Antenatal Membranous Glomerulonephritis Due to Anti–Neutral Endopeptidase Antibodies
Hanna Debiec, Ph.D., Vincent Guigonis, M.D., Béatrice Mougenot, M.D., Fabrice Decobert, M.D., Jean-Philippe Haymann, M.D., Albert Bensman, M.D., Georges Deschênes, M.D., Ph.D., and Pierre M. Ronco, M.D., Ph.D.
Membranous glomerulonephritis, a major cause of the nephroticsyndrome and chronic renal insufficiency, is associated witha wide spectrum of infections, cancers, autoimmune diseases,and drugs. The condition is characterized by an accumulationof immune deposits on the outer aspect of the glomerular basementmembrane, but the target antigens have not been identified.Major contributions to our current understanding of the diseasecome from Heymann's nephritis, a rat model of membranous glomerulonephritisinduced by immunization with an antigenic fraction of the renalbrush border.1 Studies of this experimental rat model led tothe identification of megalin, a unique constitutive antigenexpressed on the podocyte.2,3 Although megalin has been foundin human proximal tubules, it has not been found in human glomerulior in immune deposits in patients with membranous glomerulonephritis.4Dipeptidyl-peptidase IV and neutral endopeptidase are two otherantigens shared by the brush border and podocytes that are involvedin the formation of immune deposits in animal models; thesetwo proteins are expressed on the human podocyte.5,6
In this article, we report that anti–neutral endopeptidaseantibodies produced by a pregnant woman were transferred toher fetus, in which a severe form of membranous glomerulonephritisdeveloped prenatally. The mother had a deficiency of neutralendopeptidase and probably had become immunized against theantigen at the time of or after an earlier miscarriage.
Case Report
A male infant born at 38 weeks of gestation (birth weight, 3260g; length, 50 cm) presented with oligoanuria (urine volume,10 ml per 24 hours), massive proteinuria (Table 1), and respiratorydistress on the first day of life. His parents were unrelated,healthy persons without a family history of renal or autoimmunedisease. The mother, who was 24 years old, had had a miscarriageat 14 weeks of gestation 2 months before she became pregnantwith this child. Her blood pressure, findings on urinalysis,and serum creatinine concentration were normal throughout andafter the pregnancy, and she took no medications. However, antenatalultrasonography showed oligohydramnios and enlarged fetal kidneysfrom the 34th week of gestation. The mother's level of antineutrophilcytoplasmic antibodies, antinuclear antibodies, anti-DNA antibodies,and complement were normal.
Table 1. Serum Creatinine Concentration and Proteinuria in the Infant over Time.
Mechanical ventilation for hypoxemia was necessary from birthto 10 days. The infant's serum creatinine concentration was1.9 mg per deciliter (170 µmol per liter) on day 2 andpeaked at 2.7 mg per deciliter (240 µmol per liter) onday 4. Diuresis increased after the administration of intravenousfurosemide. The serum creatinine concentration subsequentlydecreased, and nephrotic-range proteinuria developed (Table 1),as did hypoalbuminemia (1.9 g per deciliter on day 7). Calcium-channelblockers and beta-blockers were needed for blood-pressure controlfrom day 5 until 6 weeks after birth. Urinary protein excretionprogressively decreased to 4.2 mg per milligram of creatinine(0.48 g per millimole) on day 52. However, at four months ofage, the blood pressure and proteinuria increased, althoughthe serum creatinine concentration remained normal (Table 1).Symptoms of serum sickness were not observed at any time.
A kidney biopsy guided by computed tomography was performedat four weeks of age. Tests for neonatal syphilis, toxoplasmosis,cytomegalovirus, and hepatitis B virus infection were negative.A Coombs' test was negative, and the levels of complement componentswere normal on day 35 (C3, 0.97 g per liter; C4, 0.25 g perliter). Low levels of circulating immune complexes (4.0 µgper milliliter) were detected in the serum on day 13, with theuse of an enzyme immunoassay kit (CIC-C1q, Quidel). This activitywas no longer detected on day 40.
Findings on clinical examination at 11 months were unremarkable,although nicardipine (2 mg per kilogram of body weight per day)was required to control blood pressure. The serum creatinineconcentration was normal, and urinary protein excretion hadmarkedly decreased (Table 1). The parents gave written informedconsent for the studies described below.
