American Journal of Patholog, Vol. 139, No. 6, December 1991 Copyright © American Association of Pathologists

Differential Expression of Basement Membrane Collagen in Membranous Nephropathy Y. Kim,*t R. Butkowski,*t B. Burke,*t M. M. Kleppel,*t J. Crosson,t A. Katz,* and A. F. Michael*t From the Departments of Pediatrics,* and Laboratory Medicine and Patbology, University of Minnesota Medical School, Minneapolis, Minnesota

Membranous nephropathy (MN) is characterized by subepithelial immune complex formation and progressive thickening of the glomerular basement membrane (GBM). Kidney tissues from 21 patients stratified according to morphology (stage I 5 patients; stage 1I 5 patients, stage 112 1 patients) were studied by immunohistochemical techniques using antibody probes to matrix components of recently described (novel) chains of type IV collagen [a3(IV)NC, a4(IV)NC, Alport antigen] and of traditional type IV collagen [alJ(IV)NC, ca2(IV)NC, 7S(WV), triple helix]; as well as laminin B2, nidogen and fibronectin. In Stage I, there were no detectable changes when compared with normal tissue. In Stage II and early Stage III, the subepithelial projections of GBM (spikes) and the thickened GBM consisted predominantly of the novel type IV collagen chains as well as laminin B2 and nidogen, with no detectable changes in traditional type IV collagen. In late Stage III, an increase in the latter was observed in the subendothelial region of the thickened GBM with narrowing of the capillary lumen. At this stage, there was close apposition of novel and traditional type IV collagen molecules. The expression of these two groups of molecules is spatially and temporally distinct during the evolution of MN It is hypothesized that immune complex formation in the subepithelial region of the GBM leads to increasedformation of the novel type IV collagen network by visceral epithelial cells resulting in the formation of spikes and thickening of GBM between and surrounding immune deposits. These changes precede and are distinct from detectable alterations in traditional type IV collagen With progression and time, the deposits become embedded in the novel collagen network and increased

subendothelial formation of traditional type iV collagen molecules occurs with narrowing of the capillary lumen. (Am J Pathol 1991, 139:1381-1388)

Membranous nephropathy (MN), a form of chronic glomerulonephritis, is characterized by the subepithelial formation of immune complexes with progressive thickening of glomerular basement membrane (GBM). The newly formed matrix has been reported to contain laminin, type IV collagen and fibronectin.15 Traditional type IV collagen, a principal component of all basement membrane, exists as a heterotrimer containing two chains of al(IV) and one chain of a2(IV). The carboxy terminal end of this molecule is composed of the noncollagenous (NC) propeptide portions of the three alpha chains. Additional collagen components that are related to type IV collagen have been identified in GBM. Upon collagenase digestion of human GBM, four NC monomers (M26, M24, M28+, M28+ + +) are released that may be separated by differences in molecular weight in SDS-PAGE and mobility in nonequilibrium isoelectrofocusing.6'7 Amino-acid sequencing has shown that M26 and M24 and their bovine equivalents are derived from al (IV) and a2(IV) chains, respectively."10 Although the precise relationship of M28. + + and M28 + peptides to a 1 (IV) and a 2(IV) has not been defined, these components have been designated as NC peptides of additional chains of type IV collagen, a3(IV) and a4(IV), re-

spectively.8'1Ol4 These novel collagens are known to play an important role in human disease. The NC domain of the a3(IV) chain is the principal target antigen of Goodpasture autoantibodies.78 By immunofluorescence of normal human kidney, the NC domains of the a3(IV) and a4(IV) chains codistribute with a recently described immunochemically distinct 26 kd NC peptide, the Alport antiSupported in part by a grant from the National Institutes of Health (Al10704) and the Viking Children's Fund. Accepted for publication August 5, 1991. Address reprint requests to Dr. Youngki Kim, Department of Pediatrics, Box 491, University of Minnesota, Minneapolis, MN 55455.

