collagen distribution in focal and segmental glomerulosclerosis: an immunofluorescence and...

JOURNAL OF PATHOLOGY, VOL. 179: 188-196 (1996)

COLLAGEN DISTRIBUTION IN FOCAL AND SEGMENTAL GLOMERULOSCLEROSIS: AN

IMMUNOFLUORESCENCE AND ULTRASTRUCTURAL IMMUNOGOLD STUDY

YI CAI*, MIKE ILL^ SICH*, A G N ~ S BEZIAU", MARY M. KLEPPEL? AND MARIE-CLAIRE GUBLER*

*INSERM C' 423, H6pital Necker-Etfants Malades, 149 Rue de SPvres, 75743 Paris Cedex 15, France; +Department of Pediutrics, University of' Minnesota Medical School, Minneupolis, U. S. A .

SUMMARY

Focal and segmental glomerulosclerosis (FSGS) is a non-specific scarring process of the glomerulus, initially described in idiopathic nephrotic syndrome. The distribution of types I, 111, IV, V, and VI collagen and of the a l , a3, a4, a5, and a6 chains of type IV collagen was studied by immunohistochemistry in sclerotic lesions of nine nephrotic children. Dual immunofluorescence and high-resolution immunogold labelling were used to determine the precise distribution of the antigens. No changes were detected in normal glomeruli of patients compared with controls. In FSGS, type 1V collagen [al(IV)2 a2(IV)], and to a lesser degree type VI, accumulates in the two components of the lesion: the enlarged mesangial matrix and the material deposited between the pushed-out podocytes and the a>aS(IV)-positive glomerular basement membrane. Staining for a6(IV) and types I, 111, and V collagen was practically negative. These results suggest that the matrix components of the sclerotic lesion are produced solely by glomerular cells. Changes in the relative distribution of type 1V collagen chains, characterized by the presence of collagen [al(IV)2 a2(TV)] in close contact with the podocytes, strongly suggest a switch in the podocyte programme of collagen synthesis.

KEV WORDS-focal and segmental glomerulosclerosis; type Iv collagen; type VI collagen; interstitial collagen

INTRODUCTION

Focal and segmental glomerulosclerosis (FSGS) is a peculiar lesion which was clearly described by Rich in 1957 in an autopsy study of 20 nephrotic children.] The lesion is focal and segmental; only some of the glomeruli are affected; these show lesions affecting segments of their glonierular tuft, with the adjacent capillary loops and the other glomeruli showing only 'minimal changes'. Typically, in the affected segment, the capillary lumen is obliterated by the accumulation of matrix material, often associated with liyaline deposits and endothelial foam cells. The podocytes covering the lesion are hyper- trophic and show intracellular vacuolation. They are often pulled away from the capillary basement membrane by a clear 'halo' corresponding to the subepithelial accumulation of multiple layers of basement membrane material, engulfing the affected glomerular capillary loops and eventually leading to capsular adhesion. These distinctive morphological features differentiate FSGS from focal glonierulonephritis and from diffuse glomerulosclerosis occurring, for example, in diabetic nephropathy and in some experimental m ~ d e l s . ~ - ~

One major histological feature in FSGS is the accumulation of extracellular matrix material. However, only a few immunohistochemical studies of matrix components have been performed in human and experimental FSGS.6 l o The aim of the present study was to analyse

Addressee for correspondence Dr M -C Gubler, INSERM U423, Tour Lavoisier heme etage Hbpital Necker -Enfants Malades. 149 Rue de Scvie\. 75743 Paris Cedex 15, France

CCC 0022-34 17/96/060 188-09 c\ 1996 by John Wiley & Sons, Ltd.

the collagenous components of the matrix in FSGS lesions. We studied, by immunofluorescence, the glomerular distribution of types 1, 111, IV, V, and VI collagen and specifically investigated the distribution of type IV collagen chains using dual immunofluorescence and high-resolution immunogold techniques. Our results show major changes in the relative distribution of these a(1V) chains in the sclerotic lesions, suggesting a switch in the podocyte programme of type IV collagen synthesis.

MATERIALS AND METHODS

Patients and venal specimens

From a series of 50 consecutive renal biopsies performed in children for diagnostic purposes and showing FSGS, kidney tissue from nine patients was selected for this study, on the basis of the presence of typical focal and segmental glomerular lesions on the frozen (eight patients) and/or paraformaldehyde-fixed LRWhite- embedded specimens (three patients). The patients were 4-14 years old at the time of renal biopsy. They pre- sented with idiopathic nephrotic syndrome which was steroid-resistant in six and steroid-dependent in three. An initial renal biopsy had shown minimal glomerular changes in one and diffuse mesangial proliferation in another.

FSGS involving 20-80 per cent of glomeruli was observed in all cases on the paraffin-embedded specimen (Fig. la). By conventional immunofluorescence, deposits of IgM, Clq, and/or C3 were present in the segmental

Received 6 June 1995 Accepted 12 December 199.5

COLLAGEN DISTRIBUTION IN FSCS 189

Fig. l-Typical FSGS. (a) Light microscopy. The lesion of focal segmental sclerosis and hydiinosis is characterized by segmental collapse of capillary lumens and mesangial sclerosis, deposition of dense ‘hyaline’ material in a subendothelial location, and the presence of a clear ‘halo’ between the lesion and the detached podocytes, which undergo severe vacuolization. Trichrome light green. x 380. (b) Electron microscopy. Paraformaldehyde-fixed, LRWhite-embedded material. The picture underlines the enlargement of mesangial strands of basement membrane material, the presence of lipid droplets within the endothelial cell, and the detachment of the podocyte (P) from the underlying GEM (arrow-head), with accumulation of lamellar basement membrane material (arrow) between the two structures. x 8500

lesions (data not shown). All components of the lesion could be identified on electron microscopy (Fig. lb).

Antibodies and reagents The polyclonal and monoclonal antibodies used in

this study are listed in Table I. Affinity-purified fluor- escein isothiocyanate (F1TC)-conjugated sheep IgG F(ab‘), fragment anti-mouse immunoglobulins (H+L) and affinity-purified FITC-coiijugated sheep IgG (F(ab’), fragment anti-rabbit immunoglobulins (H+L) were obtained from Silenus (Victoria, Australia). Affinity-purified FITC-conjugated swine anti-goat IgG

(H+L) was obtained from Caltag (South San Francisco, U.S.A.). Affinity-purified Texas red sulphonyl chloride (TRSC)-conjugated donkey IgG anti-rabbit IgG (H + L) and affinity-purified TRSC-conjugated rabbit anti- mouse IgG+IgM (H+ L) were obtained from Jackson ImmunoResearch Laboratories (West Grove, U.S.A.). Normal human serum adsorbed goat anti-mouse IgG conjugated with 5 nm colloidal gold particles and staphylococcal protein A conjugated with 20 nm colloidal gold particles were obtained from BioCell (Cardiff, U.K.). Rabbit anti-protein A was obtained from Sigma (St Louis, U.S.A.). A silver enhancing kit was obtained from BioCell (Cardiff, U.K.).

190 Y. CAI ET A L .

Table ILCharacteristics of the antibodies against various collagens

Antibodies Specificity Source

Monoclonal antibodies Mouse anti-al(IV) Mouse anti-a3(IV) Mouse Mab 85 Mouse Mab A7 Mouse Mab 5C6

a 1 (1V)NCl Wieslab AB, Lund, Sweden a3(IV)NCl Wieslab AB, Lund, Sweden u4(IV)NCl M. M. Kleppel, Minneapolis, U S A . aS(1V)NCI M. M. Kleppel, Minneapolis, U.S.A.

Collagenous domain of type VI collagen Hybridoma Bank, Iowa City, U.S.A.

Polyclonal antibodies Rabbit anti-aS(IV) Peptide sequence of aS(1V)NCI Rabbit anti-a6(IV) Peptide sequence of a6(IV)NC1I7 R. Kalluri, Philadelphia, U.S.A. Rabbit anti-collagen I Type 1 collagen Pasteur-Lyon, France Goat anti-collagen 111 Type I11 collagen Southern Biotechnology, U.S.A. Rabbit anti-collagen 1V Collagenous domain of [al(IV)2 a2(IV)] Pasteur-Lyon, France Goat anti-collagen V Type V collagen Southern Biotechnology, U.S.A. Rabbit anti-collagen VI

M. M. Kleppel, Minneapolis, U.S.A.

Type IV collagen Research Products Life Technologies, U.S.A.

