Alport syndrome

Context.-Thin basement membrane nephropathy is recognized by a diffusely thin glomerular basement membrane (GBM) ultrastructurally. In contrast to Alport syndrome (AS), there is no GBM thickening, lamellation, or granular inclusions. Morphologically, there is overlap between thin basement membrane nephropathy and AS in female patients in whom there might be only thin GBM and no pathognomonic findings of AS.

Objective.-To determine if the use of antibodies to collagen IV is helpful in making the distinction between thin basement membrane nephropathy and AS in female patients with primarily thin GBMs.

Design.-We examined renal biopsies from 9 adult female patients with thin GBMs for the presence of alpha1, alpha3,

alpha4, and alpha5 chains of type IV collagen by immunofluorescence.

Results.-In 2 patients with segmental GBM staining, no suggestion for AS was found on physical examination or in their family history. In the remaining 7 patients with normal GBM staining, 4 had family members with end-stage renal disease of unknown etiology, raising the suspicion of X-linked or autosomal-recessive AS. Three patients were presumed to have thin basement membrane nephropathy.

Conclusion.-Segmental GBM staining for alpha3, alpha4, and alpha5 chains of type IV collagen raises the suspicion of AS in the presence of adequate controls and other supporting evidence. Normal GBM staining for alpha3, alpha4, and alpha5 chains of type IV collagen, however, does not exclude AS.

(Arch Pathol Lab Med. 2001;125:631-636)

The incidence of thin glomerular basement membrane nephropathy (TBN; also called benign familial hematuria/nephritis) ranges between 5.2% and 9.2% in the general population1 and may account for up to 30% of patients presenting with asymptomatic hematuria.2 Thin glomerular basement membrane nephropathy (reviewed in references 3 and 4) is believed to have an autosomal-- dominant pattern of inheritance. A benign outcome is reported in the majority of patients, but glomerular obsolescence, proteinuria, and hypertension may occur.5,6 The predominant ultrastructural anomaly in the kidney is a diffusely thin glomerular basement membrane (GBM).2 In contrast to Alport syndrome (AS), there are no widespread areas of thickening, lamellation, or granular inclusions within the GBM,3,4 although focal splitting has been described.

Alport syndrome can be X-linked, autosomal-recessive, or possibly autosomal-dominant (reviewed in references 3 and 7-10). There are a number of phenotypic variants of AS, and the classic findings of hematuria, progressive renal failure, bilateral high-tone sensorineural hearing loss, and ocular changes are not found in all patients.7 In the X-linked form, there is a defect in the gene coding for the alpha5 chain of type IV collagen (alpha5[IV]) located on the long arm of chromosome X. Autosomal-recessive patients have mutations) in either COL4A3 or COL4A4 genes situated on chromosome 2. These 2 genes code for the alpha3 (alpha3[IV]) and alpha4 (alpha4[IV]) chains of type IV collagen, respectively. Ultrastructurally, the GBM presents alternating areas of thinning and thickening, splitting, lamellation, and granular inclusions in the lamina densa. These so-called pathognomonic findings are not always found in children and females, who may only have thin GBMs without other alterations. Therefore, it may be difficult to make a distinction between TBMN and AS in these patients on a purely morphologic basis.

Thus, the diagnosis of TBMN remains one of exclusion. Although it has been reported that glomeruli of patients with TBMN reveal no abnormalities when stained with antibodies against alpha3(IV), alpha4(IV), and alpha5(IV),4 these observations have not been published. In the present study, we sought to examine the usefulness of antibodies against alpha1, alpha3, alpha4, and alpha5 chains of type IV collagen in establishing the diagnosis of TBN in 9 adult female patients with diffusely thin GBMs.

MATERIALS AND METHODS

Patient Selection and Clinical Information

Nine female patients with diffusely thin GBM on renal biopsy were identified between 1994 and 1996 from the surgical pathology files of the University Health Network, Toronto, Ontario. Controls included 1 male patient with known X-linked AS, 1 female patient with immunoglobulin A (IgA) nephropathy, and 2 female patients with minimal-change nephrotic syndrome. Clinical charts of index patients were reviewed for age, indication for renal biopsy, and family history. Clinical follow-up was obtained.

