To Gianfranco from Rosanna

1
To Gianfrancofrom Rosanna
To a friend I will never forget
2
ESPN Palermo 2006
IgA Nephropathy may further insight help
unravelling the mystery?

Rosanna Coppo Torino, Italy
3
Pathogenesis of IgAN first hypothesis antigen
exposure and mucosal immunity response


Ag increased exposure (mucosal)
Increased IgA Synthesis (mucosal)
IgAIC
Accumulation of IgAIC in the mesangial area
IgAN and hematuria
4
Hypothesis of deposition of circulating IgAIC in
IgAN
not completely satisfying
1) Experimental models 2) Evidence in sera of
IgAN patients 3) Evidence in glomeruli of IgAN
patients
inconstantly detected
failure to find specific antigens in glomeruli
Criticism about the exclusive pathogenetical role
of deposition of circulating IgAIC in IgAN
5
Second hypothesis for the pathogenesis of
IgAN Focus on molecular structure of IgA
particularly IgA1 prevalent in mesangial
deposits in polymeric form
6
IgA1 molecule
O-linked Carbohydrate chains
Hinge region
Fab
Fab
N-terminal Carbohydrate chains
Fc
7
IgA2
CH1
IgA1
Pro Ser Thr Pro Pro Thr Pro Ser --O---
GalNAc -- ?1, 3 - Gal Pro Ser Thr
? 2, 6 ? 2, 3 Pro Neu5Ac
Neu5Ac Pro Thr Pro Ser Pro Ser
CH1
Hinge region
CH2
CH3
CH2
8
Aberrant glycosylation of serum IgA1 in
IgAN defective galactosylation and/or
sialylation of O-linked glycans
--O--- GalNAc -- ?1, 3 - Gal ? 2, 6
? 2, 3 Sialic acid Sialic
acid
1995-2000 Increased binding to GalNAc lectins
(Vicia Villosa and Helix Aspersa) Allen AC,
Feehally J. - Increased Asialo-GalNAc Hiki Y -
Galactose-deficient IgA1 Tomana M, Mestecky -
Increased exposure of GalNAc/Gal Amore and Coppo
--O--- GalNAc -- ?1, 3 - Gal ? 2, 6
? 2, 3 Sialic acid Sialic acid
--O--- GalNAc -- ?1, 3 - Gal ? 2, 6
? 2, 3 Sialic acid Sialic acid

--O--- GalNAc -- ?1, 3 - Gal ? 2, 6
? 2, 3 Sialic acid Sialic acid
9
IgA glycoforms from sera of IgAN patients
Amore A Coppo R J Am Soc Nephrol 121862-1871
2001

• Isolation of glycoforms by affinity
  chromatography on lectins with different
  specificities
• Purification by HPLC
• Analysis of 250-400 KDa HPLC peak
• - IgA and IgG (ELISA)

10
Con A IgAN
Con A Control
SBA IgAN
SBA Control
LPA IgAN
LPA Control
11
IgA content 250-400 KDa peak
plt0.05
plt0.03
plt0.02
IgA?g/peak of 250-400 KDa
SBA LPA Con A
WGA
UEA-I GalNAc Neu5Ac-2,6 GalNAc Man
GlcNAc Fuc
60
? IgA with O-linked glycan truncation in GalNAc
and Neu5Ac?2,6GalNAc Amore A Coppo R J Am
Soc Nephrol 121862-1871 2001
12
Undergalactosylated IgA1 in mesangial
deposits
Desialylated and degalactosylated IgA1 detected
in kidneys of IgAN (Feehally , Hiki)
Experimental IgAN induced by injection of
deglycosylated IgA1 (Sano)
13
Possible mechanisms leading to reduced
glycosylation of IgA1 side chain glycans -
genetic factors ? 1,3 Gal transferase -
reduced enzyme activity PMN (Allen 1997) -
Core ? 1,3 Galactosyltransferase gene
polymorphism (Amoroso 2004) - acquired
mechanisms - Th1/Th2 unbalance during
infections (Emancipator,1998)
14
Selective Th2 subset activation in IgAN
Emancipator SN
Th lymphocytes
Th2
Th1
IL-4/IL5
Modified activity of ? 1,3 Galactosyl-transferase
Aberrantly glycosylated IgA
Pre switch-B cells
15
Aberrant glycosylation of IgA1 is likely to
be secondary to aberrant immunoregulation

• O-gycosylation of serum IgD is not altered
• (Smith , Feehally and Barratt, JASN 2006)
• (IgD, produced early in B cell development and
  IgA1 , produced by mature B cells are
  O-glycosylated)

16
Aberrantly glycosylated IgA1
Self-aggregation and matrix adhesion
Escape from ASGP receptors
Reaction with circulating and mesangial matrix
glycoproteins
IgG to IgA1-GalNAc CIC
Increased binding to mesangial receptors
FAVORED MESANGIAL DEPOSITION
17
Asymptomatic mesangial IgA deposition
IgA in 16 of 510 healthy kidney donors K.
Suzuki Kidney Int 2003
18

• Still unanswered observations questions
• Why some patients have innocent IgA deposition,
  detected only at kidney donation?
• Why some IgAN progress to sclerosis and loss of
  renal function while others maintain microscopic
  hematuria but do not progress?
• Some 5-30 IgAN show remission of any clinical
  signs, but IgA deposits are stll present even in
  remission.

What regulates IgA deposition and malignancy?
19
Sera of patients with IgAN have different
amounts of aberrantly glycosylated IgA with
various degrees of desialylation and
degalactosylation
20
A.Amore and R.Coppo
May different properties of aberrantly
glycosylated IgA1 modulate mesangial
cell functions?
21
Aberrantly glycosylated IgA1
Complement activation
nephritogenesis
IgAN
22
Complement activation aberrantly glycosylated
IgA gt native IgA
C3 neoepitope fixation (C3b, iC3b, C3dg MoAb)
Coppo Amore Kidney Int 2004 65 1544-1547)
Controls IgAN patients
plt0.02
Native IgA
plt0.02
Desialylated IgA
plt0.05
plt0.05
OD 405 nm
DeGalactosylated IgA
Alternative pathway
Classical pathway
IgA exposing GalNAc (deSia/deGal IgA)
23
Risk factor for IgAN Spatial rearrangement
exposing C3 in renal deposits (Onetti Muda,
Faraggiana, 1999)
IgA yellow C3 red
Co-localization of desialylated IgA1 and
C3 Confocal microscopy (Onetti Muda, Amore, 2004)
24
Aberrantly glycosylated IgA1
Complement Activation flogosis
Relationship with mesangial matrix Integrins
nephritogenesis
IgAN
25
Expression of ?v integrin in mesangial cells
treated with Aberrantly glycosyated IgA Peruzzi
, Amore, Coppo et al Kidney Int 2000 58
2331-2340
26
?v integrin expression in mesangial cells IgA
glycoforms from IgAN gt IgA from healthy controls
Peruzzi , Amore, Coppo et al Kidney Int 2000
58 2331-2340
Man
Gal NAc
plt0.01
plt0.01
Neu5Ac 2,6 GalNAc
Number of ?v molecule/MC
plt0.05
27
Integrin expression on mesangial cells
aberrantly glycosylated IgA gt native IgA
Aberrantly glycosylated IgA
Mesangial cell
synthetic activity
?v integrin
IgAN
Mesangial matrix
Cell cycle, proliferation/apoptosis
Increased integrin expression is mostly
associated with sclerosis
28
Aberrantly glycosylated IgA1
Complement Activation flogosis
Relationship with mesangial matrix Integrins
nephritogenesis
Proliferation/apoptosis
IgAN
29

IgAN
PROLIFERATION Inflammation but tissue repair as
well
APOPTOSIS Control of flogosis but also cell
death and sclerosis
30
Apoptotic mesangial cell incubated
with deSia/deGal IgA1
31
IgA glycoforms isolated from IgAN patients
and controls MC proliferation and
MC apoptosis in cycling cells (Amore A e
Coppo R JASN 12 1862-1871, 2001)

Proliferation
Controls IgAN
Neu5Ac 2,6 GalNAc
Gal NAc
Apoptosis p53
Apoptosis TUNEL
32
Release of mediators Aberrantly glycosylated
IgA VEGF and iNOS A.Amore, R.Coppo
et al (Am J Kidney Dis 2000 36 1242-1252)
1 2 3 4 5
iNOS
VEGF
GAPDH
GAPDH
VEGF
NOS activity
plt0.01
pg/?g cell proteins
plt0.01
33
Aberrantly glycosylated IgA1
Complement Activation flogosis
Release of flogistic/sclerogenic mediators from
mesangial cells
Relationship with mesangial matrix Integrins
nephritogenesis
Proliferation/apoptosis
IgAN
34
NFkB Activation
Aberrantly glycosylated IgA
NEMO
IKKß
IKKa
U
26S proteasome degradation
P
IkB phosphorilation
U
U
IkB
IkB
IkB
p65
p50
Transcriptional activation of genes
for innate/adaptative immune response,
inflammation, cell proliferation/apoptosis
Inactive NFkB
p65
p50
Active NFkB
p65
nucleus
p50
ADHESION MOLECULES VCAM-1 ICAM-1 E-selectin
CYTOKINES CHEMOKINES IL1, IL6, IL8 TNF MCP-1
PROCOAGULANTS thrombomodulin tPA PAI-I
VASOCONSTRICTORS Endothelin 1 tromboxane
35
Unconditioned human mesangial cells stained with
anti NF-kB ( anti p50 and anti p65 Ab)
36
Mesangial cells treated with deSialylated/deGala
ctosylated IgA (90 min) stained with anti NF-kB
( anti p50 and anti p65 Ab) and anti-alpha actin
37
Mesangial cells treated with deSialylated/deGala
ctosylated IgA (90 min) stained with anti NF-kB
( anti p50 and anti p65 Ab) and anti-alpha actin
38
Mesangial cells treated with deSialylated/deGala
ctosylated IgA (90 min) stained with anti NF-kB
( anti p50 and anti p65 Ab) and anti-alpha actin

39
Aberrantly glycosylated IgA1 have systemic and
glomerular effects ( Coppo and Amore Kidney Int
2004)
Complement Activation flogosis
Release of flogistic/sclerogenic mediators
(iNOS, TGFbeta, CTGF)
Relationship with mesangial matrix Integrins
nephritogenesis
Proliferation/apoptosis
IgAN
Disease progression
Sclerogenic factors
40
May the glycosylation pattern of IgA1 molecules
influence the clinical features of IgAN?
41
Aberrantly glycosylated IgA1 in IgAN
patientswith different risk factors for
progressionIgA1 binding to Vicia Villosa or to
Helix AspersaIgA1 defective in Gal and Neu5Ac
plt0.0001
plt0.001
Microscopic gt1 g/day
IgAN hematuria proteinuria
progressed to Tx
42
   Risk factors for IgAN recurrence after
transplantation Table IV Predictive value of
serologic factors and genetic polymorphism on the
IgA recurrence at multivariate logistic
regression analysis. (Coppo et al submitted)
43
quality/amount aberrantly glycosylated IgA1
genetic conditioning of the immune response
capacity of activating complement
genetic conditioning of the renal response
activation of mediators
IgA nephropathy
44
Searching for IgAN candidate genes

• Familial IgAN 3 genetic loci
• IGAN1 on chromosome 6q 22-23
• IGAN2 on chromosome 4q 26-31
• IGAN3 on chromosome 17q 12-22
• Genes of these regions are under investigation

45
Gene polymprphisms and intrarenal expression of
gene transcripts is there any role in
expression and progression of IgAN ?

