Distribution of {alpha}-chains of type IV collagen in glomerular basement membranes with ultrastructural alterations suggestive of Alport syndrome

Paola Barsotti1,, Andrea Onetti Muda1, Gianna Mazzucco2, Laura Massella3, Bruno Basolo4, Mario De Marchi5, Gianfranco Rizzoni3, Guido Monga6 and Tullio Faraggiana1

1 Dipartimento di Medicina Sperimentale e Patologia, Università ‘La Sapienza’, Roma 2 Dipartimento di Scienze Biomediche ed Oncologia, Università di Torino, Torino 3 Divisione di Nefrologia, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma 4 Divisione di Nefrologia e Dialisi, Ospedale San Giovanni Bosco, Torino 5 Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Orbassano 6 Dipartimento di Scienze Mediche, Università di Torino, Novara, Italy



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. In Alport syndrome (AS) impaired production and/or assembly of col IV {alpha}-chain isoforms results in abnormal structure of glomerular basement membrane (GBM), haematuria and, frequently, progressive renal disease. We investigated the relationship between col IV {alpha}-chains expression and morphology of GBM, as a possible key to the better understanding of the pathogenesis of renal disease in AS.

Methods. GBM distribution of col IV {alpha}1-, {alpha}3-, and {alpha}5-chain was investigated by immunohistochemistry in 32 patients (21 males and 11 females, mean age at biopsy of 11.5 years) with ultrastructural findings suggestive of AS. Ten patients had a proven COL4A5 mutation. Based on the severity of ultrastructural findings, the biopsies were grouped in three (I–III) electron microscopy (EM) classes. Significant EM changes of GBM (thinning, thickening, splitting, basket weaving of the lamina densa) were singularly evaluated using a semiquantitative scale (0–3).

Results. Col IV {alpha}1-chain was demonstrated in GBM of all patients. Three patterns of staining for col IV{alpha}3- and {alpha}5-chains were observed: positive, negative, and {alpha}3(IV)-positive/{alpha}5(IV)-negative. By {chi}2-test, EM class III lesions and complete loss of {alpha}3(IV)- and {alpha}5(IV)-antigen were significantly more frequent (P<0.05 and P<0.01) in male patients, but no significant relation was observed between EM classes and immunohistochemical patterns. GBM alterations did not correlate with staining for {alpha}5(IV)-chain. Intensity of {alpha}3(IV)-chain staining, however, had a negative correlation (P<0.05) with the severity of GBM basket weaving.

Conclusions. Our results suggest that the {alpha}3(IV)-chain-containing col IV-network plays a fundamental role in structural and, possibly, functional organization of GBM. Absence of {alpha}3(IV)-chain in GBM could indicate a more severe renal disease in AS.

Keywords: Alport syndrome; COL4A5 gene mutations; electron microscopy; glomerular basement membrane; immunohistochemistry; type IV collagen



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Alport syndrome (AS) is a hereditary disease of basement membranes with prevalent X-linked inheritance (85%). Families with autosomic recessive or, rarely, dominant inheritance and sporadic cases of AS were also reported [1]. Basement membrane damage is mostly evident in renal glomeruli [1]. It results from a defect of genes encoding for isoforms of type IV collagen {alpha}-chains of glomerular basement membrane (GBM), namely COL4A5 gene, localized on chromosome X and encoding for col IV {alpha}5-chain (X-linked AS), and COL4A3 and COL4A4 genes, located on chromosome 2 and encoding for the col IV {alpha}3- and {alpha}4- chains (autosomic AS) [1,2]. A wide variety of mutations have been described in X-linked, autosomic, or sporadic cases of AS [1,2].

AS has variable clinical expression: it consists of childhood haematuria and progressive renal failure, predominant in the affected males, often associated with high-tone sensorineural deafness and minor ocular lesions. Severity of the renal disease varies among different families [1,2].

Examination of renal biopsy by electron microscopy (EM) is a critical step in the diagnosis of AS. Characteristic ultrastructural lesions are described as ‘basket weaving of the GBM’ and consist of irregular splitting of the lamina densa with electrolucent lacunae that often contain dense granules [3,4]. Diffuse thinning of the GBM with minor structural abnormalities may also be seen in heterozygote females [5] and in a few males [6].

