1. Volume 87, Issue 3, Pages 602-609 (March 2015)
Genetic homogeneity but IgG subclass–dependent clinical variability of alloimmune membranous nephropathy with anti-neutral endopeptidase antibodies 
Marina Vivarelli, Francesco Emma, Thimothée Pellé, Christopher Gerken, Stefania Pedicelli, Francesca Diomedi-Camassei, Günter Klaus, Siegfried Waldegger, Pierre Ronco, Hanna Debiec 
Kidney International 
Volume 87, Issue 3, Pages (March 2015)
DOI: /ki
Copyright © 2015 International Society of Nephrology Terms and Conditions

2. Figure 1
Morphological changes and characterization of immune deposits in renal biopsy specimen. (a and b) Biopsy taken at 12 days of life. Glomeruli appear ischemic and shrunken, with severe lesions of interlobular arteries (a) and tubular atrophy (b). (c) Immunofluorescence study showing granular deposits of IgG, and complement components including C3, C5b-9, and C1q. Immunostaining for IgG subclasses shows the presence of IgG1 but not IgG4 in deposits. (d) Representative segment of the capillary wall analyzed by electron microscopy. Electron-dense deposits (arrowheads) with annular formations are seen on the outer aspect of the glomerular basement membrane. Original magnification, × 150 in (a); × 300 in (b). Bar = 15μm in (c) and 1000nm in (d).
Kidney International  , DOI: ( /ki )
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3. Figure 2
Detection of anti-NEP antibodies. (a) Sera from the German and Italian (shown) mothers were tested on cryosections of human, rabbit, and rat kidneys. The same pattern of staining is observed in the human kidney and in the rabbit kidney, whereas in the rat kidney staining is restricted to cells of Bowman’s capsule and to the brush border of deep cortical segments of the proximal tubule. Original magnification, human × 200; rabbit and rat × 300. (b) Western blot of rh NEP with sera obtained from the German mother 6 months after delivery (G), the Italian mother shortly after delivery (I), and the Italian child 12 days, 1 month, 2 months, and 3 months after birth. (c) Western blot analysis of the distribution of NEP-specific IgG subclasses in sera from the German mother (upper left), the Italian mother (upper right), the Italian child at 12 days (lower left), and the Italian child at 2 months (lower right). (d) Enzyme-linked immunosorbent assay test of the distribution of NEP-specific IgG subclasses in sera from a control, the German mother, the Italian mother, and the Italian child at 12 days. Values are the mean ±s.d. of three independent experiments performed in duplicate. NEP, neutral endopeptidase.
Kidney International  , DOI: ( /ki )
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4. Figure 3
Detection of NEP in glomerular immune deposits. (a) Glomeruli double-labeled with anti-NEP (red) and anti-human IgG1 (green): the right panel shows the merged image. (b) Quantitative analysis of the fluorescence recorded across sections of a representative capillary loop (arrowheads). Note the superimposition of the two signals, which indicates that subepithelial immune deposits contain NEP (red) and IgG1 (green). (c) Immunofluorescence staining for NEP. Note that NEP is detected only in the glomeruli but not in the proximal tubules. Bar=10μm.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

5. Figure 4
Effect of maternal IgG on NEP enzymatic activity. (a) The upper panel is a western blot analysis of NEP IgG subclass reactive with rh NEP in sera from the Italian (I) and German (G) mothers and from the Moroccan (M) mother IIM8(8). The western blots were performed with the affinity-purified anti-NEP antibodies from patients’ sera. (b) The lower panel shows antibody inhibition of NEP enzymatic activity. Recombinant human NEP was preincubated or not (control) with thiorphan (Thior), an NEP-specific inhibitor, or with an equal amount (1μg/ml) of affinity-purified anti-NEP IgG antibodies isolated from the Italian (I), German (G), and Moroccan (M) mothers. NEP enzymatic activity was then determined. Values are the mean±s.d. of three independent experiments performed in duplicate.
Kidney International  , DOI: ( /ki )
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6. Figure 5
Detection of MME mutation and simple test to detect NEP deficiency. (a) Pedigree of the Italian family. Black, mutation in exon 7. (b) Mutation in exon 7; single-nucleotide deletion at position 466 (466delC, arrow). (c) Western blot analysis of urine samples detected with anti-NEP antibody. Patients with homozygous mutation in exon 7 have no NEP in the urine.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

7. Figure 6
Diagnosis of maternal-fetal alloimmune glomerulopathy. Anti-NEP antibodies should be searched (1) in children (first case in family) with congenital nephrotic syndrome (CNS) who experience an unexplained improvement in their proteinuria and renal function, (2) in children with CNS and a family history positive for alloimmune glomerulopathy or for unexplained transient CNS in older siblings, and (3) in their mothers. Note that in many cases of maternal-fetal alloimmune glomerulopathy, an initial miscarriage will be reported by the mother. NEP deficiency should be tested by western blotting of urine and genetic screening in families with this disease to detect NEP-deficient individuals among female siblings. Women in whom NEP deficiency is detected should be carefully monitored in subsequent pregnancies, as therapy with intravenous immunoglobulins or plasmapheresis may significantly improve renal outcome in their offspring. The dotted arrow between ‘Child 1’ and ‘Renal biopsy’ indicates that a renal biopsy should be performed only in the infants who do not promptly recover. Hx, history.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions