin with its specific antigencompletely abolished this staining in the one severecase so studied. There was a widening (Fig. SA) andapparent splitting (Fig. 5B) of the GBM with anaccentuation of the inner layer when anti-GBM andanticollagen antisera were used. One of these tissuesstudied by indirect immunofluorescence with the Good-pasture's antibody revealed similar staining reactionsof the GBM.

Five other tissues showed results generally indis-tinguishable from other end-stage kidneys describedbelow. Other tissues had strong autofluorescence of

FIGURE 4 Membranous nephropathy. Immunofluorescentstaining with anti-GBM shows thickened capillary loops.Magnification 600 X.

Immunohistopathology of Glomerular Antigens 1149

FIGURE 5 Diabetes Mellitus. Immunofluorescent staining with anti-GBM (A and B) andantiactomyosin (C and D) antisera. Anti-GBM staining in early (A) and late (B) diabeticnephropathy demonstrates thickening of basement membranes (white arrows) and unstainedexpanded mesangium (black arrows). Antiactomyosin staining of early (C) and late (D)diabetic nephropathy demonstrates progressive expansion of positively stained mesangium.Magnification 800 X.

GBM (five patients) and mesangial nodular scleroticareas (seven patients), which precluded satisfactoryevaluation with our antisera.

Kidneys from three patients with early diabetes werealso studied. The renal tissue of a 4-yr-old boy wasstudied at the onset of idiopathic nephrotic syndrome;juvenile diabetes mellitus was discovered 1 wk laterand has continued for 2 yr after steroid have been dis-continued. Routine light and fluorescent microscopywere normal, as were our stains for glomerular anti-gens. Two other patients, aged 15 and 21, with diabetesmellitus of 3 and 10 yr duration, had no evidence of

basement membrane thickening or pronounced mesan-gial expansion by light microscopy. Electron micros-copy in one showed focal areas of GBM thickeningand minimal enlargement of the mesangium by elec-tron microscopy (performed by Dr. Z. Posalaky, St.Paul-Ramsey Hospital). Anti-GBM and anticollagenantisera demonstrated focal areas of apparent basementmembrane thickening in these two patients, but accen-tuation of the inner aspect of the GBMwas not dis-cernible. Antiactomyosin staining of the mesangiumwas markedly expanded.

Glomerular "hyaline" droplets, although identified by

1150 1. I. Scheinman, A. J. Fish, and A. F. Michael

light microscopy in several of our patients with diabeticnephropathy, could not be identified with our three anti-sera. Intracapillary glomerular lesions characteristic of"Kimmelstiel-Wilson nodules" in mesangial areas wereoften strongly autofluorescent. However, patients withnodular patterns of mesangial expansion by light mi-croscopy did not demonstrate corresponding nodularstructures within the expanded mesangial areas stainedwith antiactomyosin.

Proliferative glomerulonephritis. Tissues of threepatients with poststreptococcal and one with rapidlyprogressive glomerulonephritis showed an extensive dis-tribution of staining with antiactomyosin that extendedto the periphery of glomerular lobules, and often lostthe mesangial pattern (Fig. 6). However, the widthof the stained mesangium stalk was not increased. Theglomeruli of seven patients with nephritis due to ana-phylactoid purpura and focal mesangial proliferativenephritis had similar findings, although the staining ofthe mesangium by antiactomyosin was expanded some-what more in width in five of these patients, corre-sponding to the widened mesangial areas seen by lightmicroscopy. Anti-GBM and anticollagen staining ofthe GBMwas normal in these 11 patients, althoughoccasionally the continuity of the GBMcould not beclearly discerned.

Fibroepithelial crescents in one patient with rapidlyprogressive nephritis, in two of the children with ana-phylactoid purpura, and in one with mesangial pro-liferative nephritis were minimally stained with ourantisera; there was a weak reticular staining, appar-ently surrounding cells, seen with anticollagen and, stillweaker, with antiactomyosin. Focal sclerotic areas ofglomeruli in a patient with mesangial proliferativenephritis were not stained with any of our antisera.

Miscellaneous renal disease. Patients with nephroticsyndrome had no clear abnormalities by immunofluo-rescent staining for glomerular antigens except focalareas of decreased GBM staining with anti-GBM inthree patients. Focal sclerotic areas of glomeruli werenot stained by any of the three antisera in the sixpatients with segmental hyalinosis.

The hypertrophied glomeruli of a child with cyanoticcongenital heart disease were stained more extensivelythan normal with all three antisera, rendering clear de-marcation of GBM and mesangial zones impossible.The end-stage kidneys of the patient with renal hypo-plasia and oligonephronia had normal glomerular an-tigens.