GBM of male patients with proven AS is generally unreactive with antibodies against {alpha}3(IV)-, {alpha}4(IV)- and {alpha}5(IV)-chain by immunofluorescence (IF) [712]. But variably positive reactions for {alpha}3(IV)-, {alpha}4(IV)- and {alpha}5(IV)-chain were reported in female and occasionally male patients [8,11,12].

In the last few years several families affected by AS have been investigated for COL4A5 mutations [1]. Nevertheless, a clear relationship among the heterogeneous mutations of COL4A5 gene, the abnormal products of the transcripts and the abnormalities of GBM substructure remains to be defined.

In the present study, we describe the {alpha}1(IV)-, {alpha}3(IV)- and {alpha}5(IV)-chain distribution in the GBM of 32 patients with EM findings suggestive of AS and COL4A5 mutation (10/25 patients). Our special aim was to investigate the relationship between col IV composition and the fine structure of GBM, to better understand the pathogenesis of renal disease in AS.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
From our files, 32 renal biopsies obtained from patients with persistent haematuria were selected according to the following criteria:

(i) EM findings suggestive of AS: that is, a report of variable (from light/focal to severe/diffuse) combinations of thinning, thickening, splitting and basket weaving of the lamina densa of GBM.
(ii) Normal or non-specific light microscopy findings, with negative or not significant IF.
(iii) Residual cryopreseved unfixed renal tissue available for immunohistochemical studies for col IV {alpha}-chain isoforms.

The selected biopsies had been obtained from 32 patients (21 males and 11 females; mean age at biopsy: 11.5 years, range: 2–47 years). They had a history of childhood haematuria, with or without proteinuria and/or renal failure. Neurosensory, high frequency deafness was present in seven males and two females. A positive family history of renal disease, with or without end-stage renal disease (ESRD) or hearing loss, consistent with X-linked transmission, was present in 15 patients; four families with the juvenile type of AS were identified. The disease appeared as ‘sporadic’ in nine cases. Twenty-five patients had been previously referred to the Italian Group for the Study of Alport Syndrome, where they were screened for COL4A5 gene rearrangements and analysed by SSCP for mutations of all the 51 exons of the Col4A5 gene [13,14]. Ten COL4A5 mutations were identified: four of these were ‘de novo’ mutations.

In conclusion, based on the criteria for the diagnosis of AS, as recently revised by Gregory et al. [15], the 32 selected cases included: 13 patients with definite diagnosis of AS (four or more AS criteria) and 10 patients with probable diagnosis of AS (three criteria). The remaining nine cases who satisfied only two criteria, represented sporadic cases, with negative or uninvestigable familial history, with persistent haematuria and GBM ultrastructural abnormalities suggestive of AS.

Controls consisted of:

(i) Normal human renal tissue fragments obtained at surgery for renal carcinoma (three cases) and
(ii) renal biopsies from patients with: IgA nephropathy (two cases), membranous nephropathy (two cases), focal segmental glomerulosclerosis, type I mesangiocapillary glomerulonephritis, and benign nephrosclerosis (one case each).

Electron microscopy
The GBM structure was evaluated using the EM material that had been routinely prepared for EM studies of renal biopsies. Briefly, small renal tissue fragments were fixed in phosphate (0.1 M, pH 7.2) buffered aldehyde solution, postfixed in 1% osmium tetroxide and embedded in Epon.

For each case, ultra thin sections were observed at EM and microphotographs of GBM were obtained from three glomeruli.

The characteristic lesions of the GBM that were evaluated were thinning (GBM <200 nm) thickening, splitting and basket weaving of the lamina densa (Figure 1Go). Because GBM thickness is age-related, thinning was not considered in children under 3 years of age [16].



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Fig. 1. Electron micrographs of GBM from patients with AS showing: (A) thinning (patient number 7), (B) thinning and splitting (patient number 19), (C) basket weaving (patient number 4), (D) thickening and basket weaving (patient number 7) of the lamina densa. UrPb, x34 000.

