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m e dic i n e
Original Article
Autoantibodies Targeting Nephrin
in Podocytopathies
F.E. Hengel, S. Dehde, M. Lassé, G. Zahner, L. Seifert, A. Schnarre, O. Kretz,
F. Demir, H.O. Pinnschmidt, F. Grahammer, R. Lucas, L.M. Mehner,
T. Zimmermann, A.M. Billing, J. Oh, A. Mitrotti, P. Pontrelli, H. Debiec,
C. Dossier, M. Colucci, F. Emma, W.E. Smoyer, A. Weins, F. Schaefer,
N. Alachkar, A. Diemert, J. Hogan, E. Hoxha, T. Wiech, M.M. Rinschen,
P. Ronco, M. Vivarelli, L. Gesualdo, N.M. Tomas, and T.B. Huber,
for the International Society of Glomerular Disease
A BS T R AC T
BACKGROUND
Minimal change disease and primary focal segmental glomerulosclerosis in adults,
along with idiopathic nephrotic syndrome in children, are immune-mediated podocytopathies that lead to nephrotic syndrome. Autoantibodies targeting nephrin have
been found in patients with minimal change disease, but their clinical and pathophysiological roles are unclear.
METHODS
We conducted a multicenter study to analyze antinephrin autoantibodies in adults
with glomerular diseases, including minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, antineutrophil cytoplasmic antibody–associated glomerulonephritis, and lupus nephritis, as well as in
children with idiopathic nephrotic syndrome and in controls. We also created an
experimental mouse model through active immunization with recombinant murine
nephrin.
The authors’ full names, academic degrees, and affiliations are listed in the Appendix. Drs. Tomas and Huber can be contacted at n.tomas@uke.de and t.huber@
uke.de, respectively, or at the III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52,
D-20246 Hamburg, Germany.
Drs. Ronco, Vivarelli, Gesualdo, Tomas, and
Huber contributed equally to this article.
This article was published on May 25, 2024,
at NEJM.org.
Copyright © 2024 Massachusetts Medical Society.
RESULTS
The study included 539 patients (357 adults and 182 children) and 117 controls.
Among the adults, antinephrin autoantibodies were found in 46 of the 105 patients
(44%) with minimal change disease, 7 of 74 (9%) with primary focal segmental
glomerulosclerosis, and only in rare cases among the patients with other conditions.
Of the 182 children with idiopathic nephrotic syndrome, 94 (52%) had detectable
antinephrin autoantibodies. In the subgroup of patients with active minimal change
disease or idiopathic nephrotic syndrome who were not receiving immunosuppressive treatment, the prevalence of antinephrin autoantibodies was as high as 69%
and 90%, respectively. At study inclusion and during follow-up, antinephrin autoantibody levels were correlated with disease activity. Experimental immunization
induced a nephrotic syndrome, a minimal change disease–like phenotype, IgG
localization to the podocyte slit diaphragm, nephrin phosphorylation, and severe
cytoskeletal changes in mice.
CONCLUSIONS
In this study, circulating antinephrin autoantibodies were common in patients
with minimal change disease or idiopathic nephrotic syndrome and appeared to
be markers of disease activity. Their binding at the slit diaphragm induced podocyte dysfunction and nephrotic syndrome, which highlights their pathophysiological significance. (Funded by Deutsche Forschungsgemeinschaft and others.)
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n e w e ng l a n d j o u r na l
idney diseases that lead to nephrotic syndrome are characterized by
excessive amounts of protein in urine.
This phenomenon is usually caused by kidney
damage — specifically, damage to podocytes,
which are specialized epithelial cells of the glomerular filtration barrier that regulate the filtration process.1-3 Podocyte dysfunction has been
associated with various factors, including genetic mutations, allergies, infections, lymphoid
neoplasms, certain drugs, and autoimmune diseases.4 Severe podocytopathies that commonly
result in nephrotic syndrome include minimal
change disease, primary focal segmental glomerulosclerosis (FSGS), and membranous nephropathy,
which all are descriptive terms based on histologic patterns. Because children with proteinuria
rarely undergo a kidney biopsy, they usually receive a diagnosis of “idiopathic nephrotic syndrome.” Although recent advances have clarified
the pathophysiological characteristics of membranous nephropathy,5 the underlying causes of
minimal change disease, primary FSGS, and idiopathic nephrotic syndrome are unclear. Given their
overlapping histological characteristics, such as
extensive effacement of podocyte foot processes,
these conditions are often assumed to represent
different manifestations within a disease spectrum,6,7 which highlights the complexity of these
kidney diseases and the need for individualized
and pathobiology-based approaches to diagnosis
and treatment.8
In this respect, the recent observation of antibodies against nephrin in some patients with
minimal change disease and recurrent FSGS has
the potential to improve our understanding,
classification, and treatment of minimal change
disease and FSGS.9,10 Nephrin is a key protein of
the complex podocyte slit-diaphragm architecture and has extensive signaling functions; severe podocyte injury occurs on genetic mutation
or experimental nephrin knockout.11-13
In this multicenter study, we developed new
methods of detecting antinephrin autoantibodies
and examined these autoantibodies in adults with
biopsy-confirmed glomerular diseases, children
with idiopathic nephrotic syndrome, and controls.