Methods
Analysis of Renal-Biopsy Specimen and Immunohistochemical Studies
The infant's renal-biopsy specimen was prepared for light, immunofluorescence,and electron microscopy by standard techniques. Serum samplesfrom both the mother and the infant were assayed by indirectimmunofluorescence after incubation with cryostat sections ofkidney. Serum samples had been obtained from the mother sevenmonths before her earlier miscarriage and at three months ofgestation during the pregnancy with this child; we obtainedadditional samples five weeks and seven months after delivery.Serum samples were obtained from the infant 13 and 40 days afterbirth. We also examined biopsy specimens containing normal glomerulifrom seven different patients ranging in age from 4 to 70 years,as well as normal rabbit and rat kidneys.
Western Blotting, Immunoprecipitation, and Enzymatic-Activity Analyses
Fractions of membrane were prepared from rat renal brush borderand rabbit kidney cortex, as previously described.7,8 Fractionsof this membrane, cultured human podocytes,9 and granulocytesfrom the mother and father were lysed, separated by 7 percentsodium dodecyl sulfate–polyacrylamide-gel electrophoresis(SDS-PAGE) under reducing conditions, and analyzed by Westernblotting with serum from the mother or control serum and withmonoclonal (Novocastra) or rabbit polyclonal (Santa Cruz Biotechnology)anti–neutral endopeptidase antibodies. Circulating immunecomplexes were isolated by precipitation of the serum sampleobtained from the infant on day 13 with polyethylene glycol-6000and were analyzed by Western blotting with anti–neutralendopeptidase antibodies.10
For immunoprecipitation experiments, rat renal brush-bordermembranes were lysed in immunoprecipitation buffer and centrifuged.The supernatant was incubated with serum from the mother orcontrol serum. The antigen–antibody complexes were isolatedby an immunoprecipitation system (Immuno-catcher, Cytosignal)with the use of protein A–G resin. The bound complexesand unbound material were analyzed by SDS-PAGE and, after blotting,were incubated with anti–neutral endopeptidase antibody.
To measure the enzymatic activity of the antigen identifiedwith the use of maternal antibodies, IgG fractions from themother's serum or control serum were bound to CNBr-activatedSepharose 4B (Pharmacia Biotech) as recommended by the manufacturer,incubated with rat renal brush-border lysates, and washed, andthen the antigen was eluted with 0.05 M diethylamine at pH 11.0and immediately neutralized.11 The enzymatic activity of neutralendopeptidase was measured by a coupled assay with the use ofSuc-Ala-Ala-Phe-pNA (Bachem Bioscience) and aminopeptidase N(Roche Diagnostics).12
Transfer of Disease to Rabbits
Two female New Zealand white rabbits were injected intravenouslywith 10 mg of IgG from the infant's mother or father preparedon a Sepharose 4B–coupled protein A column (Pharmacia).Four days later, the animals were killed, and their kidneyswere processed as described above. Three other rabbits wereinjected with 5 mg of IgG from the infant's mother or fatherand followed for up to six weeks.
Analysis of the Composition of the Glomerular Immune Deposits by Confocal Microscopy
Cryosections of the biopsy specimen from the infant and of thekidneys from the injected rabbits were first incubated withfluorescein-isothiocyanate–conjugated antihuman IgG antibodies,then with goat polyclonal anti–neutral endopeptidase antibodies(Santa Cruz), followed by rhodamine-conjugated antigoat IgGantibodies (Chemicon). After being washed, sections were examinedunder a confocal microscope.
Flow Cytometric Analysis of Neutral Endopeptidase Expression on Granulocytes
After lysis of red cells, granulocytes from each parent wereincubated with a monoclonal antibody against neutral endopeptidaseor with serum from both parents, then incubated with fluorescein-isothiocyanate–conjugatedsecondary antibodies. Results were analyzed on a flow cytometer(Elite, Beckman Coulter).