 
The biopsies were grouped in three classes according to the pattern of distribution and the severity of the GBM lesions, with particular emphasis on the lesions which are generally considered to be more relevant for AS diagnosis, such as GBM splitting and basket weaving [1]:

– EM class I: rare GBM tracts showing mild splitting or basket weaving of the lamina densa with, or without, prevalent GBM thinning.
– EM class II: segmental expression of moderate to severe splitting and/or basket weaving of the lamina densa, with variable thinning and/or thickening of the GBM.
– EM class III: diffuse expression of moderate to severe basket weaving of the lamina densa, with variable thinning and/or thickening of the GBM.

In a separate analysis, the four distinct alterations of GBM characteristic of AS were evaluated using a semiquantitative scale: 0=absent; 1=occasionally present (<30%); 2=present (30–70%); 3=widespread (>70%).

The evaluation of EM lesions was blindly performed by at least two of us. The only case with discordant classification was discussed and reclassified.

Immunohistochemistry
Monoclonal antibodies to {alpha}1-chain (MoAb 6) and {alpha}3-chain (MoAb 17) of type IV collagen [17] were kindly provided by Dr J. Wieslander. MoAb A7 against {alpha}5(IV)-chain [9] was a generous gift from Dr C. E. Kasthan.

Immunohistochemical studies were performed using indirect immunofluorescence method on frozen specimens routinely prepared for IF of kidney biopsies and stored at -75°C. Cryostate sections 5-µm thick were incubated in the primary antibodies against {alpha}1(IV) and {alpha}3(IV) at a working dilution of 1:20 and 1:100 in 0.5% BSA in PBS (PBS/BSA). Additional sections from each case were denatured by exposure to 6 M urea in 0.1 M glycine/HCl buffer (pH 3.5) to unmask the hidden epitope of Col-IV {alpha}5 chain [18] and incubated in the anti {alpha}5(IV) antibody. The working solution was 1:4 in PBS/BSA. As secondary antibody, FTIC-conjugated rabbit antibody against mouse immunoglobulins was used, at a working dilution of 1:20 in PBS/BSA.

Standard immunohistochemical controls were performed, replacing the primary antibodies with non-immune mouse serum or unrelated monoclonal antibodies.

Intensity of the glomerular staining was evaluated using a semiquantitative scale (0=absent; 1=light; 2=moderate; 3=strong).

Statistical analysis
The data were analysed using {chi}2-test and Spearman's rank-correlation test. Statistical significance level was defined as P<0.05.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Most of the selected renal biopsies were characterised by normal or slightly altered histopathology. Minor, not specific abnormalities included: focal involvement of glomeruli by slight increase of mesangial matrix and/or slight increase of mesangial cells, occasional completely sclerosed glomeruli, red blood cells in tubular lumina and minor signs of tubular atrophy. Only few cases showed chronic glomerular and tubulointerstitial damage. Interstitial foam cells were seen in about a quarter of the biopsies. Immunofluorescence was negative or showed minor deposits of IgM and/or C3.

Electron microscopy
An EM picture suggestive of AS had been the main criterion used to select the renal biopsies for the present study. Thus, all the patients showed altered GBM. Global evaluation of the biopsies allowed division of them into three groups (Table 1Go).


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Table 1. Frequency of EM classes and expression of col IV-{alpha} chains in 32 patients with EM findings suggestive of AS

 
– Nineteen biopsies showed diffuse, moderate to severe basket weaving of the lamina densa, with variable thinning and/or thickening of the GBM (EM class III). These biopsies had been considered ‘diagnostic’ for AS. In this group, there was a high prevalence of male patients (16/19 cases).
– Four biopsies, three of them from female patients, showed segmental, moderate to severe splitting and/or basket weaving of the lamina densa, with variable thinning and/or thickening of the GBM (EM class II). They had been considered ‘highly suggestive’ or ‘diagnostic’ for AS.
– Nine cases (four male and five female) showed prevalent thinning and/or occasional, mild splitting or basket weaving of the GBM (EM class I). AS diagnosis, which had been considered as ‘possible’ or ‘doubtful’ in 8/9 cases, had been originally excluded in patient number 25.

