Experience with Affymetrix Experience with Affymetrix microarrays in kidney transplantationmicroarrays in kidney transplantation

Gunilla EineckeGunilla Einecke

University of AlbertaUniversity of Alberta

Edmonton, CanadaEdmonton, Canada

Our StudyOur Study

Microarray analysis of kidney transplantsMicroarray analysis of kidney transplants

• Establish additional diagnostic criteria for disease Establish additional diagnostic criteria for disease processesprocesses

• Better understand mechanisms that lead to organ Better understand mechanisms that lead to organ damagedamage

• Develop monitoring toolsDevelop monitoring tools

ModelsModels

• Mouse kidney transplant model that Mouse kidney transplant model that simulates the human disease processsimulates the human disease process

• Human kidney transplant biopsiesHuman kidney transplant biopsies

TransplantNative kidney

The mouse modelThe mouse model

Mouse modelMouse model

• Normal CBA kidneysNormal CBA kidneys

• Isografts (CBA/CBA) at days (3,4,) 5, 7, 21Isografts (CBA/CBA) at days (3,4,) 5, 7, 21

• Allografts (CBA/B6) at days (3,4,) 5, 7, 14, 21, 42Allografts (CBA/B6) at days (3,4,) 5, 7, 14, 21, 42

Kidney Transplants (Time course)

• Mixed lymphocyte culture (day 4)Mixed lymphocyte culture (day 4)

• Cloned CTLCloned CTL

• Cultured macrophage lineage (RAW 264.7 cells)Cultured macrophage lineage (RAW 264.7 cells)

Cell cultures

HistologyHistology

WT D7 WT D21

IsoD21 WT D5

• Rapid evolution of MHC/ edema/ infiltration with CD8Rapid evolution of MHC/ edema/ infiltration with CD8++ CTL CTL• Slow evolution of tubulitisSlow evolution of tubulitis

Tubulitis + Tubulitis ++

No Tubulitis

Database of human kidney transplant Database of human kidney transplant biopsiesbiopsies

• Normal donor kidneys (n = 8)Normal donor kidneys (n = 8)

• Well-functioning transplants without rejection (n = 10)Well-functioning transplants without rejection (n = 10)

• Kidneys undergoing acute rejection (n = 7)Kidneys undergoing acute rejection (n = 7)

• Transplants with renal dysfunction without rejection (n = 4)Transplants with renal dysfunction without rejection (n = 4)

• Affymetrix HG-U95Av2 GeneChipAffymetrix HG-U95Av2 GeneChip

Flechner SM, Kurian SM, Head SR, Sharp SM, Whisenant TC, Zhang J, Chismar JD, Horvath S, Mondala T, Gilmartin T, Cook DJ, Kay SA, Walker JR, Salomon DR:Kidney transplant rejection and tissue injury by gene profiling of biopsies and peripheral blood lymphocytes.Am J Transplant 2004 Sep; 4(9): 1475-89

Human kidney transplant biopsies Human kidney transplant biopsies from Edmontonfrom Edmonton

• Nephrectomy samples (n = 3)Nephrectomy samples (n = 3)

• Implant biopsies (n=4)Implant biopsies (n=4)

• Biopsies for cause (n=33)Biopsies for cause (n=33)

• Affymetrix HG-U133 2.0 Plus GeneChipAffymetrix HG-U133 2.0 Plus GeneChip

The key elements in rejection The key elements in rejection observed in the graftobserved in the graft

• Effector T cell infiltration activationEffector T cell infiltration activation

• Macrophage infiltration/activationMacrophage infiltration/activation

• Late clonal B cell/plasma cellsLate clonal B cell/plasma cells

• IFN-IFN- effects: local and systemic effects: local and systemic

• ChemokinesChemokines

• Interstitial remodeling effectsInterstitial remodeling effects

• Epithelial mesenchymal transdifferentiationEpithelial mesenchymal transdifferentiation

• Decreased expression of kidney genesDecreased expression of kidney genes

The key elements in rejection The key elements in rejection observed in the graftobserved in the graft