Methods Indirect immunofluorescence-An indirect immuno-

fluorescence study could be performed in eight patients. Renal samples were snap-frozen in liquid nitrogen and embedded in OCT (Miles Laboratories Inc., Naperville, IL, U.S.A.). Three-micrometre cryostat sections were incubated with appropriate dilutions of primary antibodies. Incubation with fluoresceinated secondary antibodies was then performed. For dual fluorochrome labelling of (a) al(IV) and aS(IV), (b) [al(IV)2 a2(IV)] and a3(IV), (c) al(IV) and type V collagen, and (d) a1 (IV) and type VI collagen, the slides were simultaneously incubated with the two primary antibodies. They were then incubated with the corresponding secondary antibodies simultaneously. Sections were examined with a Leitz Orthoplan microscope equipped with appropriate filters. Before incubation with mouse Mab 85 and Mab A7, rabbit anti-uS(IV), or goat anti-collagen V, the slides were pretreated with 0.1 M glycine, 6~ urea, pH 3.5, for 10 min, to reveal hidden antigenic determinants.

Tissue sections for control experiments were directly incubated with the secondary antibodies, or serially incubated with normal rabbit, normal goat or mouse serum instead of the primary antibodies, followed by incubation with the corresponding secondary antibodies. They showed no fluorescence. Eight normal kidneys not used for transplantation were tested as controls for evaluation of the normal distribution of the antigens.

Immunoelectron microscopy-Renal biopsy samples from three patients were fixed in 4 per cent buffered paraformaldehyde and embedded in LRWhite. Ultrathin sections were cut, mounted on nickel grids coated with collodion and carbon films, and processed for irnmunocytochemistry.

For imrnunolabelling with polyclonal rabbit anti- collagen IV antibodies, the protein A-gold labelling method was used, as described by Desjardins et al.” For immunolabelling with monoclonal antibodies (mouse Mab 85 and Mab A7), tissue sections were pretreated

with 0.1 M glycine, 6 M urea, pH 3.5, for 20 min. They were then incubated with primary antibody for 60 min at room temperature and further incubated with goat anti-mouse IgG conjugated with 5 nm colloidal gold particles for 60 min. The grids were transferred to a drop of silver enhancing solution for 2 min, post-fixed with 2 per cent glutaraldehyde, and dried. Before examina- tion with the electron microscope, the sections were stained with uranyl acetate and lead citrate.

Tissues from three normal kidneys not used for transplantation were tested as controls for evaluation of the normal distribution of the antigens. Incubation of the sections with normal rabbit or mouse serum instead of the primary antibodies, or direct incubation with secondary antibody or the protein A-gold alone, was performed as for control experiments; no positive results were obtained.

RESULTS Immunofluorescence

Normal controls-In normal controls, the monoclonal anti-al(IV) antibody and the polyclonal anti-type IV collagen [al(IV)2 a2(IV)] stained all extraglomerular basement membranes. Within the glomerular tuft, they strongly stained the mesangial matrix and gave thin layer labelling of the subendothelial aspect of the GBM (Fig. 2a). Conversely, the a3(IV), a4(IV), and u5(IV) chains were co-distributed within the full thickness of the GBM and were absent from the mesangial matrix (Fig. 2b). The a6 chain of type IV collagen was detected in the capsular basement membrane, but not in the glomerular tuft (data not shown). Anti-type VI collagen antibodies stained the mesangial matrix and faintly labelled the subendothelial aspect of the GBM (Fig. 2c). No type I, 111, or V collagen was detected in the GBM or the mesangial area (Fig. 2d).