Light, Immunofluorescence, and Electron Microscopy

Renal biopsies were routinely processed for light, immunofluorescence, and electron microscopy. The glass slides were reviewed for all indexed cases and controls. All cases were examined by electron microscopy. The GBM was measured in all cases using the method of harmonic mean of orthogonal intercepts.11 A measurement of less than 264 run was required to make a diagnosis of TBMN.2

Antibodies

Marine monoclonal antibodies anti-alphal (IV) (MABl), anti-alpha3(IV) (MAB3), and anti-alpha5(WV) (MAB5-A7) were purchased from Wieslab AB (Lund, Sweden). The monoclonal antibody against alpha4(IV) (MAB85) was graciously provided by Clifford Kashtan (University of Minnesota, Minneapolis).

Monoclonal antibodies against alpha1(IV) (MABl), and ^sub 1^3(IV) (MAB3) were obtained from mice immunized with the noncollagenous C-terminal domain (NC1) of bovine GBM.12 The monoclonal antibody against alpha5(IV) (MABS-A7) was obtained from immunization of mice with the collagenase-resistant residue of human GBM.13,14 The monoclonal antibody MAB85 (anti-alpha4[IV]) was obtained from mice immunized with human NC1.15

Only the MAB85 was diluted 1:1 for immunohistochemistry. The other antibodies were not diluted.

Immunofluorescence for alpha1(IV), alpha3(IV), alpha4(IV), and alpha5(IV) on Frozen Tissue

The slides were stained for alpha1, alpha3, alpha4, and alpha5 chains of type IV collagen according to the method proposed by Yoshioka et al,16 with slight variation. Briefly, slides were fixed in acetone for 5 minutes. After washing in phosphate-buffered saline, the slides were treated with a solution of 6 mol/L urea, 0.1 mol/L glycine at pH 3.5 for 1 hour at 4 deg C. Incubation with the primary antibodies for 45 minutes at room temperature followed. After a phosphate-buffered saline wash, fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Dako Corporation, Carpinteria, Calif) was applied for 30 minutes. The slides were mounted in a permanent mounting solution (Gelvatol, Air Products and Chemicals, Allentown, Pa). The primary antibody was omitted in negative controls.

RESULTS

Clinical Summary

The 9 female patients with diffusely thin GBMs were between 23 and 43 years old (average, 34 years) (Table 1). Seven patients presented with microscopic hematuria, with or without low-grade proteinuria; 3 of those patients were identified during assessment for living-related kidney donation. Two patients were being investigated because of proteinuria of 2.0 g/24 h or more.

A family history of hematuria only was obtained in 1 patient. Two additional patients had a family history that included both hematuria and renal failure of unknown etiology. Another 2 patients had relatives with end-stage renal disease of unknown etiology, and 1 patient had a son diagnosed with reflux nephropathy. There was no known family history of hematuria or renal disease in 3 patients; 1 of these patients had been adopted.

Clinical follow-up was obtained for 8 patients and varied from 4 months to 5 years (average, 26 months). Of the 8 patients for whom follow-up was available, 7 had stable renal function with persistent hematuria and 1 patient had progressive renal insufficiency.

Light Microscopy

No glomerular changes other than global and/or segmental glomerulosclerosis were observed (Table 2). Global glomerulosclerosis involved less than 10% of glomeruli in 3 patients, and 12% to 30% of glomeruli in 4 patients. Two of those 4 patients also had a few glomeruli with segmental scars (Figure 1). Interstitial fibrosis of moderate severity was seen in only 1 patient (patient 4) and was minimal or mild, and usually focal, in all other patients.

Immunofluorescence Microscopy

Immunofluorescence microscopy was negative for IgG, IgM, IgA, C3, C4, and kappa and lambda light chains in all cases.

Immunofluorescence with antibodies against alpha1(IV), alpha3(IV), alpha4(IV), and alpha5(IV) (Table 3) showed diffuse and strong linear staining along GBM in 7 patients (Figure 2); this pattern was similar to that seen in positive controls (Figure 2). Patients 2 and 9 demonstrated segmental staining of GBM with alpha3, alpha4, and alpha5 chains of type IV collagen (Figure 2). Of those 2 patients, only patient 9 exhibited diffuse and strong linear staining for alpha1(IV) (Figure 3). Weak and uneven staining for alpha1(IV) was observed in the biopsy from patient 2. Staining for alpha3, alpha4, and alpha5 chains of type IV collagen was lacking altogether in the male patient with X-linked AS. Diffuse and strong linear staining for alpha1(IV) was present in that patient (results not shown).