Results
Analysis of the Renal-Biopsy Specimen from the Infant
The renal-biopsy specimen from the infant showed a severe, unusualform of membranous glomerulonephritis. Capillary tufts werecollapsed in the majority of the 40 glomeruli (Figure 1A). MostBowman's spaces were also distended. In all glomeruli, therewas a thickening of the capillary walls; such thickening wasmost apparent in noncollapsed glomerular tufts (Figure 1B),in which capillary loops showed spikes. Marked tubular atrophy(Figure 1A) and severe lesions of the interlobular arteriesand arterioles (Figure 1B) were also observed.
Figure 1. Renal-Biopsy Specimen Obtained from the Infant at Four Weeks.
Panel A shows collapsed capillary tufts, with prominent tubular atrophy and mild interstitial cellular infiltration and fibrosis (trichrome stain, x170). Panel B shows thickening of capillary walls in a noncollapsed glomerulus, conspicuous lesions of an interlobular artery, and severe alterations of the epithelium of the proximal tubule (trichrome stain, x430). Panel C shows a frozen section incubated with fluorescein-isothiocyanate–labeled antihuman IgG antibody, revealing heavy epimembranous granular deposits (x400). Panel D shows a representative segment of the capillary wall analyzed by electron microscopy (bar, 200 nm).
Immunofluorescence studies showed marked subepithelial depositsof IgG (Figure 1C) and C3 (not shown) in all glomeruli. No immunedeposits were seen in proximal tubules and vessels. Examinationby electron microscopy revealed diffuse alterations of glomerularcapillary walls and a marked atrophy of the brush border. Abundant,electron-dense deposits were seen on the outer aspect of theglomerular basement membrane. These deposits contained annularformations (Figure 1D), often overlaid by an expansion of thelamina densa. There were neither subendothelial nor mesangialdeposits.
Analysis of Antibodies in Serum Samples from the Mother and the Infant
Because of the early development of membranous glomerulonephritisin this infant, we suspected pregnancy-induced immunizationof the mother with transplacental passage of nephritogenic antibodies.This hypothesis was first tested by indirect immunofluorescenceexamination (Figure 2A, 2B, 2C, and 2D). A serum sample obtainedfrom the mother nine months before she became pregnant withthis child (seven months before she had a miscarriage) was negative(Figure 2A). Serum samples obtained at 3 months of gestation(not shown) and at 5 weeks (Figure 2B) and 7 months (not shown)after delivery showed reactivity on the glomerular capillarywalls and the brush border on all kidney biopsy specimens, asdid the serum obtained from the infant 13 days after birth (Figure 2C).No reactivity was detected in the infant's serum 40 daysafter birth (Figure 2D).
Figure 2. Immunohistochemical and Immunochemical Analyses of the Target Antigen Recognized by the Mother's Antibodies.
The reactivity of serum samples was analyzed by indirect immunofluorescence staining of biopsy specimens from human kidneys (Panels A, B, C, and D) and from normal rabbit (Panel E) and rat (Panel F) kidneys (x312). Serum samples were obtained from the mother nine months before she became pregnant with this child (Panel A) and five weeks after delivery (Panels B, E, and F). Serum samples were obtained from the infant 13 days after birth (Panel C) and 40 days after birth (Panel D). Serum dilutions are 1:50 in Panels A and D, 1:1000 in Panels B and F, and 1:100 in Panels C and E. Panel G shows immunoblots of protein extracts from rat brush border, rabbit cortex, and human podocytes (lanes 1, 2, and 3, respectively) incubated with anti–neutral endopeptidase monoclonal antibody (anti-NEP mAb), serum obtained from the mother five weeks after delivery (dilution, 1:4000), and control serum (dilution, 1:250). The same band of approximately 90 kD was detected in all tissue extracts incubated either with anti–neutral endopeptidase antibody or with the mother's serum. Panel H shows the results of immunoprecipitation (IP) studies. Rat renal brush-border membranes were lysed and incubated with the mother's serum obtained five weeks after delivery or control human serum. Antigen–antibody complexes were immunoprecipitated and were analyzed by Western blotting with anti–neutral endopeptidase monoclonal antibody. A 90-kD band was detected primarily in the immunoprecipitate obtained with the mother's serum (lane 2), whereas in the control, all anti–neutral endopeptidase immunoreactivity rested in the flow-through fraction (lane 1). Panel I shows an immunoblot of circulating immune complexes (CIC) isolated from the infant's serum on day 13, incubated with anti–neutral endopeptidase antibody.
