Abnormal expression of glomerular basement membrane laminins in membranous glomerulonephritis
Evelyne Fischer1,2,3,
Béatrice Mougenot1,4,
Patrice Callard4,
Pierre Ronco1,2 and
Jérôme Rossert,1,2
1 INSERM U489, and Departments of
2 Nephrology and
4 Pathology, Paris VI University, Assistance Publique-Hôpitaux de Paris, Paris, France,
3 Present address: Department of Nephrology, University hospital of Strasbourg, Strasbourg, France
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Abstract
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Background. Proteinuria associated with glomerular diseases is secondary to alterations of the charge-selective and/or size-selective properties of the glomerular basement membrane (GBM), but molecular alterations that are responsible for these functional changes are still poorly understood. Analysis of mice harbouring a null mutation in the gene encoding the ß2 chain of laminin has suggested that the presence of abnormal laminin chains within the GBM can be responsible for proteinuria.
Methods. We have investigated whether abnormal laminin ß chains could be detected by immunohistochemistry within the GBM of patients with proteinuria and minimal change disease (five patients), focal and segmental glomerulosclerosis (five patients), or primary membranous glomerulonephritis (10 patients). Three patients with mesangiocapillary glomerulonephritis and three patients with IgA nephropathy were also studied as controls.
Results. We showed that the GBM of all 10 patients with membranous glomerulonephritis, but not of patients with other glomerulopathies, contained laminin ß1, which is normally expressed only during metanephros development. The re-expression of the ß1 chain of laminin was not associated with that of the embryonic
1 chain of type IV collagen, or with the loss of expression of vimentin and synaptopodin, two markers of differentiated podocytes.
Conclusions. The presence of new laminin isoforms within the GBM of patients with membranous glomerulonephritis could play a role in the occurrence of proteinuria, by modifying either the sieving properties of the GBM or the interactions between podocytes and the GBM.
Keywords: glomerular basement membrane; laminin; membranous glomerulonephritis
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Introduction
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The glomerular basement membrane (GBM), which has characteristic charge- and size-selective properties, also has a unique composition since the isoforms of its major components, i.e. type IV collagen, laminin and heparan sulphate proteoglycans, are different from the ones present in most other basement membranes [reviewed in refs 1,2]. Type IV collagen molecules are formed from three
(IV) chains coiled around each other in a triple helix, except at their N-terminal ends which have a globular structure [reviewed in ref. 3]. Six genetic variants of the
(IV) chains have been identified, and named
1(IV) to
6(IV) [3]. While most type IV collagen molecules contain two
1(IV) chains and one
2(IV) chain, those present within the GBM contain
3(IV),
4(IV) and
5(IV) chains, but few
1(IV) and
2(IV) chains [reviewed in ref. 3]. Laminin molecules are formed by the assembly of one
, one ß and one
chain, the C-terminal ends of these three chains being coiled in a triple helix, while their N-terminal ends form extending arms of different lengths. Different genetic variants of each of these three chains have been identified, and currently five
, three ß and two
chains are known, which can associate according to different combinations and give rise to 11 laminin heterotrimers [reviewed in ref. 4]. While most basement membranes contain the laminin ß1 chain, the GBM is rich in laminin ß2 chain, and laminin 11 (
5ß2
1) seems to be a major laminin of the mature GBM [4]. Heparan sulphate proteoglycans contribute to the negative charge of the GBM through their glycosaminoglycan side chains. Their precise nature is still unknown, but agrin seems to be a major proteoglycan of the GBM while perlecan is an abundant component of most other basement membranes [5].
In vertebrates, the development of the metanephros is associated with major modifications in the composition of the GBM [reviewed in ref. 6]. During the earliest stages of glomerulogenesis, the GBM contains fibronectin, type IV collagen molecules formed from
1(IV) and
2(IV) chains, and laminin molecules including the laminin
1 chain or
4 chain, the laminin ß1 chain and the laminin
1 chain. At the S-shape body stage, molecules containing the laminin
5 chain start to accumulate, while those including either the laminin
1 chain or the laminin
4 chain progressively disappear. At the capillary loop stage, molecules containing the laminin ß1 chain are progressively replaced by ones containing the laminin ß2 chain. At or after the capillary loop stage, molecules including the
3(IV),
4(IV) and
5(IV) collagen chains appear and progressively become the very prominent collagenous components of the GBM. Parallel to these changes, fibronectin progressively disappears from the GBM.