The score assigned to each characteristic elemental GBM lesion of each case is reported in detail in Table 2Go. As expected, the confidence of EM diagnosis depended on male gender ({chi}2=7.625, P<0.05). We observed a higher presence in male patients of basket weaving ({rho}s=0.509, P<0.01), and splitting ({rho}s=0.514, P<0.01), but not of thinning ({rho}s=-0.051, non significant (ns)) or thickening ({rho}s=0.240, ns) of the lamina densa. No significant correlation was observed between age at biopsy and EM class, neither in the whole group of patients ({rho}s=0.164, ns), nor in males ({rho}s=0.045, ns), or females ({rho}s=0.235, ns), separately considered.


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Table 2. Semiquantitative evaluation of GBM features in 32 patients with EM findings of AS

 

Immunohistochemistry
In glomeruli from normal human renal tissue, the presence of {alpha}1(IV)-chain was diffusely revealed in mesangium (3+) and, although less intense (2+), in GBM (Figure 2aGo). Staining for col IV {alpha}3 and {alpha}5 was limited to GBM: {alpha}5(IV) staining was uniformly as intense (3+) as {alpha}3(IV) staining (Figure 3aGo and eGo).



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Fig. 2. Indirect immunofluorescence for {alpha}1-chain of collagen IV in renal glomeruli from (A) a normal control and (B) a male patient (patient number 16) with class III EM alterations of GBM. In the normal glomerulus the signal is slighter on GBM than in mesangium, while in the Alport patient, GBM and mesangial matrix showed a similarly strong staining; x250.

 


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Fig. 3. Indirect immunofluorescence for {alpha}3- (AD) and {alpha}5- (EH) chain of collagen IV in renal glomeruli from a normal control (A, E) and three patients with GBM lesions highly suggestive for AS (EM class II or III) selected to represent the possible patterns of staining: {alpha}3- /{alpha}5- patient number 16 (B, F), {alpha}3+/{alpha}5- patient number 8 (C, G) and {alpha}3+/{alpha}5+ patient number 27 (D, H); x250 (A–F), x125 (G, H).

 
In the renal biopsies from the control patients the localization of the {alpha}1(IV)-, {alpha}3(IV)- and {alpha}5(IV)-chains did not differ from the normal distribution.

In all AS patients anti-{alpha}1(IV) antibody diffusely reacted with GBM; in most cases GBM appeared intensely stained, more than in normal tissue, thus equalling mesangial staining (Figure 2bGo). Anti-{alpha}3(IV) and anti-{alpha}5(IV) antibodies demonstrated highly heterogeneous patterns of reaction (Table 2Go, Figure 3bGo–dGo and fGo–hGo), that may be summarized as follows:

– Col IV {alpha}3- and {alpha}5-chains were totally absent in GBM from 17 cases. In this group there was high prevalence of male patients (15/17 cases) with clearly diagnostic EM findings (12/15 cases).
– Col IV {alpha}3- and {alpha}5-chains were present in GBM from eight patients. Three of eight cases showed abnormally reduced intensity of {alpha}5(IV) staining, with normal or slightly reduced staining for {alpha}3(IV). This group included two male and six female patients. In one female patient (number 27) the positive staining had segmental distribution. Five of eight cases showed GBM alterations diagnostic of AS.
– Col IV {alpha}3-chain was observed in GBM from seven cases, in spite of the total absence of reactive sites for anti-{alpha}5(IV) antibody. The intensity of staining was normal in five cases and highly reduced in two cases (patients 8 and 23). This group of patients included four males; two of them showed alteration of GBM diagnostic for AS

Distribution of col IV {alpha}3- and {alpha}5-chains in our AS patients was related to patient's gender, showing higher frequency of completely negative immunohistochemical staining in male patients (Table 1Go, {chi}2=10.66, P<0.005).