To identify the target antigen, two sets of experiments wereperformed. First, positive serum samples were studied by indirectimmunofluorescence microscopy on sections from rabbit and ratkidneys. The same pattern observed in the sections from humankidneys was found in the rabbit kidney (Figure 2E), whereasin the rat kidney, staining was restricted to cells of Bowman'scapsule and to the brush border of deep cortical segments ofthe proximal tubule (Figure 2F). We had previously observedidentical interspecies differences with anti–neutral endopeptidaseantibodies, whereas the distribution of dipeptidyl-peptidaseIV is not species-dependent.6 Second, antibody specificity wasanalyzed by immunochemical and enzymatic techniques. The mother'sIgG antibody (Figure 2G) and the infant's IgG antibody (at 13days, not shown) recognized a single antigen of approximately90 kD in protein extracts from rat brush border, rabbit kidneycortex, and human podocytes. This antigen had the same electrophoreticmobility as neutral endopeptidase (Figure 2G).
To confirm that neutral endopeptidase was the reactive antigen,immunoprecipitation experiments were performed by incubatingrat brush border with either the mother's serum or control serum,and the bound and unbound fractions were incubated after blottingwith anti–neutral endopeptidase antibody. Neutral endopeptidasewas identified primarily in the antigenic fraction bound tomaternal IgG, whereas it was detected only in the unbound fractionof the control immunoprecipitation (Figure 2H). Furthermore,enzymatic activity of neutral endopeptidase was detected inthe fraction eluted from material bound to maternal IgG (0.20µmol per milligram of protein per minute) but not in theone eluted from control IgG. More than 95 percent of the enzymaticactivity was blocked by 2 µM of phosphoramidon or 50 µMof thiorphan — two specific inhibitors of neutral endopeptidase.Together, these results demonstrate that neutral endopeptidaseis the target antigen of circulating antibodies. Moreover, thepresence of neutral endopeptidase in the circulating immunecomplexes isolated from the serum sample obtained from the infanton day 13 was demonstrated by Western blotting (Figure 2I).
To evaluate a potential effect of anti–neutral endopeptidaseantibodies on enzymatic activity, lysates of human podocyteswere preincubated with IgG antibodies from the mother or thefather. The endopeptidase-24.11 activity of podocyte lysateswas blocked by its specific inhibitors, thiorphan and phosphoramidon,and was also inhibited in a dose-dependent manner by IgG fromthe mother but not by IgG from the father (Table 2).
Table 2. Effect of Maternal IgG on Endopeptidase 24.11 Activity.
Colocalization of Neutral Endopeptidase and IgG in Immune Deposits
To demonstrate that neutral endopeptidase was localized in subepithelialimmune deposits, sections of the renal-biopsy specimen fromthe infant were incubated with antihuman IgG antibody (Figure 3A)and with polyclonal anti–neutral endopeptidase antibody(Figure 3B). IgG antibodies and neutral endopeptidase were colocalizedin many areas of the outer aspect of the capillary wall (Figure 3C).
Figure 3. Colocalization of Neutral Endopeptidase and IgG in Immune Deposits and Induction of Renal Disease in Rabbits by IgG from the Mother.
Panels A, B, and C (x600) show immunofluorescence staining of kidney-biopsy specimens from the infant that have been double-labeled with antihuman IgG antibodies (Panel A) and polyclonal anti–neutral endopeptidase antibody (Panel B). Panel C shows the merged image. The insets (x2000) show the colocalization of neutral endopeptidase and IgG on the outer aspect of the capillary wall. Panels D, E, F, and G (x600) show immunofluorescence staining of kidney sections from rabbits injected four days earlier with IgG fractions from the mother (Panels D, E, and F) or the father (Panel G). The sections shown in Panels D, E, and F were double-labeled with antihuman IgG antibodies (Panel D) and with polyclonal anti–neutral endopeptidase antibody (Panel E); the merged image is shown in Panel F.
