Difficulties in differentiating thin basement membrane disease from Alport syndrome

Introduction

Erythrocyturia is a frequent symptom of three glomerulopathies – thin basement membrane disease, IgA nephropathy and Alport syndrome. IgA nephropathy generally does not pose diagnostic difficulties. Diagnosis is based on histopathological examination and immunofluorescence. There may, however, occur difficulties in differentiating thin basement membrane disease from the early stages of Alport syndrome.
The typical pattern of thin basement membrane disease is a thinning of the lamina densa of the glomeruli to under 200 nm, affecting most of the nephron loop [1, 2, 3].
Characteristic for the Alport syndrome are an uneven thickness as well as structural changes of the basement membrane, more precisely the lamina densa, such as loosening and, above all, splitting [4, 5], with small dense inclusions referred to as “breadcrumbs” between the layers [6].
This syndrome can also be accompanied by light microscopic changes, such as the presence of immature glomeruli and of foam cells in the kidney interstitium, but these are not typical features of the syndrome.
In some cases of Alport syndrome only minor ultrastructural abnormalities are observed, for example a mere thinning of the lamina densa or a loosening of its structure in some segments only. These symptoms are more frequent in women. In children, initially minor changes progress with time. The aforementioned observations were confirmed by animal experiments [7, 8]. There have also been individual cases of Alport syndrome in which the symptoms did not include any changes in the basement membranes [9]. In some cases of thin basement membrane disease, on the other hand, there may occur a loosening of the structure and even a duplication of the lamina densa.
The above examples show that the differentiation of these two diseases can be difficult. This difficulty was one of the reasons to start looking for differences in genetic research [9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19]. The first were Habib et al. [9]. They were followed by other researchers: Aarons et al. [10], Lang et al. [11], Lamprecht et al. [12], Lemmink et al. [13], Pigneras et al. [14], and others [15, 16, 17, 18, 19, 20, 21, 22, 23, 24]. It was established that both diseases are accompanied by mutations in the COL4A3/COL4A4 genes. However, based on the available results of the research on the mutations in the COL4A3/COL4A4 genes, no clear diagnostic criteria could be defined in order to distinguish thin basement membrane disease, autosomal dominant Alport syndrome and autosomal recessive Alport syndrome. Of diagnostic importance is the lack of reaction with anti chains α3, α4 and α5 collagen type IV in the basement membranes of renal glomeruli and tubules (and the lack of reaction from the anti-α5 chain in the skin).
Consequently, the diagnosis of thin basement membrane disease is more difficult than might be assumed. In clinical assessments and in morphological diagnoses the disease is often not taken into consideration.
In routine diagnosis, the most readily available and surest examination procedure is electron microscopy. However, even a complete set of such tests does not always allow one to make a definite diagnosis.

Aim of the study

Based on tissue material obtained by kidney biopsies, we decided to compare the morphological patterns, especially the electron microscopic images of thin basement membrane disease and Alport syndrome, with particular focus on characteristics which might point to an early stage of Alport syndrome.

Material and methods

The research material comprised biopsy specimens of kidneys of 83 patients (57 children and 26 adults) with a diagnosis of thin basement membrane disease and of 17 patients (9 children and 8 adults) with a diagnosis of Alport syndrome. The control group (for the evaluation of the thickness of the lamina densa of the basement membranes) comprised 11 biopsy samples of 11 patients with minimal change disease.
In the group of children with thin basement membrane disease were 29 girls aged 2 to 16 years and 28 boys aged 2 to 17 years. The group of adults with the same diagnosis was composed of 14 women aged 23 to 70 years and 12 men aged 19 to 70 years. Among the 9 children with Alport syndrome were 7 boys from 7 to 17 years of age and 2 girls of 2 and 10 years. The group of adults with Alport syndrome consisted of 5 men aged 21 to 46 years and 3 women aged 24 to 49 years (Table I).
Thirteen children with thin basement membrane disease and 3 patients with Alport syndrome (including 2 children) had a family history of kidney diseases. In most cases, the family members were diagnosed with erythrocyturia. Clinical symptoms are presented in Table II.

