J Am Soc Nephrol 14: S27–S32, 2003 Antenatal Membranous with Vascular Injury Induced by Anti-Neutral Endopeptidase : Toward New Concepts in the Pathogenesis of Glomerular Diseases

HANNA DEBIEC,* VINCENT GUIGONIS,† BE´ ATRICE MOUGENOT,* JEAN-PHILIPPE HAYMANN,* ALBERT BENSMAN,† GEORGES DESCHEˆNES,† and PIERRE M. RONCO* *INSERM Unit 489 Tenon Hospital, Paris, France; and †Department of Pediatric Nephrology, Armand Trousseau Hospital, Assistance Publique-Hoˆpitaux de Paris and University of Paris 6, Paris, France.

Membranous glomerulonephritis is a major cause of nephrotic are shared by the brush border and podocytes and are syndrome and chronic renal insufficiency. This condition may also expressed on the human podocyte (13,14). We therefore be associated with a wide spectrum of infections, cancers, suggested that they might play some role in the pathogenesis of autoimmune diseases, and drugs, although primary forms of membranous glomerulonephritis in humans (13). We recently the disease remain the most common. Histologically, membra- demonstrated that anti-NEP antibodies produced by a pregnant nous glomerulonephritis is characterized by an accumulation of woman were transferred to her fetus, in which a severe form of immune deposits on the outer aspect of the glomerular base- membranous glomerulonephritis developed prenatally (15). ment membrane, but the target antigens have not been The mother had a deficiency of NEP and had become immu- identified. nized against the at the time of or after an earlier Major contributions to our current understanding of the miscarriage. This unusual case opens up new avenues of re- disease come from Heymann nephritis, a rat model of mem- search in the pathogenesis of membranous glomerulonephritis. branous glomerulonephritis induced by an antigenic fraction of the renal brush border (1). This experimental rat model led to the identification of megalin, a unique constitutive antigen Case Report expressed on the podocyte (2,3). Although megalin has been A male infant who was born at 38 wk of gestation (birth found in human proximal tubules, it has not been detected in weight, 3260 g; length, 50 cm) presented with oligoanuria human glomeruli or in immune deposits in patients with mem- (urine volume, 10 ml per 24 h), massive (Table 1), branous glomerulonephritis (4). The antigens responsible for human membranous glomerulonephritis have eluded identifi- and respiratory distress on the first day of life. His parents were cation. Hepatitis B, hepatitis C, and Helicobacter pylori anti- unrelated, healthy individuals without a family history of renal gens; tumor antigens; and thyroglobulin have been detected in or . The mother, aged 24, had had a mis- the subepithelial deposits, but there is no real proof that these carriage at 14 wk of gestation 2 mo before this pregnancy. Her antigens are pathogenic (5–7). Some similarities, such as glo- BP, urinalysis, and serum creatinine concentration were normal merular deposition of renal tubular epithelial antigens, have throughout and after the pregnancy, and she took no medica- been found between experimental Heymann nephritis and in- tions. However, antenatal echography showed oligohydram- dividual cases of membranous glomerulonephritis, but the an- nios and enlarged fetal kidneys from the 34th week of gesta- tigens could not be characterized at the molecular level (8–10). tion. Her levels of antineutrophil cytoplasmic antibodies, We first showed that antibodies directed against two enzy- antinuclear and anti-DNA antibodies, and complement were matic antigens, dipeptidyl-peptidase IV and neutral endopep- normal. tidase (NEP), could be involved in the formation of epimem- Mechanic ventilation was necessary from birth to 10 d. The branous deposits when injected into rodents (11–13). These infant’s serum creatinine concentration was 1.9 mg/dl (170 ␮mol/L) on day 2 and peaked at 2.7 mg/dl (240 ␮mol/L) on day 4. Diuresis increased after intravenous furosemide. The H.D., V.G., and B.M. contributed equally to this work. serum creatinine concentration subsequently decreased, during Correspondence to