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gen.1-'9 In contrast to traditional type IV collagen, these chains are not detected in the GBM of Alport kidney but persist in the thickened GBM in diabetic nephropathy.15'17'19 Further, there is a concordant expression of these components during glomerular ontogeny.20 These findings suggest a close association among the three parent chains of these molecules. Recently, Trygvassen and associates21"24 reported the presence of a5(IV), an additional new chain of type IV collagen with a genomic location on the Xq22 locus of the X chromosome, a region in close association with the Alport's locus. Antiserum developed to a nonconsensus peptide synthesized from the derived amino-acid sequence reacted with human GBM. The relationship of this molecule to the Alport antigen described in our laboratory has not been determined.15 In the present study, we demonstrate that the expression of novel and traditional type IV collagen is spatially and temporally distinct during the evolution of human MN.

Materials and Methods

surrounding electron dense deposits; and stage IIIB-6 patients with encircling translucent zones. All patients with stage IIIA MN also had mixed lesions of stage 11. Normal human kidneys were obtained as previously zones

described.19

Antibodies The polyclonal and monoclonal antibodies used in this study are listed in Table 1. Monoclonal antibodies against human fibronectin32 and human nidogen33 were produced in our laboratory, and monoclonal antibody against laminin B2 chain were obtained from Hybridoma Bank.31 FITC-conjugated goat anti-rat IgG and FITCconjugated rabbit anti-sheep IgG were obtained from Pel-Freeze (Rogers, AR); FITC-conjugated rabbit antigoat IgG and FITC-conjugated goat anti-human IgG from Kallestedt (Austin, TX); rhodamine isothiocyanate (RITC)conjugated goat anti-rat IgG from Accurate (San Diego, CA); FITC-conjugated and RITC-conjugated goat antimouse IgG from Caltag (South San Francisco, CA). All secondary antibodies were absorbed with normal human serum.

Kidney tissue from 21 patients with MN were obtained at the time of renal biopsy. Histologic studies by light, immunofluorescence and electron microscopy demonstrated typical changes of MN as previously described.25 26 Seventeen patients had idiopathic MN, two had systemic lupus erythematosus, and one each had penicillamine and gold-induced MN. Using the classification based on the ultrastructural findings described by Ehrenreich and Churg,25 these patients were divided into three groups: stage 1 (5 patients), stage 11 (5 patients) and stage III (11 patients). The latter group was further divided into stage IIIA-five patients without translucent

Immunohistologic Methods Immunofluorescence studies were carried out as previously described.19 Kidney tissues were snapfrozen in isopentane precooled in liquid nitrogen, sectioned at 4 ,im in a Lipshaw cryostat in a constant temperature (250C) and humidity (30%) room, and fixed for 10 minutes with acetone (for nondenatured sections) or ethanol (for sections to be denatured). The slides were stained with primary antibodies followed by FITC-conjugated second-

Table 1. Specificity and Source of Antibodies Antibodies Specificity

Monoclonal antibodies MAb 17 MAb A2 MAb 85 MAb A7 MAb 102 MAb IV-1 MAb M3F7 MAb 2E8 MAb BM 20 MAb A9 Polyclonal antibodies Sheep anti-M24 Goat anti-type IV

oL3(IV) NC c3(IV) NC a4(IV) NC Alport antigen* al(IV) NC 7S(IV) domain Triple helix (IV) Laminin B2 Fibronectin Nidogen

a2(IV) NC Pepsin-digested

References 16, 18 20 16, 27 28 18 29

Hybridoma Bank (30) Hybridoma Bank (31) 32 33

18

Southern Biotechnology

placental BM The Alport antigen is defined by a genetically discriminating alloantibody and monoclonal antibody reactive with a M26 monomer.15'17,28 Whether this is the same as a5(IV) NC1 or an homologous chain has not been determined.