Electron Microscopy

In all cases, the mesangial matrix was either normal or mildly increased and did not contain electron-dense immune-type deposits (Table 2). Effacement of foot processes of glomerular visceral epithelial cells was noted over short segments. There was diffuse thinning of GBM that was confirmed by measuring orthogonal intercepts (Figure 4). The GBM measurement in indexed patients ranged from 161 to 239 nm (average, 200 nm), as opposed to controls, in which the GBM measured from 296 to 322 run (average, 309 run). The GBM of the male patient with AS measured 324 run and presented typical changes of AS (namely, alternating areas of thin and thick GBM with splitting, lamellation, and granular inclusions). Similar changes were seen focally in only 1 indexed patient (Figure 5, A). Changes limited to splitting of short segments of GBM were seen in 3 cases (Figure 5, B). Notably, splitting of GBM over short segments was also seen in 2 cases of minimal-change nephrotic syndrome, and 1 case of IgA glomerulopathy.

COMMENT

A pathologist confronted with a renal biopsy in which diffuse GBM thinning is the predominant anomaly has to take numerous steps to try to establish an accurate diagnosis. In an attempt to rule out AS, one must, if possible, obtain a complete clinical summary, including a detailed family history, and perform electron microscopy and immunofluorescence with antibodies against alpha3, alpha4, and alpha5 of type IV collagen.17

Unfortunately, a detailed family history might be unobtainable6; for example, patient 9 in our study was adopted. If and when available, the family history might be of limited use in some patients because of the possibility of a new mutation or because the etiology of renal failure in relatives is undetermined. Four patients in our study had relatives with renal failure of unknown origin. Furthermore, in a retrospective study like ours, and often in the practice of renal pathology, a complete clinical history cannot be obtained.

Electron microscopy cannot discriminate between TBMN and AS in patients in whom only a diffusely thin GBM with minimal alterations of the lamina densa is found. Any degree of GBM splitting or lamellation is compatible with AS, even if the GBM changes are mild and present over short segments only.17 Conversely, such changes are not pathognomonic for AS and can be found in other renal diseases,18 including TBMN. Among the 4 patients in our study with segmental splitting of GBM by electron microscopy (patients 1, 5, 8, and 9), only 1 patient (patient 9) had segmental GBM staining for alpha3, alpha4, and a5 chains of type IV collagen by immunofluorescence. This pattern of staining is likely specific for the diagnosis of AS, but in the absence of other corroborative evidence (no stigmata of AS and no family history), only genetic analysis would give a definitive diagnosis.10 Another patient (patient 2), also with segmental GBM staining (but no GBM splitting), lacked clinical indices supporting a diagnosis of AS, including a family history compatible with AS. The absence of strong staining with alpha1 of type IV collagen in that patient indicates that the GBM antigens are not well preserved, and that the segmental staining observed with alpha3(IV), alpha4(IV), and alpha5(IV) is probably spurious. This case illustrates the need to perform adequate controls and to interpret the results with caution. Interestingly, the remaining 3 patients with segmental splitting of the GBM but with normal GBM staining with alpha3, alpha4, and alpha5 chains of type IV collagen all have male relatives with end-stage renal disease of unknown etiology. In these 3 patients, normal GBM staining does not exclude AS or the carrier status of the Alport mutation. Favorable lyonization may result in a normal pattern of staining in females with X-linked AS. Another possibility is that these patients are heterozygous for the mutation associated with autosomal-recessive AS (see below). Furthermore, some patients with AS have normal staining with antibodies against alpha3, alpha4, and 5 chains of type IV collagen.19 Genetic studies could help sort out the likely pattern of inheritance of renal disease in these 3 families.