Dr. Pierre Ronco, INSERM Unit 489, Hoˆpital Tenon, 4 rue which time nephrotic-range proteinuria (Table 1) with hy- de la Chine, 75020-Paris, France. Phone: ϩ33-1-56-01-66-39; Fax: ϩ33-1- 56-01-69-99; E-mail: [email protected] poalbuminemia developed (1.9 g/dl on day 7). Calcium chan- 1046-6673/1400-0027 nel blockers and beta-blockers were needed for BP control Journal of the American Society of Nephrology from day 5 until 6 wk. Urinary excretion progressively Copyright © 2003 by the American Society of Nephrology decreased to 4.2 mg/mg creatinine (0.48 g/mmol) at day 52. DOI: 10.1097/01.ASN.0000067649.64849.75 However, at 4 mo of age, the BP and proteinuria increased, S28 Journal of the American Society of Nephrology J Am Soc Nephrol 14: S27–S32, 2003

Table 1. Time course of serum creatinine and proteinuria Identification of NEP as the Target Antigen of Nephritogenic Antibodies Serum Creatinine Urinary Protein Excretion Age (mg/dl)a (mg/mg creatinine)b Because of the early development of membranous glomer- ulonephritis in this infant, we suspected pregnancy-induced 1 d ND 14.2 immunization of the mother with transplacental passage of 2 d 1.9 15.2 nephritogenic antibodies. This hypothesis was first tested by 4 d 2.7 ND indirect immunofluorescence examination of normal human 5 d 2.2 ND kidneys. A serum sample obtained 9 mo before pregnancy (7 6 d 1.6 14.0 mo before she had a miscarriage) was negative. Serum samples 22 d 1.4 28.3 obtained at 3 mo of gestation and after delivery showed reac- 31 d 1.3 16.2 tivity on the glomerular capillary walls and the brush border on 40 d 0.8 8.4 all kidney biopsy specimens, as did the serum obtained from 52 d 0.6 4.2 the infant 13 d after birth. No reactivity was detected in the 4 mo 0.5 7.9 infant’s serum 40 d after birth. 9 mo 0.6 3.9 The nature of the target antigen was suspected by indirect 11 mo 0.6 1.3 immunofluorescence examination of rabbit and rat kidney sec- tions incubated with the mother’s or the infant’s . The ND, not determined. a To convert values for serum creatinine to micromoles per liter, same pattern as in human kidneys was observed in the rabbit, multiply by 88.4. whereas in the rat, staining was restricted to the cells of b To convert values for urinary protein excretion to grams per Bowman’s capsule and to the brush border of deep cortical millimole urinary creatinine, multiply by 0.113. segments of the proximal tubule. We had previously observed identical interspecies differences with anti-NEP antibodies, whereas distribution of dipeptidyl peptidase IV is not species dependent (14). The mother’s IgG antibody and the infant’s although the serum creatinine concentration remained normal IgG antibody recognized by Western blotting a single antigen (Table 1). Symptoms of serum sickness were not observed at of approximately 90 kD in protein extracts from rat brush any time. border, rabbit kidney cortex, and cultured human podocytes. A computed tomography–guided kidney biopsy was per- This antigen had the same electrophoretic mobility as NEP that formed at 4 wk of age (Figure 1). The biopsy specimen showed was specifically immunoprecipitated from rat brush border a severe, unusual form of membranous glomerulonephritis. with the mother’s IgG, whereas it was detected only in the Capillary tufts were collapsed in the majority of the 40 glo- unbound fraction of the control immunoprecipitation. Further- meruli. Most Bowman’s spaces were also distended. In all more, NEP activity was found in the eluted fraction from glomeruli, there was a thickening of the capillary walls; such material bound to the mother’s IgG but not in the one eluted thickening was most apparent in noncollapsed glomerular tufts from control IgG. in which capillary loops showed spikes. Marked tubular atro- The anti-NEP antibodies produced by the mother, which phy and severe lesions of the interlobular arteries and arterioles were found in the infant’s serum 13 d after birth, were most were also observed. Immunofluorescence studies showed likely responsible for the infant’s membranous glomerulone- marked subepithelial deposits of IgG, C3, and C5b-9 