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ary antibodies. Dual fluorochrome labelling was performed using mouse monoclonal anti-a3(IV) NC detected by rhodamine-conjugated goat anti-mouse IgG followed by rat monoclonal anti-al(IV) NC and FITCconjugated goat anti-rat IgG absorbed with human and mouse sera. In addition, tissues were also stained with FITC-conjugated goat anti-human IgG followed by monoclonal antibodies against laminin, 7S(IV), al (IV) NC or a3(IV) NC-each detected with appropriate rhodamineconjugated secondary antibodies absorbed with mouse or rat and human serum. Appropriate controls were used as previously described.19 To retard fluorescence quenching, p-phenylenediamine in PBS glycerol was applied to fluorochrome-stained tissue sections. Ethanolfixed tissue sections were denatured by incubation in 0.1 mol/1 glycine, 6 mol/1 urea, pH 3.5 for 1 hour at 40C as previously described.' Sections were examined using epiflourescence microscope with appropriate filters (Carl Zeiss, Inc., Oberkochen, FRG).

Results The distribution of matrix components in stage MN was similar to that observed in normal human kidney (Figure 1 A, 1 B). Although monoclonal and polyclonal antibodies against various components of traditional type IV collagen [i.e., al (IV)NC, a2(IV)NC, 7S, triple helix] stained the mesangium and the endothelial aspect of GBM, monoclonal antibodies against the recently described novel chains [a3(IV) NC, a4(IV) NC and Alport antigen] reacted with the phase-dense region of GBM but not with the mesangium. In stage 11, the subepithelial projections of GBM (spikes) stained with antibodies to the novel chains, but not with antibodies to the various components of traditional type IV collagen. In stage IIIA, the novel chains were present throughout the phase-dense region of the thickened GBM and in spikes (Figure 1 C, 1 D and Figure 2). Lacunar structures were also observed along the epithelial aspect of GBM, especially in tangential sections, representing negatively stained deposits of immunoglobulin surrounded by novel chain components. In contrast, the distribution of traditional type IV collagen was normal and was not detected in subepithelial GBM projections. Similar findings were observed in stage IIIB except no spikes were observed and the thickened GBM containing novel chains appeared to totally surround the deposits. At this stage, there was an increase in the thickness and intensity of traditional type IV collagen in the subendothelial region of the GBM in close apposition to the novel chains with narrowing of the capillary lumen (Figure 1E, 1F). In all stages of MN, the distribution of nidogen (utilizing monoclonal and polyclonal antibodies) and laminin

B2 was similar to that of the novel chains whereas the distribution of fibronectin mimicked that of traditional type IV collagen (Figure 2). Dual-label studies on the same tissue section demonstrated that the novel chains were present in spikes and lacunae surrounding the epimembranous deposits containing IgG (Figure 3). Immune deposits did not react with antibody probes to the various matrix components. Neither acid-urea denaturation of the tissue nor reversal of the staining order in dual-label studies caused any appreciable change in the staining pattern for any of the antibodies.

Discussion The histological hallmark of MN is thickening of GBM with immune-complex formation in the subepithelial region of the capillary wall. Traditionally it has been held that subepithelial immune complexes stimulate visceral epithelial cells to produce matrix with progressive thickening of the capillary wall, although changes in degradation also could play a role in this process. Previous studies have suggested that the newly synthesized GBM contains type IV collagen, fibronectin, and laminin, although the latter appeared to be the predominent component.15 In a murine autoimmune model of MN, type IV collagen was not detected in GBM spikes, whereas abundant laminin was present.35 In the present study, we have shown definitively that the various chains of type IV collagen are differentially expressed during the evolution of progressive MN. The newly described chains-a3(IV)NC, a4(IV)NC, and Alport antigen (as well as nidogen and laminin B2)-are principal constituents of subepithelial projections or spikes that separate and later encircle immunoglobulin deposits. In contrast, the traditional chains of type IV collagen [al (IV) and a2(IV)] are not present in the spikelike projections, are increased late in the course of the disease in the subendothelial zone of the GBM, and contribute to narrowing of the capillary lumen. Thus the changes in the two groups of collagen molecules are spatially and temporally distinct (Figure 4). Although not proven by this study, these results suggest a sequence in the evolution of MN that is based largely on the presumption of differing sites of synthesis of these two groups of collagen molecules. It is likely that immune complex formation in the subepithelial region of the GBM leads to production of novel type IV collagen chains (and nidogen/laminin B2) by visceral epithelial cells in an attempt to enclose the immune reactants. Traditional type IV collagen does not appear to play a role in this initial process. With progression, the deposits become embedded in the novel type IV collagen network and there is relative movement to-