Proteinuria associated with glomerular diseases is secondary to alterations of the charge-selective and/or size-selective properties of the GBM, but molecular modifications that are responsible for these functional changes are still poorly understood [7]. Animal studies have suggested that the sieving properties of the GBM depend on its precise composition in laminin isoforms. When a null mutation is introduced in the gene coding for the laminin ß2 chain, knockout mice develop heavy proteinuria immediately after birth [8]. One week after birth, the GBM of these animals appears to be structurally normal, as judged by light and electron microscopy, and the only modifications that have been detected at this time are the presence of laminin molecules containing the ß1 chain [8]. These experimental results prompted us to study whether primary glomerulonephritides responsible for heavy proteinuria were associated with modifications of laminin isoforms present within the GBM.
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Subjects and Methods
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Patients and renal biopsies
Immunohistochemical studies of kidney biopsies were performed in 20 patients with heavy proteinuria who underwent a renal biopsy within the last 3 years, in addition to routine diagnostic examination (Table 1
). Ten patients had membranous glomerulonephritis (MGN), five had focal and segmental glomerulosclerosis (FGS), and five had minimal change disease (MCD). All patients had proteinuria of at least 2.0 g/day (mean±SD: 4.7±2.7 g/day; range: 211 g/day), associated with serum albumin of <30 g/l. Serum creatinine was in the normal range (mean±SD: 76±16 µmol/l; range: 50100 µmol/l), except for one patient with MGN (serum creatinine: 150 µmol/l) and three patients with FSG (serum creatinine: 140, 160 and 180 µmol/l, respectively). All glomerulopathies were considered as idiopathic, and in particular no patient had anti-nuclear antibodies, HIV infection, viral hepatitis or was taking a drug known to induce MGN. No patient had received immunosuppressive drugs before renal biopsy, except two patients with MCD who had been treated with corticosteroids for 1 and 3 months, respectively, at the time of biopsy.
The expression of the laminin ß1 and ß2 chains was also studied in kidney biopsies from three patients with mesangiocapillary glomerulonephritis (MCGN), and from three patients with IgA nephropathy (IgAN).
Non-tumoral portions of kidneys obtained from two patients undergoing a nephrectomy for renal cell carcinoma were used as controls. In both cases, no glomerular lesion could be detected by light microscopy or by immunofluorescence.
Antibodies
Monoclonal antibodies recognizing the NC1 domain of the
1(IV) collagen chain (MAB1), of the
3(IV) collagen chain (MAB3) and of the
5(IV) collagen chain (MAB5), respectively, were obtained from Wieslab (Lund, Sweden). A monoclonal antibody directed against the laminin ß2 chain (C4) was obtained from Developmental Studies Hybridoma Bank (Iowa City, IA). A monoclonal antibody against the laminin ß1 chain (4E10) was purchased from GIBCO Life Technologies (Rockville, MD). A monoclonal anti-fibronectin antibody was obtained from Biohit (Bonnelles, France). A monoclonal anti-vimentin antibody (VIM-13.2) was obtained from Sigma (St Louis, MO). A monoclonal antibody directed against synaptopodin (G1D4) was obtained from Research Diagnostics Inc. (Flanders, NJ). Finally, an affinity purified fluorescein isothiocyanate (FITC) conjugated goat antibody specific for mouse IgG was purchased from Biosys (Karben, Germany).
Immunohistochemistry
Cryostat serial sections (2.5 µm) were air-dried, fixed in acetone for 3 min, washed once in phosphate-buffered saline (PBS) pH 7.4, and incubated in a moist chamber with the appropriate dilution of primary antibodies (MAB1 1 : 50, MAB3 1 : 50, MAB5 1 : 50, 4E10 1 : 20, C4 1 : 100, VIM-13.2 1 : 150, G1D4 1 : 1). Before adding MAB5, sections were denatured by incubation for 5 min in a solution containing 0.1 M glycine and 6 M urea, and rinsed once with distilled water. After 2 h of incubation, slides were washed three times in PBS and incubated with the FITC secondary antibody for 30 min in a moist chamber. They were then rinsed three times in PBS and mounted using 0.5 M TrisHCl pH 8.0, 50% glycerol. Labelling was examined using a Zeiss Axiophot 2 microscope. In each case, a control staining was done by omitting the first antibody. For 4E10, staining of the GBM was scored semi-quantitatively, using a 0 to +++ scale, by an investigator unaware of the diagnosis.