in all phritis, given that the injection of rabbits with the serum IgG glomeruli. No immune deposits were seen in proximal tubules fraction from the mother induced intraglomerular deposits and and vessels. Electron microscopy examination revealed diffuse proteinuria, whereas injection with the IgG fraction from the alterations of glomerular capillary walls and a marked atrophy father did not (Figure 2, D and G). Furthermore, NEP was of the brush border. Abundant, electron-dense deposits were localized by confocal microscopy in immune deposits both in seen on the outer aspect of the glomerular basement mem- the infant and in the rabbits injected with the mother’s IgG brane. These deposits contained annular formations, often (Figure 2). overlaid by an expansion of the lamina densa. There were A similar case of transplacental induction of membranous neither subendothelial nor mesangial deposits. nephropathy in a neonate was reported in 1990 (16). Recent Tests for neonatal syphilis, toxoplasmosis, cytomegalovirus, studies from our laboratory indicate that the mother’s serum, and hepatitis B virus infection were negative. The Coombs’ provided by Dr. De Heer (Leyden, The Netherlands), contain test was negative, and complement components were normal at anti-NEP antibodies (Debiec et al., unpublished data). day 35 (C3, 0.97 g/L; C4, 0.25 g/L). Low levels of circulating immune complexes (4.0 ␮g/ml) were detected in the serum at Mechanisms of the Deposition of Immune Complexes in day 13, using an enzyme immunoassay kit. This activity was the Infant’s Glomeruli no longer detected at day 40. Membranous glomerulonephritis has long been considered Clinical examination at 11 mo was unremarkable, although as a model of immune glomerulopathy because of the deposi- nicardipine (2 mg/kg body wt per d) was required to control tion of preformed circulating immune complexes. Initial stud- BP. The serum creatinine concentration was normal, and uri- ies of the active model of Heymann nephritis suggested that the nary protein excretion had markedly decreased (Table 1). subepithelial deposits resulted from glomerular trapping of J Am Soc Nephrol 14: S27–S32, 2003 Antenatal Membranous Glomerulonephritis Induced by Anti-NEP Antibodies S29

Figure 1. Infant’s kidney biopsy specimen obtained at 4 wk. (A) Collapsed capillary tufts, with prominent tubular atrophy and mild interstitial cellular infiltration and fibrosis. (B) Thickening of capillary walls in a noncollapsed glomerulus, conspicuous lesions of an interlobular artery, and severe alterations of the proximal tubule epithelium. (C) Frozen section incubated with FITC-labeled anti-human IgG antibody revealing heavy epimembranous granular deposits. (D) Representative segment of the capillary wall analyzed by electron microscopy (bar, 200 nm). From Debiec et al., N Engl J Med 346: 2053–2060, 2002 (Copyright 2002 Massachusetts Medical Society. All rights reserved). Magnification: ϫ170 in A (trichrome), ϫ430 in B (trichrome), ϫ400 in C. circulating immune complexes formed by circulating brush were low, manifestations of serum sickness were absent, and border–related antigens and the corresponding antibodies. This subendothelial and mesangial immune deposits were not seen. hypothesis was based on the observation that the glomerular Immune complexes could also be formed in situ at the sole of disease was induced by fractions of membrane prepared from podocyte foot processes where NEP is expressed (17). The two rat renal brush border, not from glomerular extracts. Subse- mechanisms are not mutually exclusive. quently, the development of passive Heymann nephritis led to Contrary to megalin, NEP is expressed in a diffuse pattern the suggestion that subepithelial Ig deposits could be formed on the membrane of podocytes, as is angiotensin-converting without the intervention of circulating immune complexes. The enzyme on the plasma membrane of mature oocytes (18). In latter hypothesis was confirmed by the demonstration of mega- vivo interaction of angiotensin-converting enzyme with diva- lin expression in the coated pits of rat podocytes, particularly