Induction of Renal Disease in Rabbit by the IgG Fraction from the Mother
Kidneys from three rabbits injected with the IgG fraction fromthe mother showed glomerular deposits of this IgG along capillarywalls (Figure 3D). When the same section was incubated withanti–neutral endopeptidase antibody (Figure 3E), we observeda clear colocalization of the injected IgG with neutral endopeptidase(Figure 3F). The rabbit that received the higher dose of IgGshowed respiratory distress and was killed when death was imminent.No deposits were seen in glomeruli of two control rabbits injectedwith IgG from the father (Figure 3G). Tests conducted betweenfour days and six weeks after injection revealed that proteinuria(5.8 to 7.6 mg of protein per milligram of creatinine [0.65to 0.86 g per millimole]) had developed in all three rabbitsthat were injected with IgG from the mother, whereas the rabbitsthat were injected with IgG from the father had urinary proteinexcretion of 1.1 to 1.8 mg per milligram of creatinine (0.12to 0.20 g per millimole).
Analysis of Neutral Endopeptidase Expression in the Parents
Because the mother had no apparent renal abnormalities despitehigh serum titers of anti–neutral endopeptidase antibody,we hypothesized that she might be deficient in neutral endopeptidase,and we therefore analyzed neutral endopeptidase expression ingranulocytes from both parents. Fluorescence-activated cell-sorteranalysis of the mother's granulocytes incubated with eitheranti–neutral endopeptidase monoclonal antibody or withthe serum obtained from the mother five weeks after deliveryshowed no neutral endopeptidase at the cell membrane (Figure 4Aand Figure 4B). Cell extracts prepared from the mother'sgranulocytes did not react with either monoclonal or polyclonalantibodies against neutral endopeptidase on Western blotting(Figure 4C). Moreover, the mother's serum reacted with the father'sgranulocytes but not with her own granulocytes, suggesting analloimmunization process (Figure 4B and Figure 4C).
Figure 4. Analysis of Neutral Endopeptidase Expression in the Parents.
Fluorescence-activated cell-sorter analysis (Panels A and B) and immunoblotting (Panel C) show a lack of expression of neutral endopeptidase in the mother's granulocytes. The mother's granulocytes were incubated with anti–neutral endopeptidase monoclonal antibody (anti-NEP mAb) (Panel A) or with positive serum from the mother (Panel B). Panel A shows the lack of neutral endopeptidase staining in the mother's granulocytes (red), whereas the father's granulocytes (black) are positive. Panel B shows that the mother's serum reacted with the father's granulocytes (black) but not with the mother's granulocytes (red). In Panel C, the reactivity of the mother's granulocyte extract (lane 1) is compared with that of the father's granulocyte extract (lane 2) with the use of monoclonal antibodies or polyclonal antibodies (pAb) against neutral endopeptidase and with the mother's or father's serum.
Discussion
In this infant born with severe membranous glomerulonephritis,nephropathy appears to have been due to anti–neutral endopeptidaseantibodies from the mother. These antibodies were found in theinfant's serum 13 days after birth but disappeared thereafter,suggesting passive transplacental immunization. They were mostlikely responsible for the infant's membranous glomerulonephritis,given that the injection of rabbits with the IgG fraction ofserum from the mother induced intraglomerular immune depositsand proteinuria, whereas injection with the IgG fraction ofserum from the father did not. Furthermore, neutral endopeptidasewas localized in immune deposits both in the infant and in therabbits injected with the mother's IgG.
The antigens responsible for human membranous glomerulonephritishave eluded identification. Hepatitis B, hepatitis C, and Helicobacterpylori antigens, tumor antigens, and thyroglobulin have beendetected in the subepithelial deposits, but there is no realproof that these antigens are pathogenic.13,14,15 Some similarities,such as glomerular deposition of renal tubular epithelial antigens,have been found between experimental Heymann's nephritis andindividual cases of membranous glomerulonephritis, but the antigenscould not be characterized at the molecular level.16,17,18 Neutralendopeptidase is thus the first podocytic antigen that has beenfound to be responsible for human membranous glomerulonephritis.Neutral endopeptidase (also called neprilysin, enkephalinase,CD10, or EC 3.4.24.11) is a 90-to-110-kD zinc-dependent metallopeptidase,identical to the common acute lymphoblastic leukemia antigen.19,20It is expressed in brain tissue, on polymorphonuclear leukocytesand lymphoid progenitor cells, and on epithelial cells withinnonlymphoid organs, such as the kidneys, the liver, the breasts,and the lungs.21,22 It is also found in the serum and the urine.23,24This enzyme is involved in the metabolism of a number of regulatorypeptides, and plays an important role in turning off peptidesignaling at the cell surface.25 In the human kidney, neutralendopeptidase is found on the brush border, podocytes, and vascularsmooth-muscle cells.6,26
Circulating immune complexes containing neutral endopeptidasewere found in the infant's serum on day 13. However, their contributionto the formation of subepithelial immune deposits is uncertain,because the levels of circulating immune complexes were low,there were no manifestations of serum sickness, and no subendothelialand mesangial immune deposits were seen. Immune complexes couldalso be formed in situ at the "sole" of podocyte foot processeswhere neutral endopeptidase is expressed.27 The two mechanismsare not mutually exclusive.