We further assessed the relationship between
these antibodies and disease activity during clinical follow-up and explored the pathophysiologi-
m e dic i n e
cal mode of action of antinephrin autoantibodies
on podocytes in an experimental setting.
Me thods
Patients
Serum or plasma samples from adults with biopsyproven glomerular disease were obtained from
the Hamburg Glomerulonephritis Registry and
the University of Bari Aldo Moro. These cohorts
included patients with minimal change disease,
FSGS, membranous nephropathy, IgA nephropathy, antineutrophil cytoplasmic antibody (ANCA)–
associated glomerulonephritis, or lupus nephritis.
Figure 1 (facing page). Prevalence of Antinephrin
Autoantibodies in Patients with Proteinuric Glomerular
Diseases and in Controls.
Panel A shows a characteristic Western blot result of
immunoprecipitates (recombinant human nephrin
ectodomain and human serum) detected with a nephrin-specific antibody. The input lane shows the human
nephrin ectodomain used for immunoprecipitation.
IgAN denotes IgA nephropathy, FSGS focal segmental
glomerulosclerosis, and MCD minimal change disease.
Panel B shows the prevalence of antinephrin autoantibodies in the investigated adult cohorts, in patients
with MCD and nephrotic-range proteinuria (defined by
a urinary albumin-to-creatinine or protein-to-creatinine ratio [with albumin, protein, and creatinine measured in grams] of >3.5 in adults), and in patients with
MCD and nephrotic-range proteinuria who were not
receiving immunosuppressive treatment (IS) at the
time of sample collection. ANCA-GN denotes antineutrophil cytoplasmic antibody–associated glomerulonephritis. Panel C shows the prevalence of antinephrin
autoantibodies in the investigated cohorts of children
with idiopathic nephrotic syndrome (INS), in the subgroup of children with INS and nephrotic-range proteinuria (defined by a protein-to-creatinine ratio [with
protein and creatinine measured in grams] of >2 in
children), and in the subgroup of children with INS
and nephrotic-range proteinuria who were not receiving IS at the time of sample collection, as well as the
prevalence among pediatric controls. Panel D shows
the urinary albumin- or protein-to-creatinine ratios,
serum albumin levels, serum cholesterol levels, and
estimated glomerular filtration rates (GFRs) in antinephrin-negative and antinephrin-positive adult patients with MCD or primary FSGS at the time of study
inclusion. Data are shown as scatterplots; long horizontal bars indicate the median, and I bars indicate
the 95% confidence interval. The widths of the confidence intervals have not been adjusted for multiplicity
and may not be used in place of hypothesis testing.
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Autoantibodies Targeting Nephrin in Podocytopathies
Samples from 67 healthy blood donors were included as additional controls. Of the 115 patients
with FSGS, 75 had primary FSGS. Samples from
children with idiopathic nephrotic syndrome
were obtained from the Division of Nephrology
at Bambino Gesù Children’s Hospital, the University of Bari Aldo Moro, and the NEPHROVIR
cohort, which includes patients from pediatric
centers in the Paris area and is coordinated at
the Division of Pediatric Nephrology at Robert
Debré University Hospital, Assistance Publique
Hôpitaux de Paris. We included serum samples
from 50 children from the Prenatal Identification
of Children’s Health (PRINCE) study as pediatric
controls.14
All the patients or their legal guardians gave
written informed consent, and the study was
conducted in accordance with federal, state, and
institutional guidelines and approved by the local
ethics committee of the Chamber of Physicians in
Hamburg, the Policlinic of Bari Hospital, the
ethics committee of Saint Louis Ile de France IV,
A Detection of Antinephrin Autoantibodies by Immunoprecipitation
Input
Control
175 —
130 —
B Antinephrin Autoantibody Prevalence among Adults
94/182 78/98 35/39 1/50
Antinephrin
positive
Percentage of Patients
36/59 24/35
Antinephrin
negative
46/105 7/74 1/40 1/50 0/48 0/20 0/20 0/67
Percentage of Patients
among Children
Antinephrin positive
Antinephrin negative
C Antinephrin Autoantibody Prevalence
N phr
eg in
An ativ
tin e
Po hrin
N phr
eg in
An ativ
tin e
Po hrin
Estimated GFR
(ml/min/1.73 m2)
N phr
eg in
An ativ
tin e
Po hrin
Serum Cholesterol
(mg/dl)
Serum Albumin
(g/dl)
N phr
eg in
An ativ
tin e
Po hrin
Urinary Albumin- or Proteinto-Creatinine Ratio
D Clinical Measures in Antinephrin Autoantibody–Negative and –Positive Patients with MCD or Primary FSGS
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n e w e ng l a n d j o u r na l
and the Bambino Gesù Children’s Hospital Ethical Committee in Rome. Further details of the
patients are provided in the Supplementary Appendix and the protocol, available with the full
text of this article at NEJM.org. The authors vouch
for the accuracy and completeness of the data and
for the fidelity of the study to the protocol.