Nonsclerotic glomeruli of nephrotic patients-In normal glomeruli and in normal parts of glomeruli affected with segmental lesions, no change was observed in the

COLLAGEN DISTRIBUTION IN FSGS 191

Fig. 2-Normal kidney. Immunofluorescence microscopy. (a) [al(IV)2 a2(IV)]. There is strong staining of extraglomerular basement membranes, Bowman’s capsule, and the mesangial matrix (MM). Thin linear staining of the subendothelial aspect of the GBM can be seen. x 350. (b) aS(IV). The antibody produces ribbon-like staining of the entire thickness of the GBM, but the MM is unstained. x 350. (c) Type V1 collagen. Strong staining of the MM and faint staining of the subendotheiial aspect of the GBM are seen. x 350. (d) Type 111 collagen. The renal interstitium is stained, but not the glomerular structure. x 280

distribution of type IV collagen chains or of types V and VI collagen. Trace staining for types I and I11 collagen was very focally found in the mesangial area or at the vascular pole, but labelling was usually negative.

Focal and segmental glomerular sclerosis-In focal and segmental lesions, antibodies against the al(1V) chain and the type IV collagen molecule [al(IV)2 a2(IV)] strongly labelled the expanded mesangial matrix. In addition, they gave intense labelling of the periphery of the lesion and of the capsular adhesion (Fig. 3a). With anti-a3(IV), a4(IV), and aS(1V) antibodies, normal ribbon-like staining of the GBM was observed (Fig. 3c).

In completely sclerotic glomeruli, ring-like staining of the periphery of the retracted glomerular tuft was observed with anti-a1 (IV) and anti-type IV collagen [al(IV)2 a2(IV)] antibodies, contrasting with the decreased mesangial labelling with the same antibodies (Fig. 3b). Antibodies against the a3, a4, and a5 chains of type IV collagen normally labelled the GBM. Focal wrinkling of the basement membrane was associated with a noticeable increase in labelling intensity. Anti- a6(IV) antibody staining remained negative in the glomerular tuft.

Dual fluorochrome labelling confirmed that the relative distribution of type IV collagen chains was completely changed in sclerotic lesions. It was characterized by the apposition of type IV collagen [al(IV)2 a2(IV)] between the podocytes and the a3-a4-a5(IV)- positive GBM (Figs 4a4d) .

A moderate increase in type VI collagen staining of the enlarged mesangial matrix and focal expansion at the periphery of affected segments was associated with changes in the distribution of type IV collagen [al(IV)2 a2(IV)] (Fig. 3d). Traces of type V collagen were very focally detected in sclerotic lesions, whereas no type I or type I11 collagen was observed.

Immunoelectron microscopy Normal glomeruli-With anti-type IV collagen

[al(IV)2 a2(IV)] antibodies, the mesangial matrix was strongly labelled whereas GBM labelling was restricted to the subendothelial region of the basement membrane. Labelling for the a4(IV) and aS(1V) chains was observed over the entire thickness of the GBM, but not over the mesangial matrix (data not shown).

Non-sclerotic glomeruli of nephrotic patients-In nonsclerotic glomeruli and in normal parts of glomeruli affected with segmental lesions, the distribution of type IV collagen [al(IV)2 a2(IV)] and that of the a4(IV) and a5(IV) chains were identical to that in controls (Figs 5 and 6).

Focal and segmental glomerulosclerosis-In the FSGS lesion, the striking feature was the extensive type IV collagen [al(IV)2 a2(IV)] labelling of the layers of basement membrane material accumulated between the GBM and the detached podocytes (Figs 5a and 5b). The

192 Y. CAI ET AL.

Fig. 3-FSGS. Immunofluorescence microscopy. (a) [al(IV)2 n2(IV)]. In addition to the normal distribution, strong staining of the peripheral region of the sclerotic lesion is seen, corresponding to the clear 'halo'. x 270. (b) [al(IV)2 a2(IV)]. In a completely sclerotic glomernlus. the periphery of the tuft is labelled, with the adhesion to the Bowman's capsule. The central part of the tuft is poorly labelled. x 280. (c) a4(IV). The staining is normal along the GBM and not increased in the sclerotic lesion. x 210. (d) Type VI collagen. The staining is increased over the expanded mesangium and along sclerotic segments of the glomerulus. x 350

mesangial matrix was also labelled with the same antibodies. By contrast, the distribution of the a4(IV) and aS(IV) chains was restricted to the GBM and no labelling of the lamellar material surrounding the lesion was observed (Fig. 6). The mesangial matrix remained unlabelled with the anti-a4(IV) and anti-aS(1V) antibodies.

