IgA Nephropathy (Berger’s Disease)
Mechanisms of Disease
Bruce A. Julian, MDUniversity of Alabama at Birmingham
Department of MedicineBirmingham, AL, USA
Jean Bergercirca 1968
IgA NephropathyThe most common primary glomerulonephritis in the world
IgA1 (with C3, IgG, or IgM) Mesangial Immunodeposits
Periodic acid-Schiff stainImmunofluorescence
Expansion of Extracellular MatrixProliferation of Mesangial Cells
IgA NephropathyElectron Microscopy
Mesangial Deposits
IgA NephropathyCause is Extra-Renal
Kidney is under attack from systemic process
Kidney donor,Subclinical IgAN
Non-IgAN recipients
Clearance of IgA, within weeks
Kidney donor,Healthy
IgAN recipients
IgAN recurrence ~50%,at 2 years
I.
II.
IgA NephropathyPossible Factors Initiating Disease
Genetic factorsAberrancy of structure of IgA1 molecules
Glycosylation aberrancy -> aggregation, CIC formation,
Increased levels of IgA and IgA-containing complexesOverproduction or defective clearance
Endogenous or exogenous antigensCIC formation, IgA renal deposition
Immunological defects(allergy, complement, coagulation, ...)
Modified from Julian BA, et al., Adv Nephrol 1999
IgA NephropathyThe most common primary glomerulonephritis in the
worldPrevalence: 25-50 / 100,000
40-50%
15-20%Rare
1-2%
20-30%
5%
3-10%
2-10%
Percentage of native-kidney biopsies showing IgA nephropathy
Familial IgA NephropathyLoci Linked with Familial IgAN
Chr 6q22-23 (IgAN1)Some Kentucky (USA) and Italian
families
Chr 4q26-31Some Italian families
Chr 7q12-22Some Italian families
Chr 2q36Canadian Caucasian family
Other loci (not defined)Lebanese family
The mode of presentation and clinical course of patients with familial IgAN do not differ from that of patients with the sporadic forms.
IgA nephropathy is a phenotype that likely results from interactions of multiple susceptibility and progression genes and environmental influences.
Nonetheless, despite the lack of success in characterizing the IgAN gene(s), the familial aggregation of IgA nephropathy (10%-15% cases)suggests that some disease mechanisms are genetically determined.
IgA NephropathyGenetic Influence(s) in the Clinical Expression
Human IgA1Structure and glycosylation
O-linked glycans
N-linked glycans
J Biol Chem 273, 2260, 1998
CH2α1
Hinge Regions of Human IgA1 and IgA2
-Pro-Val-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Cys-
(CHO) CHO CHO CHO (CHO)
225 228 230 232 236
-Pro-Val-Pro------------------------------------------------------------------------------Pro-Pro-Pro-Pro-Cys-
IgA1
IgA2m(1)CH2α2
CH1α1
CH1α2
Cα2
Cα3
Cα2
Cα3
VH VH
VL
VLCL CL
Cα1 Cα1
α2,6 β1,3 β1,3
α2,3
β1,3
α2,3
α2,6β1,3 α2,6
Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr
Human IgA1Structure and Glycosylation
Hinge-regionO-linked glycans
N-linked glycansMolecular forms:MonomerDimer (J chain)Higher polymer (J chain)Secretory IgA (J chain + SC)
IgA1 Hinge-region O-glycansPossible Structures
-Ser/Thr-
GalNAc
Ser/Thr
GalNAc
Gal
Ser/Thr
GalNAc
SAα2,6
β1,3Gal
α2,6
α2,3
β1,3
-Ser/Thr-
SA
SA
Most common forms
normal serum IgA1
Greater frequency
IgAN serum IgA1
SA = sialic acid
Ser/Thr
GalNAc
IgA1 Hinge-region O-glycansPossible Structures & Lectin Binding
-Ser/Thr-
GalNAc
Ser/Thr
GalNAc
Gal
Ser/Thr
GalNAc
SAα2,6
β1,3Gal
α2,6
α2,3
β1,3
-Ser/Thr-
SA
SA
Most common forms
normal serum IgA1
Greater frequency
IgAN serum IgA1
Lectin PNA-reactive
Lectin HAA-reactive
SA = sialic acid
Lectin jacalin-reactive
LectinSNA-reactiveSer/Thr
GalNAc
Controls Patients0
1000
2000
3000
4000
5000
6000
7000
8000
9000
HA
A-Ig
A1
Leve
lun
its/m
l
n = 152 n = 145
IgA NephropathySerum Levels of Galactose-deficient IgA
Caucasian Adult Patients with IgAN and Healthy Controls
Moldoveanu Z, et al. Kidney Int 2007
Levels in 74% of patients >90th percentile of healthy controls
Serum galactose-deficient IgA is within circulating immune complexes bound to IgG.