linear repeated-measures models were fitted to
natural log–transformed urine protein data for
adults and children who tested positive for antinephrin autoantibodies and had available followup samples and data. Further details are provided
in the Supplementary Appendix.
R e sult s
Immunoprecipitation
Antinephrin autoantibody status was determined
by immunoprecipitation9 with the use of the recombinant human nephrin ectodomain (amino
acids A36 through L1052). The protein was incubated overnight with human serum or plasma
before protein G was added. Immunoprecipitates
were collected, electrophoresed, blotted, and analyzed for nephrin with an antinephrin antibody
(AF4269; R&D Systems). Samples were analyzed
in a blinded fashion, with unblinding of disease
diagnosis and clinical data occurring after the
determination of antinephrin autoantibody status.
For quantitative readout of antinephrin autoantibodies, a hybrid assay involving immunoprecipitation followed by quantification of immunoprecipitated nephrin was established. Further details
are provided in the Supplementary Appendix.
Experiments in Animals
All experiments complied with national and institutional ethical standards and animal care
guidelines and were approved by the Veterinarian Agency of Hamburg and the local committee
for animal care. Male wild-type BALB/c mice were
immunized subcutaneously with recombinant
murine nephrin and complete Freund’s adjuvant.
Control animals received complete Freund’s adjuvant mixed with phosphate-buffered saline. Further details of the experiments in animals are
provided in the Supplementary Appendix.
Statistical Analysis
Because of the exploratory nature of the analyses
in patients, no P values are provided and clinical
data are shown as scatterplots with medians and
95% confidence intervals. The widths of confidence intervals have not been adjusted for multiplicity and may not be used in place of hypothesis testing. To quantify the association between
antinephrin autoantibody status (positive or negative, as measured by immunoprecipitation) and
disease activity (measured as the urinary albumin-to-creatinine or protein-to-creatinine ratio),
m e dic i n e
Prevalence of Antinephrin Autoantibodies
among Patients with Proteinuric Glomerular
Diseases
We initially examined serum or plasma samples
from 357 adult patients with biopsy-proven glomerular diseases (Table S1 in the Supplementary
Appendix). Because we were not able to detect
circulating antinephrin autoantibodies using direct Western blotting or enzyme-linked immunosorbent assay (ELISA) (Fig. S1), we optimized the
assay for the detection of antinephrin autoantibodies using immunoprecipitation (Fig. 1A and
Fig. S2). We found that 46 of 105 patients (44%)
with minimal change disease, 7 of 74 (9%) with
primary FSGS, 1 of 40 (2%) with nonprimary
FSGS, and 1 of 50 (2%) with membranous nephropathy tested positive for circulating antinephrin autoantibodies, whereas all 48 patients
with IgA nephropathy, all 20 patients with ANCAassociated glomerulonephritis, all 20 patients
with lupus nephritis, and all 67 healthy controls
remained negative (Fig. 1B, left panel). Antinephrin autoantibodies were detected in 36 of 59
patients (61%) with minimal change disease who
had nephrotic-range proteinuria (defined by an
albumin-to-creatinine or protein-to-creatinine
ratio [with albumin, protein, and creatinine measured in grams] of >3.5 in adults and, in accordance with the 2022 Pediatric Nephrology Association Guidelines, >2 in children), as well as
in 24 of 35 patients (69%) with minimal change
disease who had nephrotic-range proteinuria and
were not receiving immunosuppressive treatment
at the time of sample collection (Fig. 1B, right
panel).