The infant's nephropathy had several unusual features. The depositscontained annular formations. Similar structures were previouslyfound in a case of neonatal membranous glomerulonephritis thatwas associated with the transplacental transfer of maternalantibodies of undefined specificity,28 as well as in the zonapellucida of rabbit oocytes after the injection of antibodiesagainst angiotensin-converting enzyme.29 These circular particlesmay contain fragments of cells or basement membrane or the membrane-attackcomplex C5b-9. We also observed unusual alterations of the glomerularbasement membrane that may have resulted from an early insultby antibodies to the embryonic kidney, during a time when intensiveremodeling of the basement membrane was occurring. Even morestriking were the findings of severe arterial lesions withoutimmune deposits and of the collapse of glomerular capillarytufts, suggestive of major renal ischemia during prenatal development.These lesions may result from the enzymatic activity of neutralendopeptidase as it cleaves vasoactive mediators, includingbradykinin, atriopeptin, and endothelins, and thus may modifylocal blood flow.25,26 Because maternal antibodies inhibitedneutral endopeptidase activity, their transplacental passagemight increase concentrations of vasoconstrictor peptides inthe vascular wall. Furthermore, binding of antibodies to granulocytesmight trigger the activation of granulocytes and the releaseof vasoactive mediators, as suggested by the poor toleranceof rabbits for higher doses of the mother's IgG.
The mother's neutral endopeptidase deficiency was confirmedby fluorescence-activated cell sorting and Western blottingof granulocytes. Despite the absence of neutral endopeptidase,the mother was healthy, as were mice with a targeted disruptionof the neutral endopeptidase gene, suggesting enzymatic redundancy.30Neutral endopeptidase deficiency caused the alloimmunizationin the mother that most likely occurred at the time of her miscarriage,since a plasma sample obtained earlier did not show anti–neutralendopeptidase antibodies. Renal injury mediated by alloimmuneresponses to major renal antigens was first described in thetubular basement membrane of rats.31 A previously reported caseof neonatal membranous glomerulonephritis may also have involvedneutral endopeptidase deficiency and alloimmunization, becausethere were no renal abnormalities in the mother.28 It is likelythat additional persons with neutral endopeptidase deficiencywill be identified and that additional cases of acute renalfailure and membranous glomerulonephritis in neonates may beascribed to anti–neutral endopeptidase antibodies.
Supported by grants from INSERM and the University of Paris6.
We are indebted to the members of the family for their participationin the study; to Philippe Fontanges for assistance with confocalmicroscopy; to Marie-Christine Verpont for assistance with electronmicroscopy; to Madeleine Delauche and Béatrice Baudouinfor technical assistance; and to Catherine Bazaud for assistancein the preparation of the manuscript.
Source Information
From INSERM Unité 489, Tenon Hospital (H.D., B.M., J.-P.H., P.M.R.), and the Department of Pediatric Nephrology, Armand Trousseau Hospital (V.G., F.D., A.B., G.D.), Assistance Publique–Hôpitaux de Paris and University of Paris 6, Paris. Drs. Debiec, Guigonis, and Mougenot contributed equally to the article.
Address reprint requests to Dr. Ronco at INSERM Unité 489, Hôpital Tenon, 4 rue de la Chine, 75020 Paris, France, or at pierre.ronco{at}tnn.ap-hop-paris.fr.
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