• CD 34, mesgin, CXCR3, CD34
• RAS system ACE, AGT, ATR1, AT2R
• iNOS, p53
• CD80/CD86
• ICAM-I, ELAM-1, VCAM-1
• Genes encoding for cytokines and chemokynes
• PDGF, IL-1, IL6, IL10, TNFa, MCP-1, bFGF, HGF,
  TGFß

often conflicting resultsno definite conclusion
46
May the variability of clinical features and
natural history of IgAN be accounted only for
different IgA glycosylation?
47
What regulates IgA deposition and malignancy?
Genetic conditioning of IgA1 glycosyltransferases
or acquired differences in Th2 lymphocyte
subsets may lead to different glycosylation
patterns which condition different renal
toxicity
48
What regulates IgA deposition and malignancy?
Genetic conditioning of IgA1glycosyltransferases
or of Th2 lymphocyte subsets may lead
to different glycosylation patterns which
condition different renal toxicity
The toxicity of aberrantly glycosylated IgA1 is
related to the patients infammatory milieu
which is not only expressed in the kidney but
also at systemic level
49

insight into the hypothesis of a systemic
inflammatory milieu in IgAN
50
IgACE first multicentric, prospective,
double-blind, RCT of ACE-I in moderately
proteinuric IgAN in the young
EC Biomed Concerted Action Project
BMH4-97-2487(DG 12-SSMI) 25 Centers from Italy,
France, Germany, Sweden, Portugal

• ACE-I (Benazepril 0.2 mg/Kg/day)
• in children and young lt 35 y.o. IgAN with
• proteinuria gt1 and lt 3.5 g/1.73 m2/day (stable
  over 3 months run-in phase) CrCl gt50
  ml/min/1.73m2
• Effects on
• risk of renal damage progression
• 30 decrease in CrCl and/or worsening to
  nephrotic proteinuria
• and
• proteinuria remission
• partial remission lt 0.5 g/1.73m2/day,
• complete remission lt0.16 g/1.73m2/day

51
Outcome progression of renal damage
End-point 30 loss of initial CrCl
and/or worsening of proteinuria to nephrotic
range (gt3.5 g/1.73m2/day)
IgACE trial Changes in CrCl slopes over the
follow-up
P 0.034
P0.180
52
Partial remission of proteinuria(U proteinslt
0.50 g/day/1.73m2)
Total remission of proteinuria(U proteinslt 0.16
g/day/1.73m2)
ACE-I
ACE-I
P0.0150
P0.0002
53
AOPPAdvanced oxidation protein products as a
novel marker of oxidative stress in
uremia Witko-Sarsat V, Descamps-Latsha et al K
I (1995) Advanced oxidation protein products
as risk factors for atherosclerotic
cardiovascular events in nondiabetic predialysis
patientsDescamps-Latscha B, Witko-Sarsat V,
London GM et al Am J Kidney Dis. 2005
Advanced oxidation protein products a new marker
for progressive IgANDescamps-Latscha B, London
GM et al KI 2004
54
AOPP levels in IgAN patients enrolled in the
IgACE BMH4-97-2487 trial Treated with
ACE-Inhibitors (ACE-I) or placebo (PL)
Placebo
ACE-I
mMol/L
control range
55
AOPP levels in IgAN patients enrolled in the
IgACE BMH4-97-2487 trial Treated with
ACE-Inhibitors (ACE-I) or placebo (PL)
-30
plt0.01
-70
mMol/L
plt0.001
plt0.01
control range
56
OXIDATIVE STRESS
IL-6
monocytes
NF-kB
mRNA
CRP Liver synthesis
57
We investigated microinflammation markers (CRP
and IL-6) in the 57 IgAN patients enrolled in the
IgACE trial (ACE-I vs Placebo) median FU 45
months Mancuso, Coppo, Peruzzi, Amore ASN 2006
58
Correlation between IL-6 and CRP at IgACE trial
start-up
CRP (mg/dl)
59
Microinflammatory syndrome in IgAN patients
CRP IL-6 serum levels
placebo
placebo
plt0.0005
plt0.02
ACE-I
ACE-I
plt0.02
plt0.03
CRP mg/dL IL-6 pg/mL
Mancuso, Coppo, Peruzzi, Amore ASN 2006
60
IgAN patients treated with ACE-I had a
significant reduction in CRP and IL-6
levels (while both increased in the placebo group
after 45 months of follow-up)
CRP and IL-6 modifications were significantly
correlated with the clinical outcome
61
In IgAN there are serologic features of
microinflammation which can be reversed by
treatment