The expanded mesangium in amyloid nephropathy,positively stained by thioflavine T, was not stainedwith antiactomyosin which stained only narrow areas ofthe mesangium surrounding the areas of amyloid depo-sition.

FIGURE 6. Acute poststreptococcal glomerulonephritis. Im-munofluorescent staining with antiactomyosin is more ex-tensive than normal, though of lower intensity than that seenin diabetes mellitus. The staining is present throughout theglomerulus; its precise location is unknown. Increasedstaining in the interstitium is visible, without staining ofTBM. Magnification 700 X.

End-stage kidney. Nephrectomy specimens of 12end-stage kidneys showed large numbers of completelyhyalinized glomeruli, which failed to react with any ofour three antisera. Anticollagen showed an extensivestaining reaction in the interstitium, while hyalinizedglomeruli were negative. The anticollagen antiserumsupplied by Dr. Michaeli showed the same findings infour specimens examined by indirect immunofluores-cence. The antiactomyosin antiserum showed some in-creased reactivity in the expanded interstitium whileanti-GBM remained negative. In the interstitium ofthe child with renal hypoplasia with oligonephronia,a greatly increased distribution and intensity of stain-ing by antiactomyosin was noted, even more than thatto anticollagen antiserum.

DISCUSSION

In renal diseases with distinctive histopathologic char-acteristics there are alterations in antigenic componentswhich react with antibody to human GBM, acid-solubleskin collagen, and uterine smooth muscle actomyosin.Within the normal glomerulus, anti-GBM and anticol-lagen antibodies react with GBMwhereas antiactomy-osin localizes to the mesangium.

Immunohistopathology of Glomerular Antigens 1151

The most striking increase in actomyosin was ob-served in the nephropathy of diabetes mellitus wherethe distribution and quantity was correlated with ex-pansion of the mesangium characteristic of diabeticnephropathy. In contrast, the widened mesangial ma-trix observed in membranoproliferative glomerulone-phritis did not contain identifiable actomyosin or colla-gen antigens. Although large amounts of actomyosin inthe sclerotic mesangium of diabetic glomeruli couldreflect the presence of residual cellular debris, this find-ing was not observed in other diseases with glomerularsclerosis.

It is thus possible that the increased actomyosin-likematerial found in the mesangium in diabetes mellitusdoes not represent a functioning contractile system, butinstead a specific defect in the normal mechanisms forremoval of cellular debris from the mesangium; pre-liminary data suggests a defective clearance of ex-ogenously administered aggregated gamma globulinfrom the mesangium in alloxan diabetic rats (18). Ifthe increased actomyosin-like protein found in themesangium in diabetes mellitus represents a primaryphenomenon, its role in the pathogenesis of diabeticnephropathy may be related to diabetic vascular diseaseelsewhere. The early development of atherosclerosis indiabetes mellitus may be related to proliferation or al-teration in smooth muscle proteins. While hypertensionmay stimulate vascular smooth muscle proliferation(19), our two early cases of diabetes mellitus had noevidence of hypertension, but definite increase in mesan-gial actomyosin. Defects in leukocyte function in dia-betes mellitus (20) may also be related to alterations insmooth muscle proteins. Contractile protein is promi-nent in leukocytes (21) and apparently responsible fortheir motility (22) which is altered in the diabeticstate (20).

In other disease states, such as proliferative glo-merulonephritis and many cases of membranous ne-phropathy, actomyosin is mildly increased in distribu-tion by immunofluorescence; while this may be relatedto expansion of mesangial components (23), the ap-pearance of actomyosin in epithelial cells is equallylikely and would correspond to the increase in smoothmuscle-like structures in epithelial cells seen by DeMartino, Accinni, and Procicchiani (24) in prolifera-tive glomerulonephritis. However, the immunofluores-cent alterations seen in disease states may not repre-sent the same determinants of actomyosin responsiblefor the normal mesangial staining.

The presence of actomyosin antigen(s) in the glo-merular mesangium suggests but does not prove thatfunctioning contractile elements are present in thislocus. Ultrastructural localization of thin filaments(24), presumably actin (25), in the glomerulus does

not show the mesangial preponderance that we demon-strated by immunofluorescence. Smooth muscle myosin,considered by some (3) to be the predominant antigenicdeterminant of actomyosin, is poorly demonstrated byelectron microscopy.

