—  SPECIALTY CONFERENCE  —

Renal Pathology

Case 5 - Membranous Glomerulopathy and Anti-TBM Nephritis

Vivette D. D'Agati
Columbia University
College of Physicians and Surgeons
New York, NY


Click on each slide thumbnail image for an enlarged view
Clinical History
A 3 year-old African-American male (date of birth 4/2/94) was referred to a pediatric nephrologist in May l997 for work-up of polydipsia, renal glycosuria and proteinuria. The child's height was in the 50th percentile and his weight was in the 25th percentile for age. Physical examination was unremarkable, with BP 96/56. Urinalysis disclosed pH 7.0, 1+ protein, 3+ glucose (quantitated urine glucose 500 mg/dl with simultaneous blood glucose 91 mg/dl), 3-4 rbc/hpf, 0 wbc. Blood chemistries included Na 139 mEq/L, K 5.0 mEq/L, HCO3 18 mEq/L, BUN 14 mg/dl, and creatinine 0.8 mg/dl. By ultrasound, the kidneys were normal sized with increased echogenicity. Further work-up disclosed creatinine clearance 79 ml/min/1.73 m2, Uprot/Ucreat ratio of 0.8, serum albumin 4.8 g/dL, cholesterol 168 mg/dL, calcium 10.0 mg/dL, phosphorus 5.1 mg/dL (fractional excretion of PO4 17%), uric acid 2.7 mg/dL, amylase 195 (normal <80). The following were all negative or normal: liver function tests, pyruvate, lactate, CH50, C3, C4, C5, C2, C1q, ANA, RF, anti-DNA, SSA, SSB, ENA, anti-Smith, anti-RNP, lead (Pb) levels, sickle prep, slit lamp examination. A renal biopsy was performed at an outside institution in October 1997. Because of inadequate tissue for immunofluorescence, a second renal biopsy was performed in February 1998.


Case 5 - Figure 1 - Low-power view showing patchy tubular atrophy and interstitial fibrosis with a mild interstitial inflammatory infiltrate of mononuclear leukocytes. (Masson's trichrome)
Case 5 - Figure 2 - High-power view of the atrophic tubules shows inconspicuous tubular basement membranes and dense intervening interstitial fibrosis. (Masson's trichrome)
Case 5 - Figure 3 - A representative glomerulus has internal vacuolizations of the glomerular basement membranes typical of membranous glomerulopathy. (Jones methenamine silver)


Case 5 - Figure 4 - Immunofluorescence staining for IgG reveals 2+ finely granular staining of the glomerular basement membranes in an apparently subepithelial distribution. By contrast linear staining can be seen in Bowman's capsule and the adjacent tubular basement membranes.
Case 5 - Figure 5 - Immunofluorescence staining for IgG shows diffuse intense (3+) linear staining of all the tubular basement membranes, including those of atrophic and non-atrophic tubules.
Case 5 - Figure 6 - Indirect immunofluorescence was performed by application of patient serum to cryostat sections of normal kidney, followed by FITC-rabbit antihuman IgG. There is linear staining of tubular basement membranes and Bowman's capsule, but not the glomerular basement membranes or mesangium. These findings provide conclusive evidence of the presence of circulating anti-TBM antibody.


Case 5 - Figure 7 - Electron micrograph showing electron dense subepithelial and intramembranous deposits embedded in the glomerular basement membranes, consistent with stage 1-3 membranous alterations.
Case 5 - Figure 8 - Electron micrograph showing the absence of any electron dense deposits involving the tubular basement membranes, which appear thinned.

Differential Diagnosis of Clinical Features
This 3 year-old boy presented with renal glycosuria, metabolic acidosis, polyuria, subnephrotic proteinuria and renal insufficiency. The presence of renal glycosuria and metabolic acidosis, without evidence of hypophosphatemia or hyperphosphaturia, led to a clinical diagnosis of partial Fanconi syndrome. The manifestations of Fanconi syndrome are caused by proximal tubular dysfunction and may include hypophosphatemia and hyperphosphaturia, growth failure, rickets, polyuria with episodes of dehydration, aminoaciduria, glycosuria (with normal serum glucose), hyperchloremic metabolic acidosis, hypokalemia, hypouricemia and hyperuricosuria, urinary sodium and potassium wasting, and hypercalciuria.

The differential diagnosis of Fanconi syndrome in the young child primarily includes inherited or congenital disorders. Among these, cystinosis is the most common. Others include galactosemia, Wilson's disease, tyrosinemia, hereditary fructose intolerance, glycogen storage diseases, mitochondrial diseases (such as Kearns-Sayre syndrome), Lowe's syndrome, and anti-TBM nephritis. Heavy metal exposure, such as lead poisoning, should also be considered in the differential diagnosis of a child with Fanconi syndrome. By contrast, the differential diagnosis of Fanconi syndrome in the adult primarily includes acquired disorders that affect the tubulo-interstitial compartment, such as interstitial nephritis, Sjogren's disease, toxic nephropathies related to heavy metals or drug therapy, amyloidosis, myeloma (light chain Fanconi syndrome), lymphoma and Balkan nephropathy.