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Results
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Patients with minimal change disease and control biopsies
An identical staining pattern was observed for all five patients with MCD (Figure 1
) and for the two control biopsies (data not shown), using the three anti-collagen IV antibodies and the two anti-laminin antibodies.
MAB1 strongly stained all renal basement membranes, with the exception of the GBM which was weakly stained (Figure 1A
), and it also stained the mesangium (Figure 1A
). MAB3 diffusely stained the GBM and the basement membrane of distal tubules, while it gave an occasional focal staining of the Bowmans' capsule (Figure 1B
). MAB5 stained the GBM, the Bowman's capsule, the basement membrane of distal tubules and collecting ducts (Figure 1C
). 4E10 stained all tubular basement membranes and the Bowman's capsule, but there was no staining of the GBM (Figure 1D
). In addition, it faintly stained the mesangium (Figure 1D
). C4 only stained the GBM and the basement membranes of arterial smooth muscles (Figure 1E
). In control biopsies, anti-fibronectin antibody showed the same staining pattern as MAB1 (data not shown).
Patients with focal and segmental glomerulosclerosis
In biopsies from all five patients with FGS, the staining pattern observed with MAB3, MAB5 and 4E10 was the same as the one observed in normal kidneys (Figure 2CE
). In glomeruli, MAB1 (Figure 2A
and B
) and C4 (Figure 2F
) stained the same structures as in normal kidneys but, in addition, MAB1 stained segmental areas corresponding to sclerotic lesions (Figure 2B
), and C4 stained segmental areas of the Bowman's capsule (Figure 2F
). In all three biopsies with interstitial fibrosis, some focal staining of interstitial fibrotic areas was also detected with MAB1 (Figure 2B
).
Patients with membranous glomerulopathy
All the structures that were stained with MAB1, MAB3 and MAB5 in control biopsies were also stained in biopsies from patients with MGN (Figure 3AD
). In addition, in all seven stage II MGN (patients 1, 3, 4, 5, 7, 9 and 10), a clear staining of spikes was detected using MAB3 (data not shown) and MAB5 (Figure 3C
). The staining pattern observed with C4 was exactly similar to the one observed in controls (Figure 3F
). Surprisingly, in glomeruli, 4E10 stained the mesangium but also segments of the external part of the GBM (Figure 3E
, G
and H
), and this staining of the GBM could be detected in all glomeruli for all 10 patients. The staining pattern was mostly granular (Figure 3G
and H
), but some segments of the GBM showed a linear labelling (Figure 3E
and G
). The percentage of capillary loops showing such a staining pattern was quite similar when different glomeruli from a given biopsy were compared, but it was highly variable between one patient and another (Table 2
). An identical staining pattern was observed when the reaction was performed in the presence of 5% normal human serum, confirming that 4E10 did not cross-react with human IgG (data not shown). No labelling was detected when 4E10 was omitted in the first incubation step (data not shown). No clear correlation could be established between the intensity of the staining and the levels of proteinuria (compare data from Table 1
with Table 2
). The anti-fibronectin antibody stained the mesangium, but a patchy staining of the GBM could also be detected in all four patients tested (data not shown).

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Fig. 3. Immunofluorescence of kidney sections from patients with MGN using an anti- 1(IV) collagen antibody (A), an anti- 3(IV) collagen antibody (B and D), an anti- 5(IV) collagen antibody (C), an anti-laminin ß1 chain antibody (E, G and H) and an anti-laminin ß2 chain antibody (F). (D), (E) and (F) are serial sections of the same glomerulus. (E), (G) and (H) are kidney sections from three different patients. (C) Spikes were labelled with anti- 5(IV) (arrows). (E, G and H) Segments of the GBM were stained with the anti-laminin ß1 antibody; the staining pattern was mostly granular, but also linear (arrows). The staining pattern was otherwise identical to the one observed in patients with MCD (compare with Figure 1 ). (A), (B), (D), (E) and (F) magnification, x250; (C) and (G) magnification, x500; (H) magnification, x780.