Histological examinations

Part of the biopsy samples fixated in 10% neutral formalin solution were routinely processed. The material was immersed in paraffin, cut by microtome into sections of 3-5 µm and mounted on microscope slides. The preparations were stained with haematoxylin and eosin, impregnated with silver halides in accordance with Jones’ method and the PAS reaction was carried out.

Electron microscopy examinations

The material collected for ultrastructural studies was routinely fixed in a 3.6% buffered glutaraldehyde solution with pH of 7.4. Next, the material was embedded in Epon 812 epoxide resin. Photographic documentation was carried out in the column of an Opton 900 transmission electron microscope.

Immunofluorescence assays

Immunofluorescence slices of 5-6 μm were fixed in a cold 1:1 mixture of alcohol and acetone for 10 minutes, dried and rinsed three times in PBS. Next, the specimens were incubated with sera containing antibodies against IgA, IgG, IgM, complement proteins C3, C4, C1q, and fibrinogen, labelled with fluorescein 5-isothiocyanate (FITC). Following incubation, the slices were rinsed three times in PBS and covered in glycerol under cover slips. The presence, composition and locations of the deposits of immunoglobulins, complement proteins and fibrinogen were assessed by fluorescence microscopy.

Measurement of the thickness of the lamina densa

In order to assess the thickness of the lamina densa of the basement membrane, three measurements were performed in the same places by means of the computing environment MATLAB (www.mathworks.com) semiautomatic method. Measurements were carried out in different sections of the nephron loop of the glomerulus capillaries and in sections immediately neighbouring mesangial regions.
All the glomeruli in a sample (1 to 4 glomeruli) were subjected to a morphometric analysis and in each an average of 5 sections in all the vascular loops were evaluated. Microphotographs were made with the following enlargements: 3500×, 4400×, 5600×, 7000×, 8750×, 10 500×, 11 500×.

Statistical analysis

In view of the lack of a normal distribution of some variables (Shapiro-Wilk test, p < 0.05) and the small quantity of data, only non-parametric statistical methods based on median tests were applied for the statistical analyses. The differences in the thickness measurement results between the groups were analysed by means of the Kruskal-Wallis test, which is the non-parametric equivalent of the one-way analysis of variance (ANOVA). For post-hoc analysis the conservative Dunn’s multiple comparison test was applied. The Wilcoxon signed-rank test was used in order to compare the lamina densa thickness measurements obtained by the semiautomatic method in the different groups. The predetermined significance level for the analyses was p < 0.05. In the data description the levels p < 0.05 and p < 0.001 were indicated, if they occurred.

Results

Thin basement membrane disease

The light microscopic examinations did not reveal any significant changes, either in the group of children or in the group of adults with thin basement membrane disease. They were described as mesangial hypercellularity (not confirmed by the electron microscopic examination) or as non-specific abnormalities. In two children – a 2-year-old girl and a 7-year-old boy – symptoms of immature renal glomeruli were observed, and in one girl – who did not show any abnormalities of the glomeruli – the presence of foam cells and renal interstitial fibrosis. In 4 children, mesangial glomerulonephritis was suspected, due to the presence of deposits of IgA and IgM in 1 case, IgM and C1q in 1 case, IgA and M as well as C3 in 1 case, and IgA and C3 in 1 case. The deposits were vestigial, and their presence was not confirmed by the electron microscopic examination. There was also no confirmation of an increase of the number of mesangial cells. Such trace deposits were also observed in 4 adults – of IgA, IgG, IgM, C3 and C1q in 1 case, of IgA, IgM, C3 and C4 in 1 case, and of IgM and C3 in 2 cases. In these cases, the possibility of mesangial glomerulonephritis could also be excluded by electron microscopy examination due to lack of mesangial hypercellularity.
In 15 children and 8 adults exponents of focal segmental glomerulosclerosis were observed, in the case of only 4 children and 4 adults in the light microscopic examination and in all the other cases exclusively in the electron microscopic. These changes were less severe.
For all patients, the electron microscopic examination revealed typical features of thin basement membrane disease, namely a thinning of the lamina densa to under 200 nm in over 80% of the capillary loops (Fig. 1). In addition, in 16 children and 4 adults isolated minor local rarefactions of the structure of the lamina densa were discovered (Fig. 2).

