New insights into the pathogenesis of glomerular diseases : from bedside to bench

1
Novel aspects of the pathophysiology of
glomerular diseases alloimmunisation revisited
Pierre Ronco, M.D., Ph.D. INSERM Unit 702 and
Division of Nephrology, Tenon hospital, Paris,
France
ESPN, October 2006 Palermo
2
Alloimmune glomerular diseases

• Transplant glomerulopathy
• Goodpasture syndrome after transplantation in
  patients
• with Alports syndrome
• Recurrence of proteinuria after transplantation
  in patients
• with CNS of the Finnish type (anti-nephrin
  antibodies)
• Membranous nephropathy (MN) occurring after
  bone
• marrow or kidney transplantation (de novo MN)

3
Can allo immune reactions induce glomerular
diseases in nontransplanted patients ?
4
Membranous nephropathy
Major cause of nephrotic syndrome and chronic
renal failure
5
Aetiologies of membranous nephropathy

30
associated with

- infections
- cancers
-
autoimmune diseases
-
drugs

70 "
idiopathic forms
"

Target
antigens unknown

Proteinuria
is
complement
-
dependent
,
and
may
involve

production
of
oxygen
free
radicals
and
metalloproteases
6
Heymanns nephritis (HN)
Heymann (1959) immunization of rats with the
renal BB induces typical MGN with nephrotic
syndrome
7
Megalin, the target antigen of Heymanns
nephritis
gp 330
IMMUNOPRECIPITATION
Kerjaschki and Farquhar, J Exp Med 1983 157667
Ronco et al. J Immunol 1986 136125
8
In situ formation of immune deposits
Podocytes
Rat megalin
Human ?
Y
Y
Y
Y
GBM
Y
Y
Y
Y
Proteinuria
Y
Y
Endothelium
Kerjaschki et al J Exp Med 1987, 166109
9
The case Hugo D. (male gender)
34 Wks of gestation oligohydramnios and
enlarged kidneys 38 Wks birth 1st Days of
life respiratory distress and oligoanuria,
followed by nephrotic range proteinuria and
increased blood pressure 4 Wks CT-guided
kidney biopsy Negative tests for syphilis,
toxoplasmosis, cytomegalovirus and hepatitis -B
virus infections Negative Coombs test. Normal
levels of complement components at day 35
10
Infants kidney biopsy specimen
11
The cases mother

Aged
24

Miscarriage at 14 weeks of gestation, 2 months
before this pregnancy
Normal blood pressure, urinalysis, renal function
throughout and after the pregnancy


No
medication
No family history of renal or autoimmune disease
in either parent


No
consanguinity
12
What is the mechanism of antenatal glomerular
disease ?
13
(No Transcript)
14
Potential mechanisms of albuminuria
Green IgG
green - IgG
Red NEP
red - NEP
Blue C5b-9
15
Neutral endopeptidase A multifacetted enzyme
Zinc-dependent metallo-endopeptidase able to
digest biologically active peptides
Also called neprilysin, enkephalinase, CD10,
CALLA, EC 3.4.24.11, membrane metallo-endopeptida
se
Widely expressed
in brain tissue
on leukocytes and lymphoid progenitor cells
on epithelial cells in liver, breast, lung,
intestine, kidney (brush border,
podocyte, and vascular smooth muscle cells)
on syncytiotrophoblastic cells and in sperm
16
Mechanisms of immunization against NEP in the
infants mother?
17
The mother is NEP deficient
GRANULOCYTES
mother
mother
mother
mother
father
father
father
father
anti-NEP mAb
anti-NEP pAb
mothers serum
fathers serum
WESTERN BLOT ANALYSIS
Debiec et al, N Engl J Med, 2002, 346 2053
18
The objectives of the next study were
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Portugal
Netherlands
20 years CRF, proteinuria
4 years albuminuria
Morocco
MME cDNA
NEP protein
3 years albuminuria
anti-NEP antibodies
(Debiec et al. Lancet. 2004)
Children born with MN and nephrotic syndrome
20
Why didnt mother IIM8 transmit the renal disease
?
Mothers sera reactivity against NEP
Distribution of NEP-specific IgG subclasses
Debiec et al, Lancet, 2004, 3641252
21
Outcome of antenatal MN
Birth
Last follow-up
Patient
Urine protein/creat mg/mmol
Serum creat ?mol/L
Urine albumin/creat mg/mmol
Urine protein/creat mg/mmol
Serum creat ?mol/L
Age
135
72
49
IV P1
238
1717
2.0
240
400
300
III N1
anuria
ND
20
49
13
III M1
133
1785
3.0
27
III M2
168
3842
1.0
19
33
0.3