at lent antibodies induces the formation of granular immune the sole of foot processes facing the glomerular capillary wall deposits through a mechanism of “patching” and “shedding” of (2). immune complexes (18). A similar mechanism may be impli- Transient low levels of circulating immune complexes were cated in the formation of immune deposits in the infant’s detected in the infant’s serum at day 13. The immune com- glomeruli. One can speculate that the immune complexes that plexes isolated from the serum sample contained NEP, as are shed from the foot processes are sequestered between the demonstrated by Western blotting (15). However, their contri- lamina rara externa of the glomerular capillary wall and the bution to the formation of subepithelial immune deposits is podocytes’ slit diaphragms, whereas those that are shed from uncertain, because levels of circulating immune complexes the podocyte cell bodies are excreted in the infant’s urine. S30 Journal of the American Society of Nephrology J Am Soc Nephrol 14: S27–S32, 2003

Figure 2. Colocalization of neutral endopeptidase (NEP) and IgG in immune deposits and induction of renal disease in rabbit by maternal IgG. (A to C) Immunofluorescence staining of kidney sections from the infant’s biopsy specimen after double labeling with anti-human IgG antibodies (A) and polyclonal anti-NEP antibody (B). (C) The merged image. The insets show the colocalization of NEP and IgG on the outer aspect of the capillary wall. (D to G) Immunofluorescence staining of kidney sections from rabbits injected 4 d earlier with mother’s(DtoF) or father’s (G) IgG fractions, respectively. The sections in D to F were double labeled with anti-human IgG antibodies (D) and with polyclonal anti-NEP antibody (E); the merged image is shown in F. The section in G was labeled with anti-human IgG antibodies. From Debiec et al., N Engl J Med 346: 2053–2060, 2002 (Copyright 2002 Massachusetts Medical Society. All rights reserved). Magnification: ϫ600 in A–G, ϫ2000 in insets.

Mechanisms of the Immunization against NEP in the findings (16). It is likely that additional individuals who lack Infant’s Mother NEP will be identified and that additional cases of acute renal Because the mother had no apparent renal abnormalities failure and membranous glomerulonephritis in the neonate may despite high serum titers of anti-NEP antibody, we hypothe- be ascribed to anti-NEP alloantibodies. sized that she might be deficient in NEP and analyzed NEP expression in granulocytes from both parents. Fluorescence- Potential Role of the Blockade of the Enzymatic activated cell sorter analysis of the mother’s granulocytes Activity of NEP by Nephritogenic Antibodies incubated with either anti-NEP monoclonal antibody or the NEP (neprilysin, enkephalinase, CD10, EC 3.4.24.11) is a mother’s serum sampled 5 wk after delivery showed no NEP at 90- to 110-kD zinc-dependent metallopeptidase, identical to the cell membrane. Cell extracts prepared from maternal gran- the common acute lymphoblastic leukemia antigen (21,22). It ulocytes failed to react with either monoclonal or polyclonal is expressed in brain tissue; on polymorphonuclear leukocytes antibodies against NEP after Western blotting. Moreover, the and lymphoid progenitor cells; and on epithelial cells within mother’s serum reacted with the father’s granulocytes but not nonlymphoid organs such as kidney, liver, breast, and lung with her own granulocytes, suggesting an alloimmunization (23,24). It is also found in the serum and the urine (25,26). This process. Alloimmunization in the mother most likely occurred enzyme is involved in the metabolism of a number of regula- at the time of her miscarriage, given that a plasma sample tory peptides and plays an important role in turning off peptide obtained earlier did not show anti-NEP antibodies. Despite the signaling events at the cell surface (27). In the human kidney, absence of NEP, the mother was healthy, as were mice with a NEP is found on brush border, podocyte, and vascular smooth targeted disruption of the NEP gene, suggesting enzymatic muscle cells (14,28). redundancy (19). For evaluating a potential effect of anti-NEP antibodies on Renal injury mediated by alloimmune responses to major enzymatic activity, lysates of human podocytes were preincu- renal antigens was first described in tubular basement mem- bated with maternal or paternal IgG. The endopeptidase 24.11 brane in rat (20). A previously reported case of neonatal activity of podocyte lysates was blocked by its specific inhib- membranous glomerulonephritis might also involve NEP de- itors thiorphan and phosphoramidon and was also dose-depen- ficiency and alloimmunization because the mother had no renal dently inhibited by maternal but not paternal IgG. J Am Soc Nephrol 14: S27–S32, 2003 Antenatal Membranous Glomerulonephritis Induced by Anti-NEP Antibodies S31

The infant’s kidney biopsy specimen showed unusually se- component of the NAPDH oxidoreductase complex (35). In vere arterial lesions without immune deposits and a collapsing addition, major changes in the distribution of nephrin was of glomerular capillary tufts that was suggestive of major renal observed in patients with primary acquired nephrotic syndrome ischemia during prenatal development. These lesions may re- (36). In glomeruli of patients with membranous glomerulone- sult from the enzymatic activity of NEP as it cleaves vasoactive phritis, a more granular pattern or a loss of staining of nephrin mediators, including bradykinin, atriopeptin, and endothelins, was observed. In cultures of human podocytes, aggregated but and thus may modify local flows (27,28). Because the not disaggregated human IgG4, plasmalemmal insertion of the mother’s antibodies inhibited NEP activity, their transplacental membrane attack complex of complement, TNF-␣, and puro- passage might increase concentrations of vasoconstrictor pep- mycin, induced the shedding of nephrin with a loss of surface tides in the vascular wall and thus induce the proliferation of expression. This phenomenon was abrogated by cytoskeleton vascular smooth muscle cells. inhibitors. Several enzymatic antigens diffusely expressed on podo- We suggest that the binding of the nephritogenic anti-NEP cytes have been implicated in the development of membranous antibodies may result in alterations of the podocytic phenotype, glomerulonephritis in rodents: NEP, dipeptidyl-peptidase IV, such as changes in the cytoskeleton, allowing shedding of and aminopeptidase A (11–13,29). A single injection of a important podocytic from plasma membrane to an relatively large amount of an anti-aminopeptidase A monoclo- extracellular site and production of toxic mediators including nal antibody that was able to block the enzyme activity in vitro collagenases and oxygen radicals. Further research in this area induced an acute albuminuria that lasted for at least 16 d and should help elucidate the pathogenic mechanisms involved in subepithelial Ig deposits (29). Because aminopeptidase A is a the induction of albuminuria. key enzyme in the intrarenal degradation of angiotensin II, it In conclusion, we have identified the first target antigen of was hypothesized that angiotensin II mediated the induction of nephritogenic antibodies in human membranous glomerulone- this acute albuminuria. This assumption was supported by the phritis. This antigen exhibits with an enzymatic activity that observations that non–enzyme-inhibiting monoclonal antibod- could play some role in the pathogenesis of the nephropathy. ies never induced an acute albuminuria and that enalapril and We have also identified the first person with NEP deficiency. losartan reduced the acute albuminuria by Ͼ90%. However, angiotensinogen-deficient mice also developed albuminuria upon injection of a combination of enzyme-inhibiting anti- Acknowledgments aminopeptidase A monoclonal antibodies (30), thus demon- This study was funded by INSERM, the University of Paris 6, and strating that angiotensin II is not required for the induction of a private donation from Mr. And Mrs. Halpin. We thank Christine Vial for excellent secretarial assistance. albuminuria in this model. 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