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Figure 1. Immunofluorescence of glomeruli in patients with different stages of MN. The same section in each pair was stained sequentially with anti-a 3(1V)NC (rhodamine) (A, C, E) and anti-a 1(IV)NC (FITC) (B, D, F). A & B: Stage I MN; A: Anti-a 3(1V)NC stained the phase-dense region of GBM in a linear pattern without mesangial reactivity- No spikes are detected at this early stage. B: In contrast, anti-a 1(IV)NC reacted with the endothelial aspects of GBM and mesangium as in normal kidney. C & D: Stage IIIA MN; C: Anti-a 3(IV)NC reacted with the entire phase-dense region of thickened GBM. Numerous spikes ( 4 ) and lacunae ( I4) along the epithelial aspect of the GBM are seen. D: Anti-a 1(1V)NC reactivity is similar to that of stage I MN. No spikes or lacunae are observed. Note the absence of overlap in the distribution of a 3(IV)NC and a 1(IV)NC. E & F: Stage lIlB MN; E: Anti-a 3(IV)NC stained the thickened GBM in a pattern similar to that of stage IIIA exceptfor an absence ofspikes. F: In contrast, there is increased staining of the thickened GBM with anti-a 1(IV)NC. This reactivity is internal to that of a 3(IV)NC resulting in narrowing of the glomerular capillary lumen ( i ). However, there also are regions of overlapping reactivity observed with the two antibodies. The staining pattern for anti-a 4(IV)NC and anti-Alport antigen were similar to that observed for anti-a 3(IV)NC In addition, immunofluorescence using anti-a 2(IV)NC, anti-7S(1V), anti-triple helix (IV), anti-type IVcollagen, and anti-fibronectin were similar to those observed with anti-a 1(IV)NC (A & B, x 610, C-F, x 500).

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I

I

Figure 2. Immunofluorescence of kidney from a patient with stage IIIA MN Photomicrographs in the left panel depict tissue stained with anti-a4(IV)NC (A), anti-Alport antigen (B), anti-nidogen (C) and anti-laminin B2 (D). All show fluorescence of the thickened GBM with lacunae and spikes. In the right panel, tissue stained with anti-a2(IV)NC (E), anti-7S(IV) (F), anti-triple helix(IV) (G) and anti-fibronectin (H) show linearfluorescence of the endothelial aspect of the GBM and mesangial staining similar to the pattern observed in normal kidney. No spikes or lacunae are present, x550.

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Figure 3. Immunofluorescence of kidney from a patient with stage IIIA MN. A & B: The same section was stained sequentially with anti-human IgG (FITC) (A) and anti-ac3(IV)NC (rhodamine) (B). C & D: The same section was stained sequentially with anti-human IgG (FITC) (C) and anti-otl(IV)NC (rhodamine) (D). Note that a3(IV)NC is present in spikes and lacunae adjacent to and surrounding IgG deposits. In contrast, the al(IV)NC is located along the endothelial aspect of the GBM internal to the IgG deposits. Note also the absence of reactivity of IgG deposits with anti-al(IV)NC or anti-a3(1V)NC, X480.