• Anti-NEP allo-immunization can be responsible for
  apparently late-onset
• glomerulopathies

22
Outcome of second pregnancies
Mother IIM4 (Morocco)
Mother IIIP3 (Portugal)
Titer
Titer
IvIg
IvIg
160,000
IvIg
16,000
120,000
IvIg corticosteroids
80,000
8,000
40,000
4,000
2,000
8
9
7
6
5
4
2
3
4
5
6
7
Pregnancy (month)
Pregnancy (month)
Birth
Death in utero (Thrombophilia) Scarce deposits of
IgG
Nephrotic Syndrome Fanconi Syndrome Osteopenia
Nortier et al. Pediatr Nephrol 2006, 211399
23
Antigen-driven therapy in allo-immune MN
Induction of antigen-specific peripheral
tolerance
Global immunosuppressive therapy
Mother
X
IgG
Intravenous IgG Corticosteroids Plasma exchange
Nasal administration of immunodominant peptide
derived from NEP
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Mapping of T-cell epitopes on NEP
Interferon-gamma enzyme-linked immunospot ELISPOT
Synthetic NEP- derived peptides corresponding to
potential HLA binding motifs
PBMC
Y
Y
Y
Y
Y
Y
Anti-IFN? mAb
HRP-secondary antibody substrate
Spot-forming cells
25
Establishment of an experimental model of
allo-immune MN in the mouse
before
after
Indirect IF
Offsprings
Mothers
1) to test the efficacy of immuno- intervention
2) to understand natural history of disease
Induction of nasal tolerance to NEP in NEP-/-
female mice before and after pregnancy
Time-course analysis of kidney alterations
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Molecular targets in membranous nephropathy
Megalin rat (Heymanns nephritis) Kerjaschki
Farquhar, J Exp Med 1983157667 Ronco et al.
J Immunol 1986 136125
NEP human Other antigens to be identified
Drawing from Kerjaschki, Lancet Commentary 2004,
3641194
27
Adult membranous nephropathy (MN)
Is NEP responsible ? - no NEP antigen in
immune deposits in patients with  idiopathic 
MN - no extrarenal manifestations - but
anti-NEP antibodies should be searched in
patients with MN occurring after renal
transplantation (de novo MN in a NEP-deficient
recipient) or during graft vs host disease
(NEP-deficient donor) Other podocyte antigens
are still good candidates
28
Allo-Immunization A New Concept in Organ
Pathology ?
Ronco et al, Nat Clin Pract nephrol, 2006, 2594
29
Recurrent antenatal hemochromatosis

• Acute antenatal liver disease associated with
  iron
• accumulation causing death or acute liver failure
• The percentage of recurrence is more than 80
  in the
• later pregnancies, but could be significantly
  decreased by
• intravenous Ig
• Mothers are healthy
• No genetic or infectious causes
• Target antigen in the fetal liver ?

Whitington et Hibbard, Lancet 2004, 3641690
30
Tentative classification of diseases because of
fetomaternal allo-immunisation
MOTHER
FETUS
DISEASE
Access of the fetal antigens to the
maternal circulation
Immune response against fetus
PLACENTA
Anti-fetal IgG transplacental transfer
Antibody-antigen Reaction
Organ lesions
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Conclusions

• Antenatal MN induced by anti-NEP antibodies
  extends
• the concept of alloimmune disease to encompass
  nephropathies
• of the native kidney
• This concept may also apply to glomerulopathies
  in
• alloimmune settings at any life stage
• A subset of autoimmune processes that target
  the kidney
• might be triggered by an initial alloimmune
  response, followed
• by exposure of cryptic epitopes and autoimmunity
  in response
• to organ injury

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Tenon hospital
Department of Pathology
INSERM Unit 702
Béatrice
Mougenot
Laboratory of Human Genetics
Florence Coulet Florent Soubrier
Departments of Pediatric Nephrology
Trousseau hospital
Sofia hospital (Rotterdam)
Vincent Guigonis
Jeroen Nauta
Albert Bensman
Brussels Free University
Georges Deschênes
Joelle Nortier
Françoise Janssen
Thierry Schurmans
Grants from INSERM, Université Paris 6, Joan
Halpin GRIP-Genzyme, French Institute of rare
Diseases and Programme Hospitalier de Recherche
Clinique