Relationship between distribution of Col IV {alpha}-chains and fine structure of GBM
In our cases, the pattern of distribution of {alpha}3(IV)- and {alpha}5(IV)-chains was unrelated to the EM class (Table 1Go, {chi}2= 4.23, ns). Nevertheless, when each elemental EM-lesion was separately considered, the severity of the highly characteristic ‘basket weave’ lesion negatively correlated with the intensity of staining for col IV {alpha}3-chain ({rho}s=-0.361, P<0.05, Figure 4Go). No significant correlation was demonstrated between basket-weave alteration and intensity of staining for col IV {alpha}5-chain ({rho}s=0.003, ns, Figure 4Go), nor between intensity of staining for col IV {alpha}3- or {alpha}5-chain and splitting ({rho}s=-0.230, ns; {rho}s=-0.064, ns), thinning ({rho}s=0.050, ns; {rho}s=0.107, ns) or thickening ({rho}s=-0.073, ns; {rho}s=0.148, ns) of the lamina densa of GBM.



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Fig. 4. Scattergrams showing intensity of immunostaining (0–3) for {alpha}3- and {alpha}5-chain of GBM, related to the extension of GBM basket-weave lesion (BW), in 32 patients with EM findings suggestive of AS. Black dots represent the values of each patient, lines represent the mean values of each BW class.

 

Relationship of COL4A5 gene mutations with distribution of Col IV {alpha}-chains and fine structure of GBM
Mutations of COL4A5 gene had been demonstrated in 10 of 25 patients [13,14], immunohistochemical and ultrastructural data from these patients are detailed in Table 3Go.


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Table 3. Phenotype in 10 patients with COL4A5 mutations

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
AS has a heterogeneous clinical presentation. Electron microscopic study of GBM has been considered as the most reliable diagnostic tool to individualize possible Alport patients. With the exception of the few Alport families with apparently normal GBM [19], thinning, thickening, splitting of GBM and basket weave pattern of lamina densa with intramembranous dense granules have been described as specific lesions in most AS cases. Despite the high variability of distribution of GBM lesions, full expression of GBM alteration has been reported to prevail in affected males [5,20,21]. In our cases with diagnostic GBM alterations (EM classes II and III), diffuse GBM lesions (EM class III) were observed in 16 of 17 males and diagnostic elements were partially expressed (EM class II) in three of six females. Nevertheless, occasional or unspecific lesions of GBM (EM class I) were observed in four male and five female patients. In our cases, splitting and basket weaving of the lamina densa had significantly higher scores in male patients, while no significant correlation was observed between sex and thinning and/or thickening of GBM, thus indicating that the changes of GBM texture are to be considered the most relevant.

In our cases, a significant male prevalence of negative GBM staining for both {alpha}3(IV)- and {alpha}5(IV)-chain was observed, with a higher score for anti-{alpha}3(IV) and -{alpha}5(IV) staining in female gender. Variable staining in heterozygous females from X-linked AS families has been generally explained by random inactivation of X chromosome [10,11]. Some questions arise in explaining the presence of {alpha}3(IV)- and {alpha}5(IV)-chain staining in affected males. Nearly one-third (6/21) of our male cases contained detectable {alpha}5(IV)- and/or {alpha}3(IV)-antigen in their GBM. One of these male patients (patient number 1) had proven COL4A5 mutation. Few other families with proven X-linked AS and positive staining for {alpha}5(IV)- and/or {alpha}3(IV)-chain in GBM of affected males have been reported in literature [11,12]. It was hypothesized that this may depend on minor {alpha}5(IV)-chain alterations that allows synthesis of abnormal col IV molecules and their incorporation in a partially abnormal network. In these series, most of the positive males had milder lesions of GBM and/or milder clinical forms than in negative patients [11,12]. In the present group, all patients with a proven juvenile familial form of the disease (patients numbers 5, 7, 17 and 19) showed a completely negative staining for {alpha}3(IV)- and {alpha}5(IV)-chain and severe glomerular lesions.