Tomana M, et al. J Clin Invest 1999
IgA NephropathyFamilial Disease and Increased Serum Galactose-deficient IgA1 Levels
Gharavi AG, et al. J Am Soc Nephrol 2008
IgA NephropathyFamilial Pattern of Serum Galactose-deficient IgA1 Levels
Gharavi AG, et al. J Am Soc Nephrol 2008
IgAN(n = 11)
Control(n = 11)
HA
A-Ig
A1
(%) P = 0.0001
0
10
20
30
40
50
60
70
IgA1IgA1--secreting Cell Lines from IgA Nephropathy secreting Cell Lines from IgA Nephropathy PatientsPatients
Secrete Greater Amounts of GalactoseSecrete Greater Amounts of Galactose--deficient IgA1deficient IgA1
Measured as ratio of HAA lectin-reactive IgA1 (GalNAc-specific) to total IgA1,Expressed relative to standard Gal-deficient IgA1 myeloma protein
Suzuki H, et al. J Clin Invest 2008
-Ser/Thr-
GalNAc
Ser/Thr
GalNAc
Gal
1,3
HAA-lectinreactive
P < 0.001R2 = 0.883
Serum Levels of GalSerum Levels of Gal--deficient IgA1deficient IgA1Correlation with HAA Reactivity of IgA1 Secreted by Correlation with HAA Reactivity of IgA1 Secreted by
Corresponding Cell LinesCorresponding Cell LinesSu
pern
atan
t HA
A-Ig
A1
(%)
Serum HAA-IgA1
(%)0
10
20
30
40
50
60
70
0 10 20 30 40 50 60 70
Suzuki H et al., J Clin Invest 2008
IgA1 Hinge-region O-glycansEnzymes Facilitate their Construction
-Ser/Thr-
GalNAc
Ser/Thr
GalNAc
Gal
Ser/Thr
GalNAc
SAα2,6
β1,3
Ser/Thr
GalNAc
Gal
α2,6
α2,3
β1,3
-Ser/Thr-
SA
SA
β1,3 galactosyltransferase
Cosmc
α2,6 sialyltransferase
sialyltransferases
GalNAc-transferase2
Transcription of Specific GlycosyltransferasesTranscription of Specific GlycosyltransferasesIgA1IgA1--producing Cell Linesproducing Cell Lines
11 IgAN Patients and 11 Healthy Controls11 IgAN Patients and 11 Healthy Controls
Rel
ativ
e m
RN
A e
xpre
ssio
n
0.0
0.5
1.0
1.5
2.0
2.5
3.0
GalNAcT2 C1GalT1 Cosmc ST6GalNAcII
****
*ST6GalNAcI not expressed
J chain and IgA alpha chaindid not differ
IgAN patients Healthy controls
Suzuki H, et al. J Clin Invest 2008
Complex Changes in Biosynthetic Pathways of IgA1 Complex Changes in Biosynthetic Pathways of IgA1 OO--glycansglycansPatients with IgA NephropathyPatients with IgA Nephropathy
-Ser/Thr- -Ser/Thr-
GalNAc
Ser/Thr
GalNAc
Gal
Ser/Thr
GalNAc
Gal
SA
SA
Ser/Thr
GalNAc
SA
Cosmc
core 11,3-galactosyltransferase
2,6-GalNAc-sialyltransferase II
1,3 1,3
2,6
2,3
2,6Sialyltransferases
GalNAc-transferase (2)
C1GALT1
ST6GALNAC2
SA = sialic acid
Increased amount of Gal-deficient IgA1 (Ag))
Formation of circulating immune complexes
Production of anti-glycan IgG/IgA1 (Ab)
Mesangial deposition
Glomerular injury
Defect in IgA1-producing cells
Model for Pathogenesis of IgA Nephropathy
??????
