Case 5 -

Recurrent Membranous Nephropathy with Spherular Deposits Charles E. Alpers University of Washington Medical Center Seattle, WA

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Clinical History The patient is a 60 year-old female who received a 2-antigen match kidney transplant from a 36 year-old living unrelated donor. The patient's original disease was established by renal biopsy 20 years prior to transplantation. Her renal presentation was that of an asymptomatic low serum albumin and subnephrotic proteinuria detected during an evaluation for pneumonia at that time. The patient was also found to have an elevated serum creatinine of 2.5 mg/dl. Laboratory workup at that time was unrevealing, and a renal biopsy established a diagnosis of membranous nephropathy based on characteristic immunofluorescence and electron microscopic findings. Slowly progressive renal insufficiency ensued over two decades, without development of nephrotic syndrome and without treatment directed to the membranous nephropathy, leading to ESRD and transplantation. The only significant concurrent medical disease was hypertension, controlled with multidrug regimens. Noteworthy aspects of her clinical presentation were an intermittently positive ANA serology and a family history that included a brother with an elevated serum creatinine, a niece with systemic lupus, and one child (out of five) with an episode of "nephritis" at age 5 that resolved with steroid therapy and did not recur. At the time of transplantation, the patient was EBV, IgG positive but EBNA and CMV negative. The donor was EBV positive and CMV negative. Induction immunosuppression utilized steroids and thymoglobulin, with sequential introduction of mycophenolate and neoral cyclosporine. The patient had immediate graft function following transplantation, with a serum creatinine falling to 0.3 mg/dl on the day of transplantation and maintained between 0.8 and 1.0 mg/dl in the months thereafter. Her post-transplant course was initially unremarkable, complicated only by mild fluctuations in blood pressure, and she was maintained on a triple immunosuppression regimen of prednisone, cyclosporine, and mycophenolate. Three evaluations in the first year following transplantation failed to detect significant proteinuria. Approximately one year post-transplant she noted weight gain. She had a measured 24-hour urinary protein excretion of 2.0 gm. Her serum creatinine remained in the 1.1 mg/dl range. The renal biopsy available to you was performed 14 months following transplantation to evaluate the basis for her increasing proteinuria. Case 5 - Figure 1 - This is a low-power view of the renal biopsy showing patent glomeruli and a well-preserved tubulointerstitial parenchyma. Jones' silver methenamine stain. Case 5 - Figure 2 - This intermediate power micrograph shows intact glomeruli and arterial vessels without evidence of vasculitis or significant sclerosis. There is a very mild and focal interstitial mononuclear inflammatory cell infiltrate, without features of prominent interstitial edema or tubulitis. Jones' silver methenamine stain. Case 5 - Figure 3 - This representative glomerulus shows well-preserved glomerular architecture without significant alterations to the mesangium. A few circulating leukocytes are present within glomerular capillary loops. Case 5 - Figure 4 - A high-power micrograph shows irregular thickening of the glomerular capillary loops with foci of rarefactions and a hint of "spike" formation. Jones' silver methenamine stain. Case 5 - Figure 5 - This H&E stained section of the same glomerulus shows the presence of occasional leukocytes within glomerular capillary loops but otherwise relatively normal cellularity of the glomerular tuft. Case 5 - Figure 6 - This immunofluorescence micrograph shows deposition of somewhat homogeneous, granular deposits of IgG along the glomerular capillary walls. Similar but less intense staining was seen for C3 and kappa and lambda light chains. Case 5 - Figure 7 - This electron micrograph shows extensive accumulations within the subepithelial aspect of the glomerular basement membranes of spherular particles having electron lucent cores. There are irregular accumulations of basement membrane matrix, some of which appear to separate accumulations of the spherular particles. Overlying epithelial cells show extensive effacement of foot processes. Occasional circulating leukocytes are present within glomerular capillary loops. Case 5 - Figure 8 - These are higher power electron micrographs showing in greater detail the accumulations of spherular particles in the subepithelial aspects of the basement membranes. There are focal epimembranous projections of basement membrane matrix ("spikes"), best seen here. Case 5 - Figure 9 - These are higher power electron micrographs showing in greater detail the accumulations of spherular particles in