DISCUSSION

Like every basement membrane in the body, the glomerular extracellular matrix is composed of collagens, non-collagenous glycoproteins (laminin, fibro- nectin, entactinhidogen), and proteoglycans. The major collagenous component is type IV collagen, which is associated with type VI collagen within the glomerular matrix. Different isoforms of type IV collagen have now been identified. 12-13 Their normal distribution within the two glomerular extracellular matrix domains, the GBM proper and the mesangial matrix, has been precisely d e ~ c r i b e d . " . ~ ~ . ' ~ The al(1V) and a2(IV) chains are ubiquitous. In the glomerular tuft, they are present in the mesangial matrix and along the endothelial aspect of the GBM. The a3(IV), a4(IV), and aS(1V) chains, which have a restricted tissue distribution, are present within the full thickness of the GBM. From this distribution, it has been suggested that mesangial and endothelial cells synthesize the collagen IV molecule [al(IV)2 a2(IV)],

whereas the a3(IV), a4(IV), and aS(IV) chains are produced by the podocytes. The recently identified a6(IV) chain is absent from the glomerular tuft and is only found in Bowman's c a p ~ u l e . ' ~ ~ ' ~ Type VI collagen is co-distributed with collagen IV [a1 (IV)2 a2(IV)] in the mesangial matrix and the subendothelial aspect of the GBM. I 8 This specific glomerular distribution of collagen components, also observed for laminin chains and pro- teoglycan species, underlines the high specificity of the glomerular matrix domains.19 Types I, 111, and V interstitial collagens are absent from the normal glomerular

The main feature of FSGS is sclerosis. From a morphological point of view, two different matrix components participate in the constitution of the sclerotic lesion: mesangial increase, on the one hand, and subepithelial apposition of basement membrane material, on the other. Many immunohistochemical studies of extracellular matrix components have been devoted to experimental and human glomerulosclerosis involving the glomerular tuft diffusely and occurring, for example, in diabetes, but only a few studies have been performed on the FSGS lesion. In the remnant kidney model of FSGS, a focal and then diffuse increase in mesangial staining for types I and IV collagen has been demon- ~ t r a t e d . ~ In man, accumulation of type IV collagen in the mesangial matrix and the sclerotic lesions associated with type I11 collagen deposition in large focal adhesions has been observed.6 Abnormal deposition of collagen

matrix .6,7,20,2 1


Fig. L F S G S . Dual immunofluorescence microscopy. [al(IV)2 a2(IV)] (TRSC) and a4(IV) (FITC): (a) In the sclerotic lesion, accumulation of [a1 (IV)2 a2(IV)]-positive material (red colour) is clearly seen on the external aspect of the a4(IV)-positive GBM (green colour). x 350. (b) In a completely sclerotic glomerulus, the shrunken glomerular tuft is encircled by type IV collagen [al(IV)2 a2(IV)]. The wrinkled GBM is normally labelled with the anti-a4(IV) antibody. x 210. (c, d) Same glomerular section. Accumulation of [al(IV)2 a2(IV)] in the sclerotic mesangium and at the periphery

of the sclerotic lesion, with persistence of a normal GBM distribution of the a4(IV) chain. x 350

type 1 within the sclerotic segments,s or of collagen types I and I11 within the mesangial area of one ~ a t i e n t , ~ has also been reported. More recently, it was shown that the a1 chain of type IV collagen accumulated in a subepithelial position.l0