IgA NephropathyIgA NephropathyLevels of AntigenLevels of Antigen--specific IgG in Sera specific IgG in Sera
Against Aberrantly Glycosylated IgA1 are ElevatedAgainst Aberrantly Glycosylated IgA1 are Elevated
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6O
D (4
90 n
m)
P < 0.001
IgAN(n = 16)
Control(n = 16)
Serum antigen-specific IgG
IgG concentration of each sample was normalized
Suzuki H. J Clin Invest in press
Serum IgG from IgA Nephropathy PatientsSerum IgG from IgA Nephropathy PatientsBinds More GalactoseBinds More Galactose--deficient IgA1 than does Serum IgG from deficient IgA1 than does Serum IgG from
ControlsControls
1. Degalactosylated and desialylated IgA1 (dd-IgA1) 2. Degalactosylated IgA1 3. Enzymatically galactosylated dd-IgA14. Enzymatically sialylated dd-IgA1
Binding is GlycanBinding is Glycan--specificspecific
←IgA1 heavy chain
IgAN1 IgAN2 Con1 Con2
Anti-IgG Anti-IgA
+↓
Gal-deficient IgA1 (Ag)
IgG (Ab)
+↓
+↓
+↓
+↓
GalNAcGalSA
1 2 3 4
Serum IgGfrom IgAN
HAA lectin
IgA heavy chain
⇒ anti IgG
Suzuki H. J Clin Invest in press
IgA NephropathyIgA NephropathyLevels of AntigenLevels of Antigen--specific IgG Secreted by IgGspecific IgG Secreted by IgG--producing Cells producing Cells
Against Aberrantly Glycosylated IgA1 are ElevatedAgainst Aberrantly Glycosylated IgA1 are Elevated
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
OD
(490
nm
)
P < 0.001
IgAN(n = 16)
Control(n = 16)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
(490
nm
)
P < 0.001
IgAN(n = 16)
Control(n = 16)
Serum antigen-specific IgG Antigen-specific IgG secreted by cells
IgG concentration of each sample was normalized
Suzuki H. J Clin Invest in press
Binding of CellBinding of Cell--line IgG to Aberrantly Glycosylated IgA1line IgG to Aberrantly Glycosylated IgA1Inhibited by GalNAcInhibited by GalNAc--specific Lectin (HAA)specific Lectin (HAA)
Densitometric analysis
0
20
40
60
80
100
HAA HAA
34%
100%
IgG
bin
ding
(%)
+ + +
Anti-IgA
HAA lectin
Anti-IgG
IgG from IgAN
HAA HAA IgA heavy chain
Gal-deficient IgA1 (Ag)
IgG (Ab)
HAA-lectin( )
GalNAcGal
Hinge region
Glycan-specific IgG
Suzuki H. J Clin Invest in press
IgA heavy chain
IgAN con
Anti-IgG
+↓
+↓
Gal-deficient IgA1 (Ag)
rIgG (Ab)
Recombinant IgG Recombinant IgG ((rIgG) cloned from an IgAN patient binds to GalactoserIgG) cloned from an IgAN patient binds to Galactose--deficient IgA1deficient IgA1
rIgG against Fab-IgA1
IgAN
Controls
1123 1023 3081 1139 3061 2047
9017 3070 9035 3066 30 64 8043
Analysis of Monoclonal IgG by SingleAnalysis of Monoclonal IgG by Single--cell PCRcell PCR
OD
(490
nm
)
0.0
1.0
2.0
3.0
4.0
IgAN Con
Dot-blot analysisPatient #
Patient #Suzuki H. J Clin Invest in press
Cells from IgAN patients1023 &#SXXXXXXXXXXXXXXXXXXXX1123 &#SXXXXXXXXXXXXXXXXXXX1139 &#SXXXXXXXXXXXXXXXXXXXXXXXX2047 &#SXXXXXXXXXXXXX3061 &#SXXXXXXXXXX3081 &#AXXXXXXXXXXXXXXXXXX