In IgAN there are features of activation of
circulating lymphomonocytes
insight into the hypothesis of a systemic
inflammatory milieu in IgAN
62
NF-kB DNA binding activity in lymphomonocytes
(PBMC) (electromobility supershift assay EMSA
anti p50 and p65 Ab) in IgAN patients and controls
Densitometric analysis


p50
p65

plt0.0001
1 2 1 2 1 2 1 2 1 2 1 2


IgAN patients
Healthy clontrol
1 2 1 2 1 2 1 2
1 2 1 2 1 2 1 2 1 2 1 2

Healthy subjects
1 non stimulated 2 stimulated
PBMC (Caionophore, PMA)
IgAN patients
63
Lets consider IgAN under the light of a
systemic disease
Transcription factors in peripheral
lymphomonocytes of patients with IgAN are
translocated to the nucleus, indicating
activation of gene transcription
Where this activation is coming from? What is
aimed at?
64
NF-kB
Toll-like receptors mediate NF-kB activation
oxydative stress (ROS) cytokines
bacterial, viral infections
IKK ? NEMO
IkB
IKK a
E3IkB Ub ligase
IKK ?
p50
p65
p
p
IkB
Ub-Ub-Ub-Ub
p
p50
p65
karyopherins
a ?
NF-kB responsive genes transcriptional
activation
26S proteasome
65
Toll-like receptors (TLR) as mediators of renal
disease

• TLR discriminate pathogens from self, initiating
  and modulating adaptative immunity during
  infections.
• Most TLR ligands prime subsequent T cell response
  (upregulating MHC II, activating dendritic cell
  maturation, inducing co-stimulatory molecules
  and secretion of cytokines)

Role of TLR for pathogen control and also
for autoimmunity.
66
TLR4
TLR3
NF-kB
IFN
67
TLR-4 expression in circulating lymphomonocytes
of IgAN patients (Cytofluorimetry) Coppo
Amore ASN 2006
Fluorescence units
68
TLR-4 mRNA in in circulating lymphomonocytes of
IgAN patients (TaqMan PCR)
Arbitrary Units
69
MICROBICAL AGENTS
TLR4 TLR3
Dendritic cell
IFN-a
Immuneproteasome
Switch from proteasome to immunoproteasome in
peripheral lymphomonocytes Of IgAN patients Coppo
R et al Istanbul ESPN meeting , 2005
70
Low MWP 2 Low MWP7 Multicatalytic endopeptidase
complex-like
IFN?
IFN?
TNF?
?1, ? 2. ? 5
Catalytic properties optimal for MHC-I peptide
presentation
71
Proteasome and immunoproteasome in peripheral
immunocompetent cells of IgAN
plt 0.01
plt 0.0001
plt 0.01
plt 0.03
Alpha 2 proteasome
72
Ratio Inducible/Constitutive Proteasome subunits
in patients with IgAN
plt 0.01
plt 0.001
plt 0.01
plt 0.05
Arbitrary Units/Abl gene
LMP2/ß1
MECL-1/ß2
LMP7/ß5
73
Ratio Inducible/Constitutive Proteasome subunits
in PBMC of IgAN with different treatments
(Coppo , Amore Peruzzi, ASN 2006
Arbitrary Units/Abl gene
LMP2/ß1
MECL-1/ß2
LMP7/ß5
74
quality/amount aberrantly glycosylated IgA1
environmental factors
genetic conditioning immune response
Systemic activation of innate immunity
Toll-like receptor activation, Switch to
immunoproteasomes, NF-kB transcription
factor, oxydative stress and microinflammation
capacity of activating complement
genetics conditioning renal response
activation of mediators
Disease progression
IgA nephropathy
75
unravelling the mistery of IgAN
looking open-mindly to new perspectives
will be helpful for coming out of darkness
76

• 2004 Nobel prize for Chemistry
• Hershko, A. Ciechanover, I Rose
• Ubiquitin-proteasome pathway

