iga nephropathy (berger’s disease) - hÔpital...

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


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



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


IgG (Ab)

HAA-lectin( )

GalNAcGal

Hinge region

Glycan-specific IgG


IgA heavy chain

IgAN con

Anti-IgG

+↓

+↓


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


Cells from IgAN patient&#SXXXXXXXXXXXXXXXXXXX

Mutation A

Cells from healthy control&#AXXXXXXXXXXXXXXXXXXX

Mutation S

IgA1 heavy chain


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


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%



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


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

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