Our data from patients with known COL4A5 mutations have been reported elsewhere [13,14,22]. In the present study, only a few significant examples are given, to confirm a possible relationship between severity of the mutation type and lack of antigenic properties of GBM [7,12]. First, immunohistochemistry failed to detect both {alpha}5(IV) and {alpha}3(IV) chains in GBM of the male patients with major rearrangements of COL4A5 gene (patient numbers 11 and 19), or point mutations resulting in interruption of coding sequence (patient numbers 4 and 17). In these cases the GBM findings were classified in EM class III owing to the highly rarefied lamina densa. Second, as in the cases by Naito et al. [12], a variable immunohistochemical picture was observed in the four cases with missense mutations (see Table 3Go). Alterations of GBM varied from I to III EM class. The only patient with missense mutation and GBM negative for {alpha}3- and {alpha}5(IV)-chains was a male with diagnostic EM and juvenile familial form of AS. In this case (patient number 7), the missense mutation resulted in substitution of glycine at 869 with arginine. This large, basic amino acid could seriously affect triple helical stability, or, alternatively, it could cause abnormal triple helical conformation of a specific site implicated in the supramolecular organization of GBM.

The accurate evaluation of relationship between distribution of col IV antigens and EM alterations of GBM provided some new data on GBM alteration in AS. In this group of patients, when the biopsies were simply classified according the general aspect of the GBM, no clear relationship between GBM antigenicity and GBM morphology was observed. Nevertheless, the separate evaluation of each elemental GBM alteration allows the assessment of significant correlations between basket-weave lesion and abnormal distribution of col IV {alpha}-chains in GBM. In particular, we demonstrated an inverse correlation between severity of GBM basket weaving and intensity of immunohistochemical staining for {alpha}3(IV)-chain, while we were unable to demonstrate any similar relation for {alpha}5(IV)-chain. Basket weaving of the lamina densa is the most significant EM aspect for diagnosis of AS and some authors think that GBM basket weaving may have an additional prognostic significance [5,20,21]. The relationship between GBM basket weaving and abnormalities of {alpha}3(IV)-antigen reactivity seen in our patients could indicate that {alpha}3(IV) chain plays a special stabilizating and/or protecting function in the supramolecular organization of GBM, as recently proposed in the paper by Gunwar et al. [24], which reports a novel col IV network, {alpha}3(IV) · {alpha}4(IV) · {alpha}5(IV), stabilized by disulphide cross-links between collagenous domain of {alpha}3(IV) and {alpha}4(IV) chains. These observations are in line with the severe GBM lesions and the progressive course of the disease described in autosomal recessive AS [8,23], where {alpha}3(IV)- or {alpha}4(IV)-chain are primarily affected. Moreover, human epidermal basement membrane (EBM) has been demonstrated to contain {alpha}5- but not {alpha}3- and {alpha}4-chain of type IV collagen [18]. Patients with X-linked AS lack apparent skin damages and show normal EM aspect of EBM despite the absence of {alpha}5(IV)-chain in EBM (our unpublished observations). It seems therefore reasonable to hypothesize that absence of {alpha}3(IV)- and/or {alpha}4(IV)-chain, i.e. of their disulphide cross-linking, represent the crucial step of the morph-functional damage of GBM in AS. Nevertheless, our patients number 14 and number 20, as well as a few cases in the literature [11,12], do not fit this hypothesis; it could be reasonably speculated that, in these few subjects, abnormal {alpha}5(IV)-chains, interact with {alpha}3(IV) and {alpha}4(IV) to form abnormal heterotrimers [1], without affecting the possibility of their immunohistochemical detection. However, the possibility of additional, still unclear, pathogenetic mechanism(s), where the role of {alpha}3(IV) defect has minor importance, cannot be ruled out.

In conclusion, our data confirm the relevance of basket-weave lesions of GBM in diagnosis and, possibly, prognosis of AS. Absence of {alpha}3(IV)-antigen reactivity, i.e. absence or conformational defects of {alpha}3(IV)-molecules, seems to be significant for the pathogenesis of this characteristic GBM structural alteration. Negative GBM-staining for {alpha}3(IV)-antigen could indicate a more severe renal disease in AS.



   Acknowledgments
 
This work was partially supported by M.U.R.S.T. (Italy).



   Notes
 
Correspondence and offprint requests to: Dr Paola Barsotti, Università ‘La Sapienza’, Dipartimento di Medicina Sperimentale e Patologia, Sezione di Patologia Ultrastrutturale, Viale Regina Elena, 324 (Policlinico Umberto I), I-00161 Roma, Italy. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 10. 8.99
Revision received 26. 9.00.



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