Cells from healthy controls3066 &#AXXXXXX3070 &#ASXXXXXXXXXXXXXXXX3064 &#AXXXXXXXXXXXXXXX8043 &#AXXXXXXXXXX9017 &#AXXXXXXXXXXXXXXXXXXX9035 &#AXXXXXXXXXXXXXXXXX
IgAN
Controls
1123 1023 3081 1139 3061 2047
9017 3070 9035 3066 3064 8043
Analysis of Monoclonal IgG by SingleAnalysis of Monoclonal IgG by Single--cell PCRcell PCRHow to Explain Patient #3081How to Explain Patient #3081
Notable Change from Alanine to Serine in Notable Change from Alanine to Serine in CDR3 CDR3 Region Region
Heavy Chain of IgA1Heavy Chain of IgA1--GlycanGlycan--specific IgGspecific IgG
Suzuki H. J Clin Invest in press
Cells from IgAN patient&#SXXXXXXXXXXXXXXXXXXX
Mutation A
Cells from healthy control&#AXXXXXXXXXXXXXXXXXXX
Mutation S
IgA1 heavy chain
Gal-deficient IgA1 (Ag)
rIgG (Ab) IgAN IgAN Con Con(S A) (A S)
Anti-IgG
+↓
+↓
+↓
+↓
0
20
40
60
80
100Ig
G b
indi
ng (%
)
100%
28%
80%
17%
Mutagenesis for Amino Acid SubstitutionMutagenesis for Amino Acid SubstitutionA to S Substitution: Major Role A to S Substitution: Major Role in Binding of IgG to Galactosein Binding of IgG to Galactose--deficient IgA1deficient IgA1
Suzuki H. J Clin Invest in press
Novel Assay for Serum IgA1Novel Assay for Serum IgA1--GlycanGlycan--specific IgG Antibodyspecific IgG Antibody
Healthy con
Diseasecon
IgAN rIgG
IgAN Disease Con Healthy Con0
10
20
30
40
50
60
70
80
90 (%)
Sign
al in
tens
ity /
rIgG
(%)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.00.10.20.30.40.50.60.70.80.91.0
1-Specificity
Sens
itivi
ty
Area under the curve: 0.9644
DotDot--blot analysisblot analysis
Specificity: 88% Sensitivity: 95%
IgG concentration of each sample was normalized
Suzuki H. J Clin Invest in press
Why do Humans have Circulating IgG Antibodies Specific for Galactose-Deficient IgA1 Glycans?
Antibodies synthesized in response to microorganisms that express GalNAc epitopes on surface structures
This exposure to galactose-deficient IgA1 may further boost anti-GalNAc antibodies in patients with IgA nephropathy
VirusesRespiratory syncitial virusEpstein-Barr virusHerpes viruses
BacteriaStreptococcus
Serum levels of galactose-deficient IgA1 elevated. This aberrant
IgA1 is predominantly in immune complexes.
Galactose-deficient IgA1 likely originates from an imbalance of
the activities of the key enzymes in the synthesis of O-linked
glycans in IgA1-secreting cells.
Galactose-deficient IgA1 in immune complexes is bound to anti-
glycan IgG. This binding IgG often has a unique amino-acid
sequence in the antigen-binding portion of its heavy chain.
Galactose-deficient IgA1Role in the Mechanism of Disease in IgA Nephropathy
IgA1-secreting Cells
HealthyIndividual
IgANPatient
pIgA1
LIVER
KIDNEY
Hepatocytes
Mesangium
Glomerulus
IgG or IgA1
or IgA1
Immune complex
Proposal for Pathogenesis of IgA Nephropathy
Is there evidence for role of galactose-deficient IgA1 to cause IgA nephropathy?