the subepithelial aspects of the basement membranes. There are focal epimembranous projections of basement membrane matrix ("spikes"), best seen in Figure 8. Pathologic Findings Figs. 1 and 2 (Jones' silver methenamine stains) of the allograft are low and intermediate magnification surveys of the allograft biopsy showing the absence of features of rejection (interstitial inflammation, edema, prominent tubulitis, or arterial inflammation). Figs. 3-5 (Jones' silver methenamine stains in Figs. 3 and 4) show typical glomeruli, which contain widely patent capillary loops, lack prominent inflammatory cell infiltration, hypercellularity, or evidence of sclerosis, and which demonstrate irregular thickening and vacuolization of the basement membranes. Fig. 6 shows granular deposition of IgG along the glomerular capillary walls. Similarly distributed but less intense staining was present for C3 and kappa and lambda light chains. The electron micrographs (Figs. 7-9) show intramembranous accumulations of membrane bound spherular structures of generally uniform size (approximately 85 nm in diameter) with electron lucent cores. There are also irregular accumulations of ill-defined lamellar material, most suggestive of basement membrane matrix. Discrete, homogeneously electron dense immune type deposits were not identified. Visceral epithelial cells show extensive effacement of foot processes. Mesangial areas and extra-glomerular structures were free of the deposits and without specific pathologic alterations. Diagnostic Interpretation We believe the histologic appearance and immunopathologic findings are sufficient to establish this case as an example of membranous nephropathy in an allograft kidney. Were tissue not procured for electron microscopy, it is unlikely that any other diagnosis would be given major consideration. However, the ultrastructural findings are unusual. The typical electron dense and/or electron lucent deposits of membranous nephropathy are lacking, and instead the deposits are those of aggregates of spherular vesicles of uniform size and appearance. Such deposits have been occasionally reported in single case reports and small series of patients with membranous and other glomerulopathies [1, 2, 3] . As discussed below, their pathogenesis remains unknown. The history of a prior biopsy of the native kidney performed 21 years previously prompted us to retrieve the original biopsy and tissue block. Electron microscopy of the prior biopsy was not available, so tissue obtained from the paraffin block was utilized for new ultrastructural studies. We were able to demonstrate that the membranous nephropathy in the native kidney was characterized by histologic and ultrastructural features identical to those in the allograft kidney. Our Diagnosis: Membranous nephropathy in an allograft kidney, recurrent, with atypical spherular deposits. Discussion The prevalence of de novo and recurrent membranous nephropathy in renal allografts is difficult to ascertain precisely, but with the advent of multi-insitutional transplant registries, it may become easier to obtain such data. Several recent reports from such registries indicate membranous nephropathy is the third most frequent type of glomerulonephritis resulting in graft loss [4, 5, 6] . Most of these cases of membranous nephropathy are the result of de novo disease in the allograft, which has been estimated to occur four times as often as recurrent membranous nephropathy. While there are many difficulties in establishing the incidence of recurrent membranous nephropathy due to such variables as poor biopsy documentation of the original renal disease and non-standardized biopsy practices for allograft kidneys with chronic renal dysfunction, recent data from transplant registries indicates clinically significant membranous nephropathy (presenting with significant proteinuria or nephrotic syndrome) may have a recurrence rate of 7-26% (although a few single center reports report higher incidences) [7, 8, 9, 10, 11, 12, 13] resulting in a 5-10% incidence of graft loss in all patients transplanted for this disease [7]. The overall incidence of recurrent membranous nephropathy remains low, largely because the epidemiology and clinical features of membranous nephropathy are such that relatively few young or middle-aged patients progress to ESRD with this disorder. It has been estimated that patients with membranous nephropathy constitute only a small proportion (less than 5%) of patients transplanted for glomerular disorders [7, 14] . It is reasonable to suggest that evaluation of membranous nephropathy in a kidney transplant recipient include investigation for infection by hepatitis B and C viruses. Case reports and single institutional series with such associations have been reported [15], but proof of the pathogenetic role of these viruses is currently lacking. There are no specific risk