wards the endothelium. At this stage there is an increase in traditional type IV collagen from endothelial and mesangial cells, possibly stimulated by juxtaposition of immune complexes to the subendothelial and paramesan-

gial regions. These observations are in concert with scanning electron microscopic studies of acellular GBM in MN.' In early disease, craterlike deformities on the epithelial aspect of the GBM are observed with no changes on its endothelial surface. In late MN, the epithelial surface contains irregular plaques but the endothelial surface had multiple perforations, probably reflecting extension of intramembranous immune complexes into the subendothelial space. It has been shown previously that the duplication of GBM seen in various forms of glomerulonephritis reflects newly synthesized matrix from both endothelial and mesangial cells.37 In the present study, projections of cytoplasm from endothelial and mesangial cells were observed within the split GBM of patients with IIIB MN supporting the notion that these cells are respon-

sible for the increased synthesis of traditional type IV collagen which contribute to thickening of the GBM. We have shown previously unique differences in the distribution of these chains in human renal disease. In Alport's syndrome, novel chains of type IV collagen are not detected in the GBM of affected males by analysis of tissue sections or Western blots of solubilized components, whereas the traditional chains of type IV collagen are present.15'17 Further, in diabetic nephropathy, the novel chains are a principal constituent of the thickened GBM, whereas the traditional chains are prominent within the expanded mesangium; however, with progression to sclerosis, hyalinized glomeruli contain only novel chains.19 The contributions of synthesis and degradation of matrix components in MN are unknown. Recently, Fogel et al.38 reported no increase in mRNA levels for laminin B2 as well as a1 (IV) collagen and fibronectin in whole glomeruli at various stages of passive Heymann nephritis. However, immune deposits were surrounded with lami-

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Figure 4.

B

A.

Schematic drawing of changes

in

GBM in different stages ofMN (modifiedfrom reference 26). E = endothelial cells; M = mesangial matrix, CL = capillary lumen; a3(IV), cL4(IV) and Alport antigen are depicted in red; and a2(IV) in blue, IgG deposits in black. A: In stage I, no changes in GBM is appreciated. B: In stage II, spikes consist of at3(IV), a.4(JV) and Alport antigen. C: In stage . _ deposits are embedded in the ;9W immune IIIA newly synthesized GBM which contains a3(IV) and oW4IV), and Alport antigen. D: In stage IIIB, immune deposits are totally embedded in the thickened GBM. In addition,

:a(IV)

CL

.

t

t C

ji _

ctJ

ff

nin B2 by immunoelectron microscopy. The absence of changes in mRNA for laminin B2 in the experimental model may be related to the slow process of spike formation and the insensitivity of mRNA measurements in whole glomeruli. Although the membrane attack complex of complement (C5b-9) increases collagen synthesis by glomerular visceral epithelial cells in vitro39 and C5b-9 is present in the immune deposits of early and late MN,40 it is unclear at present whether the complement system plays a determinant role in matrix metabolism in the human disease.

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Acknowledgments The authors thank Kathy Divine and Crystal Blocher for immunohistochemical staining; Marshall Hoff and David Hurd for photography; and Mary Jo Jansen for secretarial support.

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endothelial region due to increase in al(IV) and a2(IV) with extension of these compoWnents between deposits.

progression. Springer Semin Immunopathol 1982, 5:297320 Fukatsu A, Matsuo S, Killen PD, Martin GR, Andres GA, Brentjens JR: The glomerular distribution of type IV collagen and laminin in human membranous glomerulonephritis. Hum Pathol 1988,19:64-68 Butkowski RJ, Wieslander J, Wisdom BJ, Barr JF, Noelken ME, Hudson BG: Properties of the globular domain of type IV collagen and its relationship to the Goodpasture antigen. J Biol Chem 1985, 260:3739-3747 Kleppel MM, Michael AF, Fish AJ: Antibody specificity of human glomerular basement membrane type IV collagen NC1 subunits. J Biol Chem 1986, 261:16547-16552 Butkowski RJ, Langeveld JPM, Wieslander J, Hamilton J, Hudson BG: Localization of the Goodpasture epitope to a novel chain of basement membrane collagen. J Biol Chem 1987, 262:7874-7877 Langeveld JPM, Wieslander J, Timoneda J, McKinney P, Butkowski RJ, Wisdom BJ, Hudson BG: Structural heterogeneity of the non-collagenous domain of basement membrane collagen. J Biol Chem 1988, 263:10481-10488 Butkowski RJ, Shen G, Wieslander J, Michael AF, Fish AJ: Characterization of type IV collagen NC1 monomers and Goodpasture antigen in human renal basement membranes. J Lab Clin Med 1990,115:365-373 Saus J, Wieslander J, Langeveld JPM, Quinones S, Hudson BG: Identification of the Goodpasture antigen as the a 3(IV) chain of collagen IV. J Biol Chem 1988, 263:13374-13380 Hudson BG, Wieslander J, Wisdom BJ, Noelken ME: Biology of disease. Goodpasture syndrome: molecular architecture and function of basement membrane antigen. Lab Invest 1989, 61:256-269 Gunwar S, Saus J, Noelken ME, Hudson BG: Glomerular basement membrane. Identification of a fourth chain, a 4, of type IV collagen. J Biol Chem 1990, 265:5466-5469 Fagg WR, Timoneda J, Schwartz CE, Langeveld JPM, Noelken ME, Hudson BG: Glomerular basement membrane: evidence for collagenous domain of the a 3 and a 4 chains