Identifying biomarkers in children with nephrotic syndrome is particularly important because these children often do not undergo a
kidney biopsy. We therefore assessed whether
antinephrin autoantibodies can also be found in
children with idiopathic nephrotic syndrome
(Table S2). We performed immunoprecipitation
analyses using samples from 182 children with
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Autoantibodies Targeting Nephrin in Podocytopathies
idiopathic nephrotic syndrome and found that
94 (52%) had detectable antinephrin autoantibodies at the time of sample collection (Fig. 1C).
Antinephrin autoantibodies were detected in 78
of 98 patients (80%) with idiopathic nephrotic
syndrome who had nephrotic-range proteinuria
and in 35 of 39 patients (90%) with idiopathic
nephrotic syndrome who had nephrotic-range
proteinuria and were not receiving immunosuppressive treatment at the time of sample collection (Fig. 1C). Of the 50 control children, 1 was
positive for antinephrin autoantibodies by immunoprecipitation (Fig. 1C).
Antinephrin Antibody Status:
Ratio of Albumin or Protein to Creatinine:
Overall, the cohorts of patients with minimal
change disease, FSGS, and idiopathic nephrotic
syndrome had the expected ratio of male to female patients and spanned a broad age distribution similar to that in earlier studies. Adult nonWhite patients were underrepresented in this study
because of limited sample availability in the investigated European patient cohorts (Table S3).
At study inclusion, patients with antinephrinassociated minimal change disease or primary
FSGS had a more severe nephrotic syndrome than
patients with these conditions who were antinephrin-negative, although kidney function was
Antinephrin positive
0.3–3.5
Antinephrin negative
A Antinephrin Autoantibodies during Follow-up in Adults
B Antinephrin Autoantibodies
Patient
during Follow-up in Children
Patient
Follow-up Time Point
Follow-up Time Point
Figure 2. Correlation of Antinephrin Autoantibodies with Longitudinal Disease Course of Remission and Relapse.
Panel A shows a heat map indicating antinephrin antibody status (upper half of each square) and urinary albuminor protein-to-creatinine ratio (lower half of each square) at study inclusion and during follow-up in adult patients
with minimal change disease or primary FSGS who were positive for antinephrin autoantibodies in at least one serum sample. Patients 2, 12, 15, 16, and 18 had primary FSGS; the other patients shown had minimal change disease. Each row represents one patient, and each column represents one follow-up time point. The numbers on the
right indicate the total follow-up time in months. Panel B shows the heat map indicating antinephrin antibody status and urinary protein-to-creatinine ratio at study inclusion and during follow-up in children with idiopathic nephrotic syndrome who were positive for antinephrin autoantibodies in at least one serum sample.
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n e w e ng l a n d j o u r na l
Disease Activity
m e dic i n e
B Correlation of Antinephrin Autoantibody Levels with
Urinary Albumin- or Proteinto-Creatinine Ratio
Antinephrin Autoantibody Level (RU/ml)
A IP–ELISA Hybrid Assay
MCD or
Primary FSGS
Membranous
Nephropathy
10−1
10−2
r=0.64
10−3
Control
Antinephrin Autoantibody Level (RU/ml)
C Long-Term Clinical Course in a Patient with Antinephrin-Associated Podocytopathy
Prednisolone
Prednisolone
Rituximab
Rituximab
Albumin-to-creatinine ratio
Antinephrin
autoantibody
level
Antinephrin Autoantibody Level
(RU/ml)
Urinary Albumin-to-Creatinine
Ratio
Cyclosporine A
Months
D Rituximab Treatment in Patients with Antinephrin-Associated Podocytopathies
Patient A
Patient B
Prednisolone
Prednisolone
Cyclosporine A
Albumin-tocreatinine
ratio
Antinephrin
autoantibody
level
Urinary Albumin-to-Creatinine
Ratio
Antinephrin
autoantibody
level
Albumin-tocreatinine
ratio
Antinephrin Autoantibody Level
(RU/ml)
Rituximab
Antinephrin Autoantibody Level
(RU/ml)
Urinary Albumin-to-Creatinine
Ratio
Rituximab
Weeks
similar in the two groups (Fig. 1D). Findings
were similar among the children with idiopathic
nephrotic syndrome (Fig. S3). Moreover, none of
the patients with minimal change disease, primary FSGS, or idiopathic nephrotic syndrome had
type 1 diabetes, which argues against a major
role of these autoantibodies in the development of
pancreatic autoimmunity, as has been suggested
previously.15
Together, these analyses corroborate the pres-
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Autoantibodies Targeting Nephrin in Podocytopathies
Figure 3 (facing page). Quantitative Measurement of
Antinephrin Autoantibodies.