Renal Biopsy Findings
The first renal biopsy contained renal cortex and medulla with severe and diffuse tubular atrophy, moderate interstitial fibrosis and a patchy dense chronic inflammatory cell infiltrate. No tubulitis was identified. Of the 23 glomeruli present, 19 were globally sclerotic and these obsolescent glomeruli were closely clustered due to severe intervening tubular atrophy. The remaining glomeruli displayed mild retration of the tuft, which appeared normocellular. No tissue was available for immunofluorescence. By electron microscopy, peculiar intramembranous confluent electron dense deposits were detected in Bowman's capsule and tubular basement membranes (TBM), with a rare minute subepithelial electron dense deposit involving the glomerular basement membranes (GBM). These findings suggested the possibility of tubulo-interstitial nephropathy of an immune complex type, such as related to Sjogren's disease or systemic lupus erythematosus. Because IF was necessary to better characterize this tubulo-interstitial process, a repeat biopsy was performed in February 1998.

The repeat biopsy again showed patchy tubulo-interstitial scarring affecting approximately 50% of the cortex sampled. Glomeruli in the zones of tubular atrophy were globally sclerotic. Elsewhere the glomeruli displayed mild and diffuse thickening and rigidity of the glomerular basement membranes. With the silver stain, fine vacuolizations of the glomerular basement membrane were demonstrated, consistent with membranous alterations. There was dense peritubular fibrosis in the regions of tubular atrophy. No crystalline deposits typical of cystine were identified. By electron microscopy, there were now well developed membranous changes (ranging from stage 1-3) with numerous small subepithelial and intramembranous electron dense deposits indenting the outer glomerular basement membrane accompanied by severe foot process effacement. Electron dense deposits were no longer detectable involving the tubular basement membranes or Bowman's capsule, which now appeared thinned. By immunofluorescence, there was 2+ granular staining for IgG, kappa, lambda and C3 involving the glomerular basement membranes. Intense 3+ linear and diffuse staining of the tubular basement membranes and Bowman's capsule was observed for IgG, kappa and lambda.

Differential Diagnosis of Renal Biopsy Findings
These two successive biopsies performed over a four-month period illustrate an evolution from a predominantly tubulo-interstitial nephropathy in the first biopsy to well-developed membranous glomerulopathy with associated tubulo-interstitial nephropathy in the second biopsy. Whereas the membranous features are relatively easy to identify, the nature of the tubulo-interstitial process is more difficult to classify. The vague intramembranous electron densities that had been identified in the tubular basement membranes of the first biopsy were no longer detectable in the second biopsy, although the immunofluorescence displayed intense linear positivity for IgG involving TBM's.

Tubular basement membrane deposits are commonly encountered in a variety of conditions, including lupus nephritis, Sjogren's disease, membranoproliferative glomerulonephritis type 2 (dense deposit disease), light chain deposition disease, and amyloidosis. In this case, the TBM deposits lacked the usual granular texture of tubulo-interstitial deposits seen in lupus nephritis or other immune-complex-mediated diseases. The composition of the deposits (polyclonal IgG only), argued against dense deposit disease, in which the deposits usually consist exclusively of C3. Critical to a proper diagnosis is the appreciation of the linear and diffuse quality of the tubular basement membrane deposits. True linear staining is not generally observed in immune complex-mediated diseases (in which IF staining is usually granular and more irregularly/focally distributed). Linear staining is a characteristic feature of anti-TBM nephritis and is analagous to the linear staining of glomerular basement membranes observed in anti-GBM nephritis. In this case, the absence of associated linear staining of the glomerular basement membranes argued against anti-TBM deposits occurring in association with Goodpasture's disease. Moreover, the absence of corresponding electron dense deposits involving the TBM's in the second biopsy supported a diagnosis of anti-TBM disease.

Confirmation of anti-TBM nephritis in this patient was obtained by demonstration of identical linear staining of tubular basement membranes and Bowman's capsule by application of patient's serum to cryostat sections of normal kidney, followed by fluoresceinated rabbit anti-human IgG, in an indirect immunofluorescence technique. Thus the Fanconi syndrome could be attributed to proximal tubular dysfunction caused by anti-TBM nephritis, whereas the albuminuria, (which subsequently reached nephrotic range), could be explained by the co-existent membranous glomerulopathy.

Final Diagnosis
Membranous glomerulopathy and anti-TBM nephritis

Clinical course
The patient was subsequently treated for 6 months with monthly intravenous cyclophosphamide and alternate-day prednisone (3 mg/kg/dose). The titer of anti-TBM antibody fell from 1:10,000 at the start of therapy to 1:50 after six months of treatment. At the end of the 6-month period, the patient's serum creatinine had risen from 0.8 to 1.5 mg/dL, and proteinuria had increased to a Uprot/Ucreat ratio of 1.2. A third renal biopsy was performed in September, 1998. Immunofluorescence in the third biopsy showed a significant decrease in the intensity of staining of both the granular GBM and linear TBM deposits, consistent with treatment effect. However there was considerable increase in chronicity, with approximately 80% cortical scarring. The patient progressed to end-stage renal failure by December 2003 and is preparing for renal transplantation.

Discussion
Anti-TBM nephritis has been reported to occur in association with membranous glomerulopathy in young children ranging from two months to 10 years of age, with strong male predominance of 9:1 [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] . The occurrence of this condition in siblings and the demonstration of certain HLA associations (DrW8 and B7) suggest the importance of predisposing genetic factors [3, 7] . Presentation includes Fanconi syndrome, polydipsia, polyuria, nephrotic proteinuria and renal insufficiency. In some cases membranous glomerulopathy with nephrotic syndrome precedes the development of Fanconi syndrome, whereas in others (such as this case) Fanconi syndrome dominates the initial clinical course, with subsequent development of membranous glomerulopathy. Extrarenal associations are variable and include ocular manifestations [10], autoimmune enteropathy [2, 6] , neonatal insulin dependent diabetes mellitus [8], dermatitis [4] and rarely pulmonary hemorrhage [1]. In this case, the elevated serum amylase raises the question of possible pancreatic involvement. These extrarenal manifestations likely reflect the distribution of a common TIN antigen (i.e. "tubulo-interstitial nephritis antigen") in the basement membranes of the eye, intestine, and skin, as well as the possible presence of other types of autoantibodies.

Anti-TBM antibodies demonstrate species-specific cross-reactivity and react with tubular basement membranes in Brown Norway rat, mouse, guinea pig, dog, sheep and rabbit, but not Lewis rat [12]. The precise nature of the TIN antigen is unknown. By Western blot, the human anti-TBM antibody reacts predominantly with a 58 kD non-collagenous glycoprotein component of tubular basement membranes, (as well as other higher molecular weight bands up to 300 kD in some patients) [12]. The cDNA encoding the TIN antigen has been cloned and sequenced and displays 30% homology to preprocathepsin B, a member of the cysteine proteinase family of proteins [13]. The protein is found predominantly in proximal tubular basement membranes and interacts directly with laminin and type IV collagen, suggesting a role in epithelial adhesion to basement membrane [14]. Because of the co-development of membranous glomerulopathy and anti-TBM nephritis, it is possible that the TIN antibody cross-reacts with a yet unidentified secreted podocyte antigen. This scenario is analogous to that of membranous glomerulopathy occurring in association with anti-GBM nephritis. In the latter condition, membranous glomerulopathy may precede, occur simultaneously with, or follow the development of anti-GBM nephritis [15, 16] . A multi-specific immune response to antigens that are synthesized and secreted by podocytes, similar to that observed in Brown Norway rats injected with mercuric chloride, may be operant [15, 17] . In cases of anti-TBM nephritis and anti-GBM nephritis occurring in association with membranous glomerulopathy, the application of patient's serum to normal kidney by indirect immunofluorescence reveals linear staining of TBM and GBM respectively, without an observable membranous pattern of glomerular staining [11, 15] . This observation suggests that the membranous component develops by an active in vivo process of in situ immune complex formation, possibly involving capping and shedding into the subepithelial space, and fueled by upregulation of antigen synthesis by injured podocytes, as proposed for the Heymann model of membranous glomerulopathy [18]. The possibility of antibody to human podocyte gp600/megalin has been proposed in a case of childhood membranous glomerulopathy and anti-TBM nephritis associated with HLA B7 serotype [9].

Anti-TBM antibodies have been identified in several conditions other than childhood membranous glomerulopathy. In addition to anti-GBM nephritis [19], they may occur rarely in drug-induced interstitial nephritis [20], renal allografts [21], post-streptococcal glomerulonephritis [22] and lupus nephritis [23].

The prognosis for children with anti-TBM nephritis and membranous glomerulopathy is generally poor, with progression to end-stage renal disease within 2-10 years from presentation. Some cases have responded to corticosteroid therapy. Recurrence has been reported in the renal transplant. Plasmapheresis prior to transplantation may protect against recurrence of anti-TBM disease for up to 1 year post transplantation.

References

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