• Effector T cell infiltration activationEffector T cell infiltration activation

• Macrophage infiltration/activationMacrophage infiltration/activation

• Late clonal B cell/plasma cellsLate clonal B cell/plasma cells

• IFN-IFN- effects: local and systemic effects: local and systemic

• ChemokinesChemokines

• Interstitial remodeling effectsInterstitial remodeling effects

• Epithelial mesenchymal transdifferentiationEpithelial mesenchymal transdifferentiation

• Decreased expression of kidney genesDecreased expression of kidney genes

CTL associated transcripts (CATs)CTL associated transcripts (CATs)

Defining CTL associated transcriptsDefining CTL associated transcripts

Definition:

• absent in normal kidney

• high expression in cultured lymphocytes (d4MLR and cloned cultured CTL)

n = 287 transcripts

Cytotoxic T-cell associated transcripts Expression in Mouse Kidney Transplants

21 7 5 0 5 7 14 21 42

NCBAIsografts Allografts

Time after transplantation (days)

Fo

ld c

han

ge

vs.

NC

BA

1

10

100

---- - + ++ ++ ++

No tubulitis Tubulitis

Measuring the effector T cell burden -Measuring the effector T cell burden - Expression of CATs in rejecting allografts

100

80

60

40

20Ge

ne

exp

ress

ion

in %

of

ML

R

Day 42Day 21Day 14Day 7Day 5Dilution

C Tx R Tx R NR R NR C Tx NR

CATs in human kidney transplant biopsies(published Database)

C = normal donor biopsiesTx = well functioning transplantNR = dysfunction but no rejection AR = acute rejection

Refining the CATsRefining the CATs

• Transcripts with increased expression Transcripts with increased expression ((≥ 2 fold) in allografts D5≥ 2 fold) in allografts D5

• Not increased by rIFN-Not increased by rIFN-

n = 168

CATs in Human Kidney BiopsiesCATs in Human Kidney Biopsies(published Database)(published Database)

Tx Tx Tx Tx Tx Tx Tx Tx Tx TxNR NR NR NR AR AR AR AR AR AR AR

Cluster 1 Cluster 2

Tx = well functioning transplantNR = dysfunction but no rejectionAR = acute rejection

CATs in human kidney transplant biopsies – CATs in human kidney transplant biopsies – Relationship to Banff score (Inflammation)Relationship to Banff score (Inflammation)

(Samples from Edmonton)(Samples from Edmonton)

0 0 1 1 2 30 00 0 2 1 21 10 2 0 1 22 32 3 3 3 32 22 1 2 1

Cluster 1 Cluster 2 Cluster 3

2.2 1.30.8

CATs in human kidney transplant biopsies – CATs in human kidney transplant biopsies – Relationship to Banff score (Tubulitis)Relationship to Banff score (Tubulitis)

(Samples from Edmonton)(Samples from Edmonton)

0 0 0 1 0 20 01 3 1 2 30 10 0 0 0 00 10 3 0 0 11 01 1 1

1.4 0.40.6

Relationship between CAT expression and TCMRRelationship between CAT expression and TCMR(Samples from Edmonton)(Samples from Edmonton)

TCMR TCMR

Summary - CATsSummary - CATs

• CATs in rejecting mouse kidneys are highly stereotyped with stable transcript patterns over time.

• Expression of CATs precedes tubulitis and persists despite massive alterations in the pathology.

• Measurement of CATs allows to estimate the T cell burden.

• In human kidney transplant biopsies expression of CATs correlates with Banff “t” and “i” scores and the diagnosis of acute rejection.