Panel A shows antinephrin autoantibody levels in adult
patients with MCD or primary FSGS, patients with
membranous nephropathy, and in controls at study inclusion, measured with the use of an immunoprecipitation (IP)–enzyme-linked immunosorbent assay (ELISA) hybrid assay. Data are shown as scatterplots; long
horizontal bars indicate the median, and I bars indicate the confidence interval. The widths of confidence
intervals have not been adjusted for multiplicity and
may not be used in place of hypothesis testing. RU denotes relative units. Panel B shows the Spearman correlation between antinephrin autoantibody levels and
the urinary albumin- or protein-to-creatinine ratio
among patients with antinephrin-associated MCD or
primary FSGS. Panel C shows the longitudinal disease
course in one patient with antinephrin-associated
MCD (Patient 1). Panel D shows two patients, one
with antinephrin-associated MCD (Patient A, left) and
one with antinephrin-associated primary FSGS (Patient
B, right), who were prospectively treated with rituximab because of antinephrin autoantibody positivity
during disease relapse.
ence of circulating antinephrin autoantibodies
in a substantial proportion of adult patients with
minimal change disease or primary FSGS as well
as in children with a diagnosis of idiopathic nephrotic syndrome, indicating a prevalence of approximately 70% among patients with minimal
change disease and 90% among those with idiopathic nephrotic syndrome during active disease
before treatment.
Antinephrin Autoantibodies and
Longitudinal Disease Course of Remission
and Relapse
We next examined the utility of longitudinal
measurement of antinephrin autoantibodies in
patients with antinephrin-associated podocytopathies. We examined 163 follow-up serum samples from 38 adult patients with minimal change
disease or primary FSGS for circulating antinephrin autoantibodies by immunoprecipitation
in available samples, which were obtained without a prespecified follow-up time. Of these patients, 18 were positive for antinephrin autoantibodies at one or more points, 13 of whom had
minimal change disease and 5 of whom had primary FSGS (Fig. 2A and Fig. S4A). The presence
and absence of antinephrin autoantibodies were
associated with active disease and remission, respectively, in these patients (Fig. 2A), and linear
repeated-measures models revealed a strong as-
sociation between antinephrin autoantibody positivity and protein levels in urine (Table S4).
We also investigated 33 available follow-up
samples from 22 children with idiopathic nephrotic syndrome, 18 of whom were positive for
antinephrin autoantibodies at one or more time
points (Fig. 2B and Fig. S4B). Of the 13 children
with antinephrin-associated idiopathic nephrotic
syndrome for whom we had longitudinal paired
samples at disease onset before immunosuppressive treatment and after a few weeks in remission, all 13 (100%) were antinephrin-positive
at onset and 12 of 13 (92%) were antinephrinnegative in remission (Patients 6 through 18 in
Fig. 2B).
In addition to immunoprecipitation with subsequent Western blotting, we developed a standardized immunoprecipitation–ELISA hybrid assay to quantify antinephrin autoantibody levels.
At study inclusion, this assay reliably detected
circulating antinephrin autoantibodies in 59 adult
patients with minimal change disease or primary
FSGS, whereas 17 patients with membranous
nephropathy and 18 healthy controls remained
negative (Fig. 3A and Fig. S5 and Table S5). Using
this assay, we measured antinephrin autoantibody
levels in follow-up samples from the 18 adults
with antinephrin-associated minimal change
disease or primary FSGS for whom such samples
were available (Fig. S6 and Table S6). This analysis
revealed a strong correlation between all measured
antinephrin autoantibody levels and the urinary
albumin-to-creatinine ratio (r = 0.64) (Fig. 3B).
The clinical utility of antinephrin autoantibody measurements is exemplified by the clinical course in one patient with antinephrin-associated minimal change disease (Patient 1 in
Fig. 2A). This patient had three episodes of relapsing nephrotic syndrome over an observation
period of 51 months. Both antinephrin autoantibody positivity measured by immunoprecipitation
and quantitative antinephrin autoantibody levels
measured by combined immunoprecipitation–
ELISA were closely correlated with the urinary
albumin-to-creatinine ratio (Fig. 3C). During follow-up, immunosuppressive treatment with glucocorticoids and cyclosporine A led to a transient
remission, whereas CD20-mediated B-cell depletion with rituximab induced complete and sustained remission, as indicated by undetectable
antinephrin autoantibodies and no proteinuria
(albumin-to-creatinine or albumin-to-protein
ratio,