77
METHODS
Peripheral Blood mononuclear Cells (PBMC) from
20 children with IgAN and 20 healthy subjects
were isolated by gradient centrifugation
Tested by quantitative Taqman analysis for
Constitutive a2 proteasome mRNA Functional ß1, ß
2, ß 5 proteasome mRNA LMP2, LMP7 and MECL-1
immunoproteasome mRNA expression
78
(No Transcript)
79
Staphylococcus aureus cell envelope antigen is a
new candidate for the induction of IgAN Koyama A
KI 2004 66121-132
In 68 of renal biopsy specimens from IgAN
patients detection of a probable adhesin from
S.A. envelop which could bind to aberrantly
glycosylated IgA1 and deposit in the kidneys
80
Pathogenesis of IgAN
1980 Exclusive role of environmental
factors conceivable hypothesis, not proved (no
detection of specific antigens, IgAIC not
constantly detected)
Possible modulation of IgA1 glycosylation by
Th2 (genetically conditioned or due to
environmental factors).
1990 Aberrantly glycosylated No mucosal
immunity, no Ag
The eliciting agent is going to be
reconsidered
Genetic conditioning factor galactosyl-transferas
e gene polymorphism Seeked, never proved
Role of innate immunity in the pathogenesis of
IgAN
81
Immunoproteasome is hyperexpressed in peripheral
immunocompetent cells from IgAN
The antigen presentation is likely to be enhanced
82
Is it possible to modulate the systemic
inflammation in IgAN?
the effect of steroids is known
Angiotensin inhibition therapy seen as an
antiinflammatory treatment
83
Classical IKK
Alternative IKK
TNFa, IL-1, CD40L (Blys)
LPS
NOD ligands
CD40L Blys/BAFF
TLR
BCR
NOD
IRAK, TRAF6
Btk
BLNK
PLC
NIK
Syk
MAP 3K
RIP2
IP3
DAG
?
Ca
calcineurin
PKC
IKK ? NEMO
IKK complex activation
IKK a
IKK ?
84
Immuneproteasomes are activated in circulating
lymphomonocytes of IgAN patients. Meanwhile the
transcriptional factor NF-kB is also activated.
Can we consider a role for a common activating
factor?
Role of antigenic challenge in the pathogenesis
of IgAN
85
Pathogenesis of IgAN
1980 Exclusive role of environmental
factors conceivable hypothesis, not proved (no
detection of specific antigens, IgAIC not
constantly detected)
Possible modulation of IgA1 glycosylation by
Th2 (genetically conditioned or due to
environmental factors).
1990 Aberrantly glycosylated No mucosal
immunity, no Ag
The eliciting agent is going to be
reconsidered
Genetic conditioning factor galactosyl-transferas
e gene polymorphism Seeked, never proved
Role of innate immunity in the pathogenesis of
IgAN
86
Strategies to inhibit NF-kB pathway
NBD IKK? Bind dom
NSAID Thalid CyPg flavonoids
IKK ? inhibitors
IKK ? NEMO
TIRAP Hsp90 antiox
IKK a
IkB
IKK ?
p50
p65
Ub-Ub-Ub
IkBa super-repressor
p
p
IKB
p50
p
p65
Proteasome inhibitors
RelA antisense NFkB decoy RNAi
a ?
NF-kB responsive genes
26S proteasome
87
IFN? IFN ?
TNF?
Ub-Ub-Ub-Ub
protein
LMP2
LMP7
MECL-1
a ?
a ?
LMP
LMP
immunoproteasome
MECL
20S
Catalytic properties optimal for MHC-I peptide
presentation upregulation of HLA Class I
19S
26S proteasome
Degradation /activation of proteins
88
Constitutive ?2 chain of poteasome mRNA in
circulating immunocompetent cells of IgAN
I
plt0.0001
I
I
controls
IgAN
89
Cathalitic beta subunits of immunoproteasome mRNA
in circulating immunocompetent cells of IgAN
LMP2, LMP7 and endopeptidase complex-like
(MECL-1) mRNA (Taqman analysis)
plt0.0001
plt0.0001
plt0.0001
90
Proteasome cathalyze non-lysosomal degradation of
intracellular proteins, as those involved in
inflammation, cell cycle regulation, gene
expression
?1, ? 2. ? 5
91
TLR-4 levels in IgAN patients (Taq-man)
Arbitrary Units
92
Aberrantly glycosylated IgA1 levels in IgAN
patients enrolled in the IgACE BMH4-97-2487
trial treated with ACE-Inhibitors (ACE-I) or
placebo (PL)
Vicia Villosa
Helix Aspersa
plt0.001
plt0.01
plt0.01
OD 405 nm
plt0.07
93
Ciclo cellulare Cicline ed Inibitori
S Sintesi e replicazione
Apoptosi
pRb
DNA
Caspasi
G2
G1
attivazione
Mitosi
Smac/Diablo
Switch Bcl-2 Bfl-1
Regolazione dei proteasomi
inibizione
94
IL-6/CRP ratio at the end of the study
95
IL-6/CRP ratio basal vs end of the study in
Placebo group
96
IL-6/CRP ratio basal vs end of the study in ACE-I
group
CRP (mg/dl)
97
IgA C3 IgG
IgA nephropathy
98
IkB, alpha 2 proteasome, ubiquitin mRNA/GAPDH
mRNA in PBMC from IgAN patients and controls
Alpha 2 Proteasome mRNA
IkBmRNA
plt0.0001
plt0.001
ubiquitin mRNA
p ns
IgAN
Healthy subjects
99
PDGF
Deposited IgA
IL-8
MCP-1
RANTES
Fc?R
Lympho- monocyte infiltrates
TfR
O2-
Ca

Cytokines, ROS
actin
Cell contraction, proliferation, mediator
synthesis
MC proliferation/ apoptosis
PAI-1
Mesangial matrix accumulation
NO
TGF??
Sclerosis
TX LT
ET1
100
IgAN seen under the light of a systemic disease
Circulating lymphomonocyte abnormalities Th2
subset prevalence
what do they mean? Is this dysregulation of the
immune system only aimed to affecting the IgA1
glycosylation?
101
IgAN seen under the light of a systemic disease
Gene expression profiles of circulating
leukocytes correlate with disease activiy in
IgAN. Preston GA, Jennette JC, Falk RJ et al
KI2000465420-30
Circulating lymphomonocytes are activated gene
chip microarrays and TaqMan PCR showed 15 genes
trascriptionally up-regulated, differently from
FSGS, ANCA vasculitis and healthy controls.
CONCLUSION In IgAN leukocytes carry
disease-specific markers of pathogenic changes in
renal tissue
102
Lets consider IgAN under the light of a
systemic disease
Transcription factors in peripheral
lymphomonocytes of patients with IgAN are
translocated to the nucleus, indicating
activation of gene transcription
What is that activation aimed at? Is it only
matter of IgA1 glycosylation?
103
TLR activation innate immunity and glomerular
disease
Banas M , Alpers C Resenberg, Germany, Seattle
MP058
TLSP transgenic mice -/- for Fc? RIIb model of
cryoglobulinemic membranoproliferative GN (MPGN).
Mice were analysed for TLR expression during
MPGN development. TLR 1,2,4 were up-regulated in
glomeruli of animals with MPGN. TLR 4 staining
was increased in tubular cells and podocytes
Innate immune system activation via TLR
upregulation and hyperexpression in resident
renal cells play a role in membranoproliferative
GN
104
MRL 1pr/1prmice SLE proneTLR3 are espressed by
resident glomerular cells and TLR3
mRNAincreases at the 20th week, when
inflammatory glomerular and interstitial lesions
develop. Patole et al JASN 2005 161326-38
105
TLR3 in kidney of MRL 1pr/1prmice .C-D
Rhodamine-labelled polyinosinic-cytidylic acid
(pIC)RNA (TLR3 ligand) injected iv and tissue
harvested after 2 hs.C interstitial D mesangial
cells positive, antigen-presenting cells