Four patients (patients 3, 4, 6, and 7) demonstrated no ultrastructural GBM abnormalities other than diffuse thinning; all had a normal pattern of GBM staining by immunofluorescence. It is likely that these patients have TBMN. Of these 4 patients, 2 reported a family history of hematuria. Hematuria tends to be underreported unless family members are tested specifically. Renal function has remained normal and stable in these 4 patients during an average follow-up of 3.75 years, although patient 7 has a sister with end-stage renal disease of unknown etiology. In the family of this patient, the possibility of autosomal-- recessive AS has to be considered. Presumably, patient 7 would be heterozygous for the mutant gene (see below). In contrast, an extensive family history of hematuria would suggest TBMN with an autosomal dominant pattern of inheritance in patient 3.

The putative protein defect in GBM of patients with TBMN remains unknown (reviewed in references 3 and 4). Based on linkage analysis studies, Lemmink et al20 and others21 have proposed that patients with TBMN might be heterozygous for the mutant gene that causes autosomal-- recessive AS. Their findings would tend to support the hypothesis that TBMN and AS are entities belonging to the same spectrum of disease, although others have not been able to demonstrate a similar association.22 Arguing against such a hypothesis is the fact that Nomura and his colleagues23 reported no hematuria on repeated testing of heterozygous parents of a patient with autosomal-recessive AS. Furthermore, in 3 animal models of autosomal-- recessive AS, heterozygous animals have a normal phenotype.24 Thin GBM nephropathy is believed by some to have an autosomal-dominant pattern of inheritance.3,4 The issue remains unresolved. In our study, no abnormality of alpha3(IV), alpha4(IV), or alpha5(IV) could be demonstrated by immunofluorescence in 7 patients. Possibly, this method lacks sensitivity for the detection of small quantitative changes.

A more contentious issue is whether patients with TBMN should be considered for kidney donation. The long-term prognosis of patients thought to have TBMN is unknown. While a benign outcome is the rule, glomerular obsolescence, hypertension, and proteinuria have been reported.5,6 It is important to note that the studies reporting progression toward renal insufficiency in TBN might have unknowingly included unidentified AS patients. It has been suggested that in patients with a presumed diagnosis of TBMN, only a detailed family history reporting several male members with long-standing hematuria and no progression toward renal failure in advanced age is the best evidence for a benign diagnosis.7 Follow-up was too short for most of the patients included in our study to allow prognostication about renal outcome.

Thin GBM in female patients presents a common diagnostic puzzle in the practice of renal pathology. There is much overlap between TBMN and AS, especially in female patients. Our discussion emphasizes the need to correlate clinical history and pathologic findings in the interpretation of renal biopsies. The addition of immunofluorescence for alpha1, alpha3, alpha4, and alpha5 chains of type IV collagen might be useful in confirming a diagnosis of AS in patients in whom there is a documented family history.17 In other patients, abnormalities of GBM staining might raise the degree of suspicion for that diagnosis. In our study, patients presumed to have TBMN did not have detectable defects of alpha3, alpha4, and alpha5 chains of collagen type IV in the GBM. Normal GBM staining, however, does not exclude AS or the carrier status of the Alport mutation; the family history of 4 of our patients with normal GBM staining was suggestive of AS.

Studies of families with suspected TBMN are needed to determine the pattern of inheritance of this disorder. There are possibly at least 2 groups of patients; the first group might be heterozygous carriers of the mutant gene for autosomal-recessive AS, while in others TBMN might be inherited as an autosomal-dominant disease.

This study has been funded in part by the University Health Network Pathology Associates and by the University Health Network Department of Pathology, Toronto, Ontario.

The author thanks Clifford Kashtan, MD, for his generous gift of the MAB85 antibody and recognizes the excellent technical assistance of Richard Leung, BSc, ART (electron microscopy and photography) and Joanne Mariano, BSc, MLT (immunofluorescence).

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Accepted for publication December 7, 2000.

From the Department of Laboratory Medicine and Pathobiology, Toronto General Hospital, University Health Network, University of Toronto, Ontario.

Reprints: Ginette Lajoie, MD, FRCPC, Department of Pathology, Eaton Wing 4-323, Toronto General Hospital, University Health Network, 200 Elizabeth St, Toronto, Ontario, M5G 2C4 Canada (e-mail: ginette.lajoie@uhn.on.ca).

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