Alport syndrome

In the group with diagnosis of Alport syndrome, the morphological pattern – both in the light and the electron microscopic image – was clearly more diverse than in the previous group.
In 10 patients, the light microscopic examination revealed not very pronounced changes described as mesangial hypercellularity or non-specific lesions. In 4 cases focal segmental glomerulosclerosis (FSGS) was diagnosed. In all 4 cases this diagnosis was confirmed by the electron microscopic examination. The histological examinations showed in all cases numerous foam cells in the interstitium (Fig. 3).
In 1 case the immunofluorescence test revealed the presence of IgA, IgM and C3 deposits, due to which the possibility of IgA nephropathy was taken into consideration. The electron microscopic examination confirmed the presence of deposits as well as mesangial hypercellularity and resulted in a diagnosis of IgA nephropathy imposed on the changes typical for Alport syndrome.
In this group, there was a 24-year-old female patient who had been diagnosed 13 years previously with thin basement membrane disease. From the age of 2 she had erythrocyturia and proteinuria. The first biopsy confirmed only exponents of thin basement membrane disease (extensive thinning of the lamina densa). However, the light microscopic examination revealed the presence of foam cells and focal interstitial fibrosis. This patient has a family history of erythrocyturia. Her sister underwent biopsies at the age of 12 and again at the age of 17. The first biopsy revealed exponents of thin basement membrane disease and minor segmental glomerulosclerosis. The second biopsy showed that the glomerulosclerosis had not intensified and no other abnormalities were discovered apart from the thinning of the membranes.
For the entire group with a diagnosis of Alport syndrome the electron microscopic image clearly pointed to this syndrome, although the different changes were not equally developed. In all cases uneven thickness of the lamina densa was observed – with sections of less than 200 nm and significantly thickened sections (1337 nm). In most cases the capillary loops showed changes in the structure of the lamina densa – numerous loosenings over large sections, splitting and, above all, local formation of several layers, separated from each other by small dense particles (Fig. 4). The surface of the lamina densa, especially the subepithelial layer, was uneven over large sections and intermeshed in some places. Moreover, mesangial matrix expansion was found in 7 cases.
No differences of intensity of the aforementioned changes depending on the clinical symptoms or the age of the patients were noted.

Minimal change disease – control group

In all cases there were discovered ultra-structural changes that are typical for minimal change disease: disappearance of podocyte foot processes over more than 75% of the surface of the capillaries and microvilli-related surface changes in many podocytes, directed toward Bowman’s capsule.
The lamina densa of the basement membrane of most of the capillary loops was of normal thickness. Only in a few isolated sections, a thickness of maximum 200 nm was observed. None of the cases showed a loosening of the structure or a duplication of the lamina densa.

Measurement of the thickness of the lamina densa of the basement membrane

The results of the measurements of the thickness of the lamina densa revealed significant differences between thin basement membrane disease and Alport syndrome, as illustrated by Table III, which also contains the measurement results from the control group (MCD).