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Table 2. Semi-quantitative scoring of the immunostaining observed using the anti-laminin ß1 antibody (4E10), in patients with MGN
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The presence of embryonic isoforms of laminin within the GBM prompted us to study the expression of two proteins that are normally synthesized at or after the pre-capillary loop stage: vimentin and synaptopodin. Kidney biopsies from seven patients with MGN were stained using VIM-13.2 and G1D4. In all seven cases, G1D4 stained the podocytes, and VIM-13.2 stained the podocytes as well as the endothelial cells (Figure 4
).

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Fig. 4. Immunofluorescence of kidney sections from a patient with MGN using an anti-vimentin antibody (A), and an anti-synaptopodin antibody (B). Podocytes were labelled by both antibodies. Magnification, x250.
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Patients with mesangiocapillary glomerulonephritis and IgA glomerulopathy
To confirm that the GBM staining observed using 4E10 in biopsies from patients with MGN was not due to the presence of immune deposits along the GBM, kidney biopsies from three patients with MCGN were stained using 4E10 and C4. In all three cases, 4E10 stained the tubular basement membranes, the Bowman's capsule and the mesangium but it did not stain the GBM (Figure 5
). In contrast, the GBM was labelled by C4 (data not shown). Identical results were observed with kidney biopsies from three patients with IgAN (Figure 5
and data not shown).

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Fig. 5. Immunofluorescence of kidney sections from a patient with MCGN (A) and from a patient with IgAN (B) using an antibody to the ß1 chain of laminin. The GBM was not stained by this antibody. Magnification, x250.
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Discussion
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In this study, we used indirect immunofluorescence to compare the composition of the GBM in normal kidneys and in renal biopsies from patients with various primary glomerulonephritides associated with heavy proteinuria. Five MCD, five FSG and 10 MGN were studied.
Our findings concerning the distribution of the
1(IV),
3(IV) and
5(IV) collagen chains, the laminin ß1 chain, the laminin ß2 chain and fibronectin in normal glomeruli are in agreement with previous reports [911]. Our data confirm that the GBM contains large amounts of
3(IV) and
5(IV) collagen chains, and little
1(IV) collagen chain. They also confirm that laminin molecules present within the GBM contain the laminin ß2 chain, but not the laminin ß1 chain. Finally, they show that the GBM does not contain fibronectin.
In renal biopsies from patients with proteinuria, results obtained using antibodies recognizing different type IV collagen chains are consistent with earlier reports [1116]. Previous studies have shown that the collagen composition of newly formed matrix varies depending on the glomerulopathy, and that in MGN the spikes contain the
3(IV),
4(IV) and
5(IV) collagen chains [1114,16], while in FGS the matrix that accumulates between podocytes and the GBM is composed of
1/
2(IV) collagen chains [11,15]. In biopsies from patients with MGN or MCD incubated with the anti-
1(IV) antibody, staining of the GBM seemed to be restricted to the endothelial side, while in biopsies from patients with FGS the anti-
1(IV) collagen antibody stained the newly formed matrix. In patients with MGN, spikes were labelled with the anti-
3(IV) and anti-
5(IV) antibodies.