• TLR3 staining in mesangial cells
• and
• infiltrating cells

106
TLR activation innate immunity and glomerular
disease
Anders A , Schlondorff D Munich, Germany MP050
Female MRL1pr (SLE prone) and MRL wild-type
mice Injected with TLR stimuli viral dsRNA
TLR3 viral SSRNA TLR7 microbial CpG-
DNA TLR9 Ligands induced lymphoproliferation and
anti DNA Ab. Only CpG- DNA induced diffuse
proliferative LN, proteinuria, glomerular
macrophage infiltrates, IgG and C3 deposits in
MRL1pr and not in wild- type mice.
Unique role of microbial CpG-DNA to trigger SLE
nephritis via TLR9 ligation in B-cells and
dendritic cells of SLE genetic prone mice.
107
Thanks to
Licia Peruzzi, MD Giovanni Conti, MD Monica
Chiesa, PhD Paola Cirina, PhD Alessandra Mollo,
PhD Stefania Turcato, PhD Laura Ghisellini,
PhD Collaboration Andrea Onetti Muda Prof of
Pathology University La Sapienza Roma, Italy
108
Possible mechanisms leading to reduced
glycosylation of IgA1 side chain glycans

• Genetic factors ? 1,3 Gal transferase
• - reduced enzyme activity PMN (Allen
  1997, but not confirmed in 2004)
• - Core ? 1,3 Galactosyltransferase gene
  polymorphism
• (Amoroso 2004)

Acquired mechanisms - Th1/Th2 unbalance
during infections with prevalence of Th2 subset
(Emancipator, 1998)
109
Lets consider IgAN under the light of a
systemic disease
Transcription factors in peripheral
lymphomonocytes of patients with IgAN are
translocated to the nucleus, indicating
activation of gene transcription
What is that activation aimed at? Is it only
matter of IgA1 glycosylation?
110
Factors affecting different toxicity of
ciculating or deposited IgA
Transcription factors in peripheral
lymphomonocytes of patients with IgAN are
translocated to the nucleus, indicating
activation of gene transcription
What is the meaning of this activation? Where is
it coming from ? Does it only reflect a similar
activation in the kidney or does it play an
independent role?
111
NF-kB
Cytokines, bacterial, viral infections stress
TNFa, IL-1, LPS, CD40L
IKK ? NEMO
IkB
IKK a
E3IkB Ub ligase
IKK ?
p50
p65
p
p
IKB
Ub-Ub-Ub-Ub
p
p50
p65
karyopherins
a ?
26S proteasome
NF-kB responsive genes transcriptional
activation
112
We investigated immunoproteasome switch in the
65 IgAN patients treated with ACE-I or steroids
for gt 3 months
113
Proteasome structural subunits Alpha 2 mRNA in
PBMC of IgAN with different treatments (TaqMan)
Arbitrary Units/Abl gene
?2 Subunit
114
IgA
IgA1 in renal deposits suggesting a bone marrow
origin
polymeric IgA in renal deposits suggesting
a mucosal origin
Polymeric IgA1 deposited in IgAN originated in
mucosal lymphoid tissue, have a bone marrow
homing
115
Which is the origin of the aberrant glycosylated
IgA1
binding of tonsillar IgA from IgAN patients to
the glomerular mesangium of IgAN patients
(Tokuda M et al, 1996)
Tonsillar IgA1 as a source of hypoglycosylated
IgA1 in IgAN (Itoh A, Hiki Y et al, 2003 and
Hiki 2004)
Under-O-glycosylated glomerulophilic IgA1
Ag
Tonsils
Mesangial deposition
116
Which is the origin of the aberrant glycosylated
IgA1
Mucosal T lymphocytes Homing in the Bone
marrow Release in circulation and increase in T
lymphocytes expressing ?4ß1 systemic homing
receptor Batra A, Feehally J, Smith AC JASN 2002
Bone marrow
Mucosa
?4ß1
?4ß7
Increase in circulating T lymphocytes expressing
systemic homing receptor ?4ß1
Mucosal T lymphocytes Expressing Mucosal
homing Receptor ?4ß7
117
Which is the origin of the aberrant glycosylated
IgA1

• Tonsillectomy induces cessation of acute episode
  of gross hematuria and
• improves urinary abnormalities during outcome
  (Bené 1993)
• Increase in B cell producing IgA with reduction
  of B cell producing IgG
• (Bené 1983)
• Decrease in IgA serum levels in IgAN after
  tonsillectomy, without
• modification in salivary IgA (DAmelio 1982)
• Epithelial tonsils cell does not produce SC tract
• (Bené 1993)