The presence of contractile proteins in the mesan-gium may play an important role in regulating glo-merular blood flow and ultrafiltration (26). This isparticularly relevant because of the contiguity of themesangium with smooth muscle cells in afferent arteri-oles and the juxtaglomerular apparatus (27).

The absence of significant glomerular fluorescencewith antisera to GBMand collagen in membranopro-liferative glomerulonephritis suggests that these anti-gens may be altered or lost. The basement membrane-like material seen by electron microscopy in the mesan-gium in membranoproliferative glomerulonephritis maybe (antigenically) abnormal GBM, and its synthesis bymesangial cells may be related to the pathogenesis ofthe disease. The apparent splitting of the GBM ob-served by silver methenamine stain in membranopro-liferative glomerulonephritis was not seen in our studieswith anti-GBM and anticollagen sera. That splittingseen with silver methenamine stain may be related tocomponents of the glomerular capillary wall other thanGBM. In contrast, however, a laminated structure ofthe thickened GBMwas seen in diabetes mellitus andmembranous nephropathy, with the inner layer moreintensely stained by anti-GBM antiserum; it was alsofound with the eluted Goodpasture's anti-GBM anti-body. The relationship between this finding and theultrastructurally observed laminae rara and densa of theGBMis unknown.

Localization of heterologous anti-GBM serum to glo-merular and tubular basement membranes, Bowman'scapsule, and capillary basement membranes is well es-tablished. The more restricted pattern of staining seenwith Goodpasture's antibody, staining mainly GBM(1), suggests a more specific reactivity with GBMantigens. Anticollagen antibody localized to GBM asshown by Rothbard and Watson (2), but is clearlydifferentiated from anti-GBM antibody by staining ofthe interstitium and virtual absence of TBM staining.Anti-GBM and anticollagen antibody staining of theGBMwere abolished by collagenase digestion of tissuesbefore staining. These data suggest that our anti-GBMand anticollagen antibodies both may react with colla-gen-related proteins. However, the results of absorptionand tissue fixation studies clearly differentiate thesetwo antisera. Previous studies have emphasized thenumberous antibodies elicited by collagen (14), GBM(28), and other basement membranes (29). The im-munochemical relationship between different collagen-

1152 J. I. Scheinman, A. J. Fish, and A. F. Michael

like proteins and fractions of their molecules awaitsfurther clarification.

Our studies confirm the presence of large amounts ofcollagen in renal tissue with interstitial fibrosis. How-ever, we could not demonstrate collagen in hyalinizedglomeruli with our antiserum, nor with that of Dr.Michaeli. Similarly, we found almost no localization ofanticollagen or anti-GBM antibody to fibrosing loculesof glomeruli with focal sclerosis, little in fibroepithelialcrescents, and none in the expanded mesangial areasof diabetic nephropathy and membranoproliferative glo-merulonephritis. These areas may contain collagenousmaterial, but its physiochemical state may have beenaltered and antigenic determinants not available forimmunofluorescent reactions. Although the cells offibroepithelial crescents have been considered "myo-fibroblasts" (30), we found no evidence for the pres-ence of actomyosin. Although interstitial localizationof actomyosin is minimal in the normal kidney, it isclearly present in specimens from patients with renalinsufficiency and interstitial fibrosis. A similar ob-servation was made by Nagle, Kneiser, Bulger, andBenditt (31) in obstructive uropathy in the rabbitusing a human smooth muscle autoantibody (32).

ACKNOWLEDGMENTSThe technical assistance of Mrs. Lore Lang and Mrs. Car-lene Tompkins in sectioning and processing tissue for im-munofluorescent microscopy, Mr. Vincent Berg and Ms.Susan Sisson for photographic illustrations, and Mrs. ElinorBeaton for secretarial assistance are gratefully acknow-ledged. We especially wish to thank Dr. John Najarianfor supplying tissue from renal transplant and nephrectomyspecimens, Dr. Zoltan Posalaky for performing light andelectron microscopy on the autopsy tissue of a diabeticpatient, Ms. Juille Johnson for performing acrylamideelectrophoresis, Dr. Dov Michaeli for supplying his anti-collagen antiserum, and Dr. Carl Becker for supplying asample of antiserum to human uterine actomyosin and forhis advice in isolating the protein. Dr. Inga Platou's artisticillustration (after the design by Kazimierczak, by per-mission) of the antigen localization in the promerulus isgratefully acknowledged.

This work was supported by grants from the U. S.Public Health Service (HL 06314 and AJ-10704-14).

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1154 J. 1. Scheinman, A. J. Fish, and A. F. Michael