706050403020100.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
HAA
IgA
IgG
Fraction number
OD
(405
nm
)
vo 670 kDa 158 kDa
HAA binds to GalNAc
Circulating Immune Complexes with Galactose-deficient IgA1Activate Proliferation of Human Mesangial Cells in Culture
Size-exclusion chromatography
Stimulation of Proliferation of Mesangial CellsCirculating Immune Complexes Obtained during Acute and Convalescent Phases
Background levels
453525150
2
4
6
ConvalescentGross hematuria
Fraction number
Rel
ativ
e pr
olife
ratio
n
700 kDaVo
IgA NephropathyEluted Glomerular IgA is Galactose-deficient
IgA1 Myeloma Protein: HSP NephritisGalactose-deficient O-linked Glycans in the Hinge Region
0 1 2 3 4 5 6 7 8
Sign
al in
tens
ity /
rIgG
(%)
UP/Cr ratio (g/g)
P < 0.0001R2 = 0.743
0
10
20
30
40
50
60
70
80
0
10
20
30
40
50
0 10 20 30 40 50 60
UIgA-IgG IC/Cr ratio
P = 0.0082R2 = 0.485Si
gnal
inte
nsity
/ rIg
G (%
)
Serum Levels of IgA1Serum Levels of IgA1--GlycanGlycan--specific IgGspecific IgGCorrelate with Urinary ParametersCorrelate with Urinary Parameters
Contemporaneous SamplingContemporaneous Sampling
ProteinuriaProteinuria Urinary ExcretionUrinary ExcretionIgAIgA--IgG Immune ComplexesIgG Immune Complexes
Suzuki H. J Clin Invest in press
IgA1-Associated GlomerulonephritisMechanisms of Renal Injury
Pathogenesis of IgA Nephropathy2009
Substantial progress has been made in understanding genetics and pathology.
IgA nephropathy can be labeled an autoimmune disease, with galactose-deficient IgA1 as the antigen and IgG as the antibody. Still, information about specific molecular mechanisms remains incomplete.
Current “hot topics” for research in the pathogenesis and clinical expression of disease:• Specific site of IgA1 glycosylation aberrancy• Function of IgAN-linked genetic loci• Binding of IgA1-immune complexes to mesangial cells• Why some transplant patients never develop recurrent disease• Urinary / serum biomarkers for diagnosis or monitoring disease
MedicineCathy BarkerGloria GodwinMilan TomanaSue Woodford
Cell BiologyRichard Mayne
MicrobiologyRhubell BrownStacy HallWen-Qiang HuangRose KulhavyClare N. LyasJiri MesteckyZina MoldoveanuJennifer S. MooreJan NovakMilan RaskaHitoshi SuzukiHuong L. VuKazuo TakahashiTakeshi Yanagihara
PathologyBill CookLea Novak
MS core group Stephen BarnesHelen KimMarion Kirk Heath McCorkleTodd SandersonLandon Wilson
Acknowledgments UAB
FT-ICR MS Matthew B. RenfrowStephanie Wall
NIDDK funding
Aarhus University, Aarhus, Denmark Mogens Kilian, Knud Poulsen
Columbia University, New York, NY Ali G. Gharavi, Krzysztof Kiryluk
Fujita Heath University, Toyoake, Japan Yoshiyuki Hiki
Juntendo University, Tokyo, Japan Yasuhiko Tomino, Yusuke Suzuki
Natl. High Mag. Field Lab., Tallahassee, FL Alan G. Marshall
Palacky University, Olomouc, Czech Republic Milan Raska
University of Cincinnati, Cincinnati, OH Andrew B. Herr
University of Tennessee, Memphis, TN Robert J. Wyatt, Keith L. Lau, M.Colleen
Hastings
Yale University, New Haven, CT Richard P. Lifton, Isabel Beerman
AcknowledgmentsBeyond UAB