118
Heterogeneity of O-glycosylation detected by
mass spectometry in the hinge region of IgA1
(Novak 2000)

Different glycosylation of IgA1 and GBM
thickness (Berthoux 2003)
119
Which is the origin of the aberrant glycosylated
IgA1
IgA1 molecules produced by tonsilar lymphocytes
are under-galactosylated In IgA nephropathy
(Horie A, Hiki Y et al, 2001)
Tonsillar IgA1 as a possible source of
hypoglycosylated IgA1 in serum of IgA
nephropathy patients (Itoh A, Hiki Y et al, 2003)
Direct evidence of the production of IgA by
tonsillar lymphocytes and the binding of IgA to
the glomerular mesangium of IgAN patients
(Tokuda M et al, 1996)
In IgA nephropaty
Under-O-glycosylated glomerulophilic IgA1 (few
?g/ml)
Tonsil
Mesangial deposition
120
Peruzzi , Amore, Coppo et al Kidney Int 2000
58 2331-2340
?v integrin expression in mesangial cells
deSia/deGal gt native IgA
plt0.001
Number of ?v molecules/MC
121
PDGF
Mesangial deposited IgA IgAR
IL1 IL6
IL8 RANTES
MIP
Fc?
Lymphomonocyte infiltrates
Asialo Glyc R
O2- ROS
Ca
MCP-1
Cytokines
actin
Cell contraction, proliferation, mediator
synthesis
Proteases
Mesangial matrix accumulation
NO
TGF??
PAF
TX LT
ET1
122
PDGF
Deposited IgA
IL-8
RANTES
MCP-1
Fc?R
Lympho- monocyte infiltrates
AT1R
O2-
Ca

Cytokines, ROS
actin
Cell contraction, proliferation, mediator
synthesis
MC proliferation/ apoptosis
PAI-1
Mesangial matrix accumulation
NO
TGF??
Sclerosis
TX LT
ET1
123
Mesangial cell proliferation /apoptosis aberrantly
glycosylated IgA proliferation and
apoptosis in cycling cells. (Amore A, Coppo
R, Am J Kidney Dis 36 1242-1252,2000)
Proliferation
basal
Native IgA
DeSia IgA
DeSia/ DeGal IgA
Apoptosis p53
Apoptosis TUNEL
124
Putative Genes conditioning progression of IgAN

• RAS system ACE, AGT, ATR1
• Adducin
• TCR a andß
• eNOS
• PAI-1
• CD14
• Genes encoding for cytokines and chemokynes
• TNF a, IFN ?, IL1R , IL-4, uteroglobin,
  IL-10, TGF ß

often conflicting results
125
NF-kB DNA binding activity in human MC
incubated with in vitro prepared aberrantly
glycosylated IgA (deSia/deGal IgA1 or
GalNAc-IgA1) Coppo R,
Amore A . Kidney Int 2004 65 1544-1547
NF-kB
NF-kB densitometry
Free-probe
1 2
1 basal conditions 2 deSia/deGal IgA
Basal deSia/deGl IgA1
126
Aberrantly glycosylated IgA
iNOS release of NO from mesangial cells
Depressed VEGF production
Favored apoptosis
Depressed cell proliferation
MESANGIAL SCLEROSIS
127

• Still unanswered observations questions
• Why some patients have innocent IgA deposition,
  detected only at kidney donation?
• Why some IgAN progress to sclerosis and loss of
  renal function while others maintain microscopic
  hematuria but do not progress?
• Some 5-30 IgAN show remission of any clinical
  signs, but IgA deposits are stll present even in
  remission.

What regulates IgA deposition and malignancy?
128
NF-kB DNA binding activity in lymphomonocytes
(PBMC) (electromobility supershift assay EMSA
anti p50 and p65 Ab)
p50
1 non stimulated 2 stimulated
PBMC (caionophore, PMA)
1 2 1 2 1 2 1 2 1 2 1 2

IgAN patients
Healthy control
p65
1 non stimulated 2 stimulated PBMC
1 2 1 2 1 2 1 2 1 2 1 2

Healthy control
IgAN patients
129
AOPP levels in IgAN patients treated with
ACE-Inhibitors (ACE-I) and good outcome
(proteinuria remission) or with placebo (PL) and
poor outcome (GFR reduction ?30 or nephrotic
syndrome)
PL with poor outcome
mMol/L
plt0.03
ACE-I with favourable outcome
130
NF-kB DNA binding activity in lymphomonocytes
(PBMC) from IgAN patients and controls
(densitometry of electromobility supershift
assay EMSA p50 and p65 Ab)
plt0.0001
plt0.0001
p50
p65
1 2 1 2 1 2 1
2
1Non stimulated 2 Stimulated (Caionophore
PMA)
Healthy subjects
IgAN
131
IgAN patients with proteinuria gt 0.5 g/day have
higher TLR expression in circulating Leukocytes
than those with high proteinuria
Innate immunity is involved in IgAN
132
The activation of NF-kB pathway produces
circulating monocytes activation with production
of ROS
133
ROS
MAPK
p21ras
NFkB
p65
p50
p65
p50
Transcriptional activation of genes
for inflammation, cell proliferation/apoptosis
ubiquinitation
ikB
Phosphorylation IkB
nucleus
NFkB
p65
p50
ADHESION MOLECULES VCAM-1 ICAM-1 E-selectin
VASOCONSTRICTORS Endothelin 1 tromboxanes
PRO-COAGULANT FACTORS thrombomodulin tPA PAI-I
CYTOKINES CHEMOKINES IL1, IL6, IL8 TNF MCP-1
134
the proteasome
protein