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AF: Alport familial nephritis. Absence of 28 kilodalton noncollagenous monomers of type IV collagen in glomerular basement membrane. J Clin Invest 1987, 80:263-266 28. Kleppel MM, Fan WW, Kashtan CE, Michael AF: Isolation and characterization of the Alport familial nephritis antigen. Kidney Int 1990, 37:441 (abstract) 29. Scheinman JI, Tsai C: Monoclonal antibody to type IV collagen with selective basement membrane localization. Lab Invest 1984, 50:101-112 30. Foellmer HG, Madri JA, Furthmayr H: Monoclonal antibodies to type IV collagen: Probes for the study of structure and function of basement membranes. Lab Invest 1983,

48:639-649 31. Engvall E, Davis GE, Dickerson K, Ruoslahti E, Varon S, Manthorpe M: Mapping of domains in human laminin using monoclonal antibodies: Localization of the neuritepromoting site. J Cell Biol 1986, 103:2457-2465 32. Michael AF, Yang, J-Y, Falk RJ, Bennington MJ, Scheinman JI, Vemier RL, Fish AJ: Monoclonal antibodies to human renal basement membranes: Heterogenic and ontogenic changes. Kidney Int 1983, 24:74-86 33. Katz A, Fish AJ, Kleppel MM, Hagen S, Michael AF, Butkowski RJ: Renal entactin (nidogen): Isolation, characterization, and tissue distribution. Kidney Int 1991, 40:643-652 34. Yoshioka K, Michael AF, Velosa J, Fish AJ: Detection of hidden nephritogenic antigen determinants in human renal and non-renal basement membranes. Am J Pathol 1985, 121:156-165 35. Matsuo S, Brentjens JR, Andres G, Foidart J-M, Martin GR, Martinez-Hernandez A: Distribution of basement membrane antigens in glomeruli of mice with autoimmune glomerulonephritis. Am J Pathol 1986,122:36-49 36. Weidner N, Lorentz WB: Scanning electron microscopy of the acellular glomerular basement membranes in idiopathic membranous glomerulopathy. Lab Invest 1986, 54:84-92 37. Katz SM: Reduplication of the glomerular basement membrane. Arch Pathol Lab Med 1981, 105:67-70 38. Fogel MA, Boyd CD, Leardkamolkam V, Abrahamson DR, Minto AWM, Salant DJ: Glomerular basement membrane expansion in passive Heymann nephritis. Absence of increased synthesis of type IV collagen, laminin, or fibronectin. Am J Pathol 1991, 138:465-475 39. Torbohm I, Schonermark M, Wingen A-M, Berger B, Rother K, Hansch GM: C5b-8 and C5b-9 modulate the collagen release of human glomerular epithelial cells. Kidney Int 1990, 37:1098-1104 40. Falk RJ, Dalmasso AP, Kim Y, Tsai CH, Scheinman JI, Gewurz H, Michael AF: Neoantigen of the polymerized ninth component of complement. Characterization of a monoclonal antibody and immunohistochemical localization in renal disease. J Clin Invest 1983, 72:560-573