In the present study, we have shown by immunofluorescence the accumulation of type IV collagen [al(IV)2 a2(IV)] in the two components of the sclerotic lesion: the enlarged mesangial matrix and the material deposited between the GBM and the detached podocytes, or between the GBM and Bowman's capsule when there is a capsular adhesion. In contrast, no significant change was detected in the amount or in the GBM distribution of the a3-a5(IV) chains. These results were confirmed by dual immunofluorescence and ultrastructural immunogold labelling. Both techniques clearly demonstrate that the a3-a5(IV)-positive GBM is separated from the podocytes by thin layers of al-a2(IV)-positive basement membrane material. The peculiar distribution of type IV collagen chains in sclerotic lesions, characterized by the presence of collagen [al(IV)2 a2(IV)] in close contact with the podocytes, strongly suggests a switch in podocyte collagen IV production, from the a3-a5 chains to the less specific al-a2 chains. This major alteration in the podocyte programme of collagen synthesis may represent an additional marker of the severity of visceral epithelial cell damage in FSGS. It is a possible factor for irrevers- ibility of the FSGS lesion. In membranous glomerulo-

nephritis, also characterized by the subepithelial accumulation of basement membrane material, the absence of a severe degenerative change of visceral epithelial cells is associated with preservation of the relative distribution of type IV collagen chains and with the possible complete recovery of a normal GBM structure10,22 (unpublished results). No aberrant expression of the a6(IV) chain was observed in the sclerotic lesions or in the adjacent normal glomerular capillary

Type VI collagen participates to a minor degree in the formation of sclerotic lesions: it is associated with type IV collagen [al(IV)2 a2(IV)] in the expanded mesangial matrix and the subepithelial basement membrane material. Parallel expansion of types IV [al(IV)2 a2(1V)] and VI collagen within the glomerular matrix has been observed in other pathological conditions, such as membranous glomerulonephritis (unpublished results), allograft g l~merulopathy ,~~ and Alport syndrome.24 However, in the obsolescent glomeruli, surrounded by a ring of collagen IV [al(IV)2 a2(IV)], a decreased expression of this collagen in the collapsed capillary tuft strongly contrasts with the accumulation of type VI collagen. These features are not specific for FSGS, as they have been observed in the obsolescent glomeruli of various types of g l o m e r ~ l o p a t h y . ~ ~ , ~ ~

Contrary to previous reports,&1° we did not observe accumulation of interstitial collagen in the sclerotic lesions. Only trace staining for types I, 111, and V

loops.

194 Y. CAI ET AL.

Fig. 5-FSGS. Immunogold electron microscopy. Anti-type IV collagen [al(IV)2 a2(IV)] antibodies. (a) There is strong labelling of the thin layers of basement membrane-like material (arrow) accumulated between the podocyte (P) and the GBM (arrow-head). The mesangial matrix is also labelled. x 9000. (b) This picture shows the normal distribution of the antigen along the subendothelial aspect of the GBM in the normal part of the loop (arrow-head) and the subepithelial accumulation of the antigen in the adjacent sclerotic segment (arrow). CL=capillary lumen; P=podocyte. x 9000

collagen was occasionally detected in the mesangial area, collagen in sclerotic segments suggests that the matrix or at the vascular pole of glomeruli. As in a model of components of the lesion are produced solely by glomerulosclerosis in the rat,26 the absence of interstitial glomerular cells.


Fig. C F S G S . Immunogold electron microscopy (with silver amplification). Anti-u4(IV) antibody. Gold-silver particles are distributed over the normal GBM and the GBM included in the sclerotic lesion (arrow-head). No labelling is observed on the mesangial matrix or the lamellar basement membrane material (arrow), located between the a4(IV)-positive GBM and the podocyte (P). x 11 000

ACKNOWLEDGEMENTS

The monoclonal antibody Mab 5C6 against type VI collagen was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biology, University of Iowa, Iowa City, IA under contract NO1-HD-2-3 144 from the NICHD. The polyclonal anti-a6(IV) antibody was a gift from R. Kalluri, University of Pennsylvania, Philadelphia. Dr Yi Cai is a recipient of a Chinese Visiting Scholarship and a fellowship training award from the Fondation pour la Recherche Medicale, France. We thank Mr Y. Deris for micrographs, Ms B. Coupe for typing the manuscript, and Mrs D. Broneer for reviewing the manuscript.

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collagen distribution in focal and segmental glomerulosclerosis: an immunofluorescence and...

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