The unexpected finding of our study is that in patients with MGN, there were discrete modifications of the laminin molecules present within the GBM. In biopsies from all 10 patients with MGN, laminin molecules containing the ß1 chain could be detected along the external side of the GBM, in all glomeruli. These modifications of the GBM composition were specific of MGN, since they were not observed in biopsies from patients with MCD, FGS, MCGN or IgAN. In these latter cases, there was a strong staining of the tubular basement membranes and a staining of the mesangium with the anti-laminin ß1 chain antibody, but no staining of the GBM. Our patients with MGN tended to be older than the ones with FSG or MCD, but the presence of laminin ß1 within the GBM is most likely not age related, since it was observed in all patients with MGN, including two patients who were below 30 years of age, and three patients between 30 and 50 years-old. Several groups have shown that laminin is present in the newly formed matrix in patients with MGN [1114,16,17] and that there is an increased production of laminin in a rat model of MGN [18], but to the best of our knowledge, no previous study has focused on the expression of the different isoforms of the laminin ß chain during the course of MGN. Nevertheless, in an experimental model of lupus nephritis and in a model of glomerulonephritis associated with graft-versus-host disease, isoforms of laminin containing the ß1 chain were detected within the GBM [19,20]. It is of note that in both cases, the renal disease was responsible for heavy proteinuria [19,20].
At least two mechanisms can account for the presence of the laminin ß1 chain within the GBM of patients with MGN. One possibility is an abnormal regulation of the GBM assembly. In this case, there would be no modification of the production of the different laminin isoforms, but molecules containing the ß1 chain would accumulate within the GBM, while normally they do not. A more likely explanation is that GBM-producing cells synthesize abnormal laminin isoforms during the course of MGN. In this case, there would be a phenotypic modification of GBM-producing cells, leading to synthesis of the laminin ß1 chain. As the laminin ß1 chain is present within the GBM during embryonic development until the capillary loop stage [6], we thought that this phenotypic modification could correspond to a dedifferentiation process affecting visceral epithelial cells. This hypothesis was reinforced by the fact that the GBM of patients with MGN contained not only the laminin ß1 chain, but also fibronectin, which is another component of embryonic GBM [6]. Nevertheless, our results, as well as those of other groups, suggest that the GBM does not contain an increased amount of the type IV collagen chains produced during embryonic development (i.e.
1(IV) and
2(IV) collagen chains) [1117]. Furthermore, our results and those of Barisoni et al. [21] show that the podocytes of patients with MGN still contain vimentin and synaptopodin, although these proteins start to be expressed at the pre-capillary loop stage and at the capillary loop stage, respectively, and can be considered as markers of differentiated podocytes [21]. Thus, during the course of MGN, except for the expression of the laminin ß1 chain and of fibronectin, the phenotype of GBM-producing cells is different from the one of their embryonic counterpart.
The presence of the laminin ß1 chain within the GBM of patients with primary MGN raises the question of its role in the pathogenesis of proteinuria. Analysis of mice harbouring a null mutation in the laminin ß2 gene has shown that these animals develop a glomerular disease characterized by massive proteinuria immediately after birth, and that while their GBM is initially structurally intact, it contains the laminin ß1 chain instead of the laminin ß2 chain [8]. Nevertheless, in this model, it is not clear whether proteinuria is due to the absence of laminin molecules containing the ß2 chain or to the presence of laminin molecules harbouring the ß1 chain. Furthermore, in patients with MGN, the amount of laminin ß1 present within the GBM is much less important than in laminin ß2-/ mice. If the presence of the ß1 chain of laminin within the GBM plays a role in the pathogenesis of proteinuria, it could be by directly modifying the sieving properties of the GBM, or by altering podocyteGBM interactions, and thus podocyte functions. Abnormal expression of the ß1 chain of laminin can be added to the list of factors that are thought to increase glomerular permeability in MGN. These factors include a decrease in heparan sulphate side chains in GBM proteoglycans [22], an enhanced expression of MMP-9, a metalloprotease which can degrade type IV collagen [23], and an overproduction of oxygen radicals [24].
In conclusion, our data show that primary MGN responsible for heavy proteinuria are associated with discrete modifications of the composition of the GBM, and in particular with the appearance of abnormal laminin isoforms. These new isoforms could play a role in the pathogenesis of proteinuria by modifying functional properties of the GBM and/or by modifying podocyte functions.
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Acknowledgments
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We thank Madeleine Delanche (INSERM U489) for technical assistance and Dr Gübler for providing us with an anti-fibronectin antibody.
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Notes
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Correspondence and offprint requests to: Jérôme Rossert, INSERM U489 and Department of Nephrology, Tenon hospital, 4 rue de la Chine, F-75020 Paris, France. 
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Received for publication: 25.11.99
Revision received 2. 8.00.