factors (e.g., degree of antigen matching, HLA type, specific immunosuppression protocol employed) that have been shown to influence the risk of recurrence. Therapy for membranous nephropathy in kidney allografts is as unsettled as therapy for this disease in native kidneys. Our understanding of the pathogenetic basis for immune complex deposition in membranous nephropathy has remained unsatisfactory. While concepts of the formation of immune complexes with subsequent activation of complement in the subepithelial aspect of the glomerular basement membrane are now well established [16], the identification of the inciting antigens in humans generally remains to be accomplished. In recent years, based on studies of the Heymann nephritis model in rats, the existence of antigens derived from podocytes has been the leading hypothesized target of the immune response leading to membranous nephropathy. Until the recent studies of Debiec and Ronco, et al, which demonstrated rare cases in which neutral endopeptidase can serve as a target antigen, the identity of putative podocyte antigens in humans remained unknown [17, 18] . We and others have been intrigued by the report of Dales and Wallace, which provided evidence that the spherular particles seen in a case of membranous nephropathy similar to ours may be derived from nuclear pores [19]. Nuclear pores are unique and complex structures, measuring 80-120 nm in diameter, depending on cell type and species. These structures are embedded in the nuclear envelope of cells and possess central channels that allows trafficking of ions and small molecules between the nucleus and cytoplasm [20]. In the case reported by Dales and Wallace, the morphology and size of the spherules suggested that they might be nuclear pores. Analysis of the patient's serum using immunofluorescence studies showed that antibodies were present that bound to discrete structures in the nuclear membranes of several cultured mammalian cell lines, further supporting their hypothesis. We are unaware of other studies that further elucidate the origin of these structures. Our group at the University of Washington, with contributions from colleagues at Vanderbilt University and Oregon Health Sciences University, has assembled a series of 13 patients, including the patient presented here, with membranous nephropathy in native kidneys with predominant deposition of spherules. Work by Jolanta Kowalewska, Kelly Smith, and myself that currently is being prepared for publication sought to test this hypothesis further using a variety of antibody probes to nuclear pore proteins. This work will be summarized at the conference. To date, our studies have not substantiated the hypothesis that nuclear pores serve as the underlying antigenic target resulting in spherular deposits. A second possibility for the origin of these structures was suggested by studies of the group of Debiec and Ronco, cited above. Their identification and very elegant study of a case of antenatal induction of membranous nephropathy in a neonate resulted in the demonstration of neutral endopeptidase on podocytes as an antigenic target for membranous nephropathy. They showed that this was the result of transfer of maternal antibodies in utero after the mother, with functional inactivation of the expression of this antigen, became sensitized to its expression on fetal leukocytes. Noteworthy is Figure 1 of their 2002 NEJM paper first reporting such a case, in which the ultrastructural pathology of the membranous nephropathy lesion reveals accumulations of microspherules that appear morphologically similar to those of the case under discussion here [17]. Again, Dr. Kowalewska in my laboratory has performed preliminary studies, using a commercially available antibody to neutral endopeptidase, to see if the findings of the French group may be generalizable to our cases. To date, we have been unable to detect expression of the neutral endopeptidase antigen recognized by the antisera by the spherular particles in cases in our series. In summary, the origin of the spherular structures remains obscure. The finding that this rare type of membranous nephropathy has recurred in an allograft suggests that these cases form a distinct subset of a common disease with a unique but specific pathogenesis. References Burkholder PM, Hyman LR, Barber TA. Extracellular clusters of spherical microparticles in glomeruli in human renal glomerular diseases. Lab Invest 1973; 28:415-425. Akhtar M, Bunuan H, Al-Mohaya S. Renal lesion in a 50-year-old Saudi man. Ultrastruct Pathol 1983; 4:107-113. Barry M, Rennke HG. "Nuclear pore" glomerulopathy: A clinicopathological study. Modern Pathol 2004; 17:284A (abstract). Hariharan S, Johnson CP, Bresnahan BA, et al. Improved graft survival after renal transplantation in the United States, 1988 to 1996. 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