Discussion

In the specialist literature, thin basement membrane disease and Alport syndrome are often discussed together, especially due to the frequent occurrence of thin membranes in the Alport syndrome, above all in the early stages of the disease. In this phase, the morphological differentiation of the two diseases can be very difficult [25, 26]. Both cause similar clinical symptoms: erythrocyturia, erythrocyturia and proteinuria, proteinuria, nephrotic syndrome. In our material, patients with thin basement membrane disease were relatively frequently diagnosed with proteinuria. The high percentage of patients with thin basement membrane disease diagnosed with proteinuria may be explained by the fact that, in the case of proteinuria or proteinuria and erythrocyturia, material for electron microscopic tests is usually secured, whereas in the case of isolated erythrocyturia often no material is secured for such tests. If such tests were carried out in all the relevant cases, the number of diagnosed cases of thin basement membrane disease would be significantly higher and the percentage of patients with proteinuria consequently correspondingly lower. The extra-renal symptoms typical for Alport syndrome – hearing loss and eye abnormalities – are not always developed, and if they are developed they appear only later in life [27, 28].
The diagnostic difficulties in the early stages of Alport syndrome are well illustrated by the above described case of a female patient who was first diagnosed with thin basement membrane disease and developed symptoms of Alport syndrome 13 years later. The first biopsy revealed the presence of foam cells in the interstitium, but this was not a sufficient argument to suggest the possibility of the Alport syndrome. It is to be considered, though, if this change, given the simultaneous occurrence of thin membranes, should not be taken into account as possibly pointing to an early stage of Alport syndrome.
On the other hand, in 2 cases of thin basement membrane disease exponents of immature glomeruli were observed. These are characteristic for the Alport syndrome and should not be considered as a sign for a threatening development of Alport syndrome.
Also to be taken into consideration are patients with thin basement membrane disease who show subtle changes such as segmental loosening of the structure of the lamina densa or its uneven contour.
Another interesting fact in connection with the analysed material is the stronger development of other morphological changes, including light microscopic changes, in patients with Alport syndrome than in patients with thin basement membrane syndrome. It is not a significant factor, but the occurrence of such changes together with extensive thinning of the lamina densa should dictate caution concerning the prognosis.
Thin basement membrane disease does not always have a good prognosis; it is therefore unjustified to describe this disease as “mild erythrocyturia”, and patients diagnosed with this disease must remain under permanent monitoring.

Conclusions

Based on an analysis of the specialist literature data and the results of our own observations derived from the comparison of the morphological characteristics of both diseases, it can be said that in cases when thin basement membrane disease is diagnosed, the following changes should be taken into consideration: presence of even minor sections of thickened lamina densa, unevenness of the surface of the lamina densa, changes in the structure of the lamina densa, presence of interstitial changes, and development of other glomerular abnormalities, especially sclerosis.
These changes can be significant for the detection of the Alport syndrome in its early stages of development.
The research results for both diseases indicate the necessity to perform kidney biopsies in each case of erythrocyturia, which is often neglected due to the opinion that erythrocyturia is not a burdensome symptom. Absolutely necessary are also electron microscopic examinations, which should be compulsory for every kidney biopsy.

The authors declare no conflict of interest.