• 19S
• 20S

Ub-Ub-Ub-Ub

a
a
a
a
ß
ß
Tripsin Chymotripsin Glutamyl-peptidase
ß
ß
ß
ß
ß
ß
ß
ß
ß
ß
ß
ß
a
a
ß
a
a
26 S proteasome
I
I
I
I
I
135
Proteasome
E3 Ubiquitin Ligase complex
Ub-ATP
Ub-Ub-Ub-Ub
E1 Ub thioester
protein
E2 Ub carrier
a ?
20S
Trypsin and Chymotrypsin Glutamyl-peptidase
26S proteasome
19S
Degradation of proteins crucial for activation of
transcription factors (NF-kB, STAT, Smad, p53,
cdkip21) production of pro-inflammatory
cytokines cell-cycle progression / apoptosis.
Antigen processing for MHC class I
136
SPECIFICITIES OF THE IMMUNOPROTEASOME
Different professional processing of peptides
for Cytotoxic T Cell presentation
Prepare the C-terminus of peptide residues to
bind with high affinity to MHC class I cleft
Dendritic cells switch to 100 immunoproteasome
when migrate to the lymph node to stimulate T
cells
137
A switch from proteasome to immunoproteasome
occurs in peripheral immunocompetent cells from
IgAN
The antigen presentation is likely to be enhanced
?
138
MICROBICAL AGENTS
IFN-a
IFN-?
Cytotoxic Th1
139
Aberrantly glycosylated IgA1 in patients with
IgAN investigated for immuneproteasomes
IgA1 binding to Vicia Villosa
IgA1 binding to Helix Aspersa
140
In IgAN there are signs of microinflammation at
systemic level
The transcriptional factor NF-kB is also
activated.
Immunoproteasomes are activated in circulating
lymphomonocytes of IgAN patients.
Can we consider a role for a common activating
factor?
Can we envisage a role of innate immunity in the
pathogenesis of IgAN ?
141
ROC analysispredictive value for IgAN
recurrence of aberrantly glycosylated IgA1 TNF
alpha genotype GA and IL-10 genotype ACC/ATA
1.0
0.8
plt0.0001
0.5
0.3
AUC 0.860 95 CL 0.764-0.956
Sensitivity 76, Specificity 87 Accuracy 81
142
Toll-like receptors mediate NF-kB activation
LPS
TLR4
NOD ligands
MAL MyD88
NOD
TRAM
IRAK, TRAF6
TRIF
CARD
RICK
MKK
JNK
AP-1
IKK ? NEMO
IKK complex activation
NF-kB
IKK a
IKK ?
143
Enzymatically active Proteasome subunits in PBMC
of IgAN with different treatments (TaqMan) (Coppo
, Amore Peruzzi, ASN 2006
plt0.05
plt0.05
plt0.01
Arbitrary Units/Abl gene
ß1 Subunit
ß2 Subunit
ß5 Subunit
144
Inducible immunoproteasome subunits in PBMC of
IgAN with different treatments (TaqMan)
plt0.001
Arbitrary Units/Abl gene
LMP2 Subunit
LMP7 Subunit
MECL-1 Subunit
145
Xu L Kidney Int 2005 68 167-72

• Aberrantly glycosylated serum IgA1 are associated
  with pathologic phenotypes of IgAN
• The exposure of GalNAc (VV binding) is
  significantly associated with sclerosing more
  than proliferative changes in renal biopsies of
  IgAN

146
Serum fractions rich in IgA isolated from IgAN
patients proliferation
proliferation in resting mesangial
cells in cycling cells
(Tomana, Lai)
(Tomana, Amore)
Different effects are also related to the
molecular size of macromolecular IgA. 400-800
kDa depress proliferation (Amore, Tomana) gt 800
kDa enhance proliferation (Tomana)
The milieu is crucial for the collaboration among
molecules producing the final effect.
147
Heterogeneity of O-glycosylation detected by mass
spectometry in the hinge region of IgA1 (Novak
) Different glycosylation of IgA1 and GBM
thickness (Berthoux ) Different defects in IgA1
glycosylation might influence the natural
history of the single case IgAN with renal graft
loss due to progressive IgAN had IgA1 with
increased exposure of GalNAc ß1,3Gal (Feehally)
148
Outcome progression of renal damage
End-point 30 loss of initial CrCl
and/or worsening of proteinuria to nephrotic
range (gt3.5 g/1.73m2/day)
End-point 30 reduction of initial CrCl
P 0.034
P0.180
149
IgACE trial Changes in CrCl slopes over the
follow-up
34 patients Median FU 38 months
23 patients Median FU 46 months
150
(No Transcript)
151
IgACE first multicentric, prospective,
double-blind, randomized, placebo-controlled
multicenter trial of ACE-I in moderately
proteinuric IgAN in the young
Rosanna Coppo, Licia Peruzzi, Alessandro
Amore, Antonio Piccoli, Pierre Cochat, Rosario
Stone, Martin Kirschstein, Tommy Linné on
behalf the EC Biomed Concerted Action Project
BMH4-97-2487(DG 12-SSMI) and IgACE European
Collaborative Group.
152
MICROBICAL AGENTS
LPS
TLR4 TLR3
Dendritic cell
IFN-a
Aberrantly glycosylated IgA
Immuneproteasome
B Linf
CD-40
IL-6
CD40 L Blys BAFF April
TGFß IL-2
Th2
IgA