References

1. Mandache E, Gherghiceanu M. Ultrastructural defects of the glomerular basement membrane associated with primary glomerular nephropathies. Ultrastruct Pathol 2004; 28: 103-108.
2. Collar JE, Ladva S, Cains TD, Cattel V. Red cell transverse througt thin glomerular basement mambranes. Kidney Int 2001; 59: 2069-2072.
3. Lapis H, Foster K, Miner JH. Red cell transverse through thin glomerular basement membrane. Kidney Int 2002; 61: 762-763.
4. Antonovych TT, Deasy PF, Tina LU, et al. Hereditary nephritis. Early clinical, functional, and morphological studies. Pediatr Res 1969; 3: 545-556.
5. Spear GS, Slusser RJ. Alport’s syndrome. Emphasizing electron microscopic studies of the glomerulus. Am J Pathol 1972; 69: 213-224.
6. Liapis H, Gökden N, Hmiel P, Miner JH. Histopathology, ultrastructure, and clinical phenotypes in thin glomerular basement membrane disease variants. Hum Pathol. 2002; 33: 836-845.
7. Cosgrove D, Kalluri R, Miner JH, et al. Choosing a mouse model to study the molecular pathobiology of Alport glomerulonephritis. Kidney Int 2007; 71: 615-618.
8. Cangiotti AM, Sessa A, Meroni M, et al. Evolution of glomerular basement membrane lesions in a male patient with Alport syndrome: ultrastructural and morphometric study. Nephrol Dial Transplant 1996; 11: 1829-1834.
9. Habib R, Gubler MC, Hinglais N, et al. Alport’s syndrome: experience at Hôpital Necker. Kidney Int Suppl 1982; 11: S20-S28.
10. Aarons I, Smith PS, Davies RA, et al. Thin membrane nephropathy: a clinico-pathological study. Clin Nephrol 1989; 32: 151-158.
11. Lang S, Stevenson B, Risdon RA. Thin basement membrane nephropathy as a cause of recurrent haematuria in childhood. Histopathology 1990; 16: 331-337.
12. Lamprecht R, Gross O, Netzer KO, et al. Autosomal recessive Alport syndrome and benign familial hematuria: diseases of same origin? J Am Soc Nephrol 1996; 7: 1616 (abstract).
13. Lemmink HH, Nillesen WN, Mochizuki T, et al. Benign familial hematuria due to mutation of the type IV collagen alpha4 gene. J Clin Invest 1996; 98: 1114-1118.
14. Piqueras AJ, White RH, Raafat F, et al. Renal biopsy diagnosis in children presenting with hematuria. Pediatr Nephrol 1998; 12: 386-391.
15. Lajoie G. Approch to diagnosis of thin basement membrane nephropathy in femals with the use of antibodies to IV collagen. Arch Pathol Lab Med 2001; 125: 631-636.
16. Badenas C, Praga M, Tazon B, et al. Mutations in the COL4A4 and COL4A3 genes cause familial benign hematuris. J Am Soc Nephrol 2002; 13: 1248-1254.
17. Ueda T, Nakajima M, Akazawa H, et al. Quantitative analysis of glomerular type IV collagen alpha 3-5 chain expression in children with thin basement membrane disease. Nephron 2002; 92: 271-278.
18. Buzza M, Dagher H, Wang YY, et al. Mutations in the COL4A4 gene in thin basement membrane disease. Kidney Int 2003; 63: 447-453.
19. Gross O, Netzer KO, Lambrecht R, et al. Novel COL4A4 splice defect and in-frame deletion in a large consanguine family as a genetic link between benign familial haematuria and autosomal Alport syndrome. Nephrol Dial Transplant 2003; 18: 1122-1127.
20. Gregory MC. Alport syndrome and thin basement membrane nephropathy: unraveling the tangled strands of type IV collagen. Kidney Int 2004; 65: 1109-1110.
21. Akazawa H, Nakajima M, Nishiguchi M, et al. Quantitative immunoelectron-microscopic analysis of the type IV collagen a 1-6 chains in the glomerular basement membrane in childhood thin basement membrane disease. Clin Nephrol 2005; 5: 329-336.
22. Rana K, Wang YY, Powell H, et al. Persistent familial hematuria in children and the locus for thin basement membrane nephropathy. Pediatr Nephrol 2005; 20: 1729-1737.
23. Hou P, Chen Y, Ding J, et al. A novel mutation of COL4A3 presents a different contribution to Alport syndrome and thin basement membrane nephropathy. Am J Nephrol 2007; 27: 538-544.
24. Zang KW, Tonna S, Wang YY, et al. Do mutations in COL­4A1 or COL4A2 cause thin basement membrane nephropathy (TBMN)? Pediatr Nephrol 2007; 22: 645-651.
25. Foster K, Markowitz GS, D’Agati VD. Pathology of thin basement membrane nephropathy. Semin Nephrol. 2005; 25: 149-158.
26. Meleg-Smith S. Alport disease: a review of the diagnostic difficulties. Ultrastruct Pathol 2001; 25: 193-200.
27. Fogo AB, Kashgarian M. Glomerular diseases. In: Diagnostic Atlas of Renal Pathology. Elsevier Sanders 2005; 2-276
28. Churg J, Sobin LH. Chapter 17. In: Renal Disease. Igaku-Shoin Tokyo, New York 1982; 280-283.

Addresss for correspondence

Jakub Żurawski
Department of Immunobiochemistry
Chair of Biology and Environmental Sciences
Poznan University of Medical Sciences
Rokietnicka 8
60-806 Poznan, Poland
e-mail: zurawski@ump.edu.pl