Renal GenesRenal Genes

Defining Renal genesDefining Renal genes

Definition:

• Present in normal CBA kidney

• Absent in cultured effector lymphocytes (MLR and CTL line)

• Absent in cultured macrophage line (RAW 264.7 cells)

n = 1293

Renal genes altered in Renal genes altered in isograftsisografts(altered (altered ≥≥3x in isografts d5,7,21; n = 40)3x in isografts d5,7,21; n = 40)

21 7 5

NCBA5 7 14 21 420

Isografts Allografts

Time post transplant

1

10

0.1

0.01

Fo

ld c

ha

nge

vs

NC

BA

Renal genes altered in allografts Renal genes altered in allografts (altered (altered ≥≥3x in allografts d5,7,14,21,42; n = 712)3x in allografts d5,7,14,21,42; n = 712)

21

1

10

0.1

0.01

Fo

ld c

ha

nge

vs

NC

BA

7 5

NCBA5 7 14 21 420

Isografts Allografts

Time post transplant

21 7 5

NCBA

5 7 14 21 42

0

50

100

150

200

250

300

350

400

450

500

Num

ber

of g

enes

dec

reas

ed 3

x vs

NC

BA

Isografts Allografts

0

Number of renal genes decreased Number of renal genes decreased ≥≥3x post transplant 3x post transplant is much higher in allograftsis much higher in allografts

Glucose TransportersGlucose Transporters

0.020.010.100.060.010.150.220.930.720.602397Slc5a2

0.040.040.170.210.030.370.400.780.730.71572Slc5a11

0.110.130.460.640.840.710.891.051.070.82779Slc5a1

0.120.070.170.090.120.310.410.640.510.671778Slc2a5

0.130.070.290.220.150.380.370.950.770.67380Slc2a4

0.020.010.230.160.200.290.310.880.740.863192Slc2a2

MLRCTLAllo D42

Allo D21

Allo D14

Allo D7

Allo D5

Iso D21

Iso D7

Iso D5NCBATranscript

0.710.630.180.330.396280Slc5a2

0.890.500.220.420.511180Slc5a11

1.001.000.911.011.131789Slc5a1

0.450.350.250.300.433904Slc2a5

1.631.000.290.550.70473Slc2a4

0.670.620.280.380.489441Slc2a2

Left D5

ATN D7

Allo D5

Allo D4

Allo D3NCBATranscript

20

40

60

80

100

120

% o

f ex

pre

ssio

n in

NC

BA

Amino acid transportersGlucose transporters

Aquaporins

Days post transplant

5 7 21

isografts

allografts

Renal epithelial transporters decrease transiently in Renal epithelial transporters decrease transiently in isografts but decrease is much more profound and isografts but decrease is much more profound and

sustained in allograftssustained in allografts

Renal genes in human kidney transplant biopsiesRenal genes in human kidney transplant biopsies(published Database)(published Database)

Tx Tx Tx Tx Tx Tx Tx Tx Tx Tx NR NR NR NR AR AR AR AR AR AR AR

High expression Decreased expression

Renal genes in human kidney transplant biopsiesRenal genes in human kidney transplant biopsiesRelationship to antibody or T-cell mediated rejectionRelationship to antibody or T-cell mediated rejection

Cluster 1 Cluster 2 Cluster 3

TCMR TCMR TCMR TCMRABMR ABMR ABMR ABMR

8/135/20

Overlap in clusters for CTL genes and renal genesOverlap in clusters for CTL genes and renal genes(Samples from Edmonton)(Samples from Edmonton)

BD

I43

CN

61

PI2

0

SO

48

ST

26

-2

WJ

A5

5

ZA

T7

4

WN

45

RD

49

-4

ER

N3

8

MG

71

PM

76

TW

R6

5

SM

C7

7

RD

49

-3

BL

W7

5

WR

56

-1

WR

56

-2

NC

J6

6

HB

39

-1

TK

C

DM

K2

7

MJ

21

ST

26

-1

CL

G4

0

HB

39

-2

MJ

54

MD

A6

4

HN

25

RD

49

-2

TS

H6

0

TI4

7

MJ

72

BD

I43

CN

61

PI2

0

SO

48

ST

26

-2

WJ

A5

5

ZA

T7

4

WN

45

RD

49

-4

ER

N3

8

MG

71

PM

76

TW

R6

5

SM

C7

7

RD

49

-3

BL

W7

5

WR

56

-1

WR

56

-2

NC

J6

6

HB

39

-2

TK

C

DM

K2

7

MJ

21

ST

26

-1

CL

G4

0

MJ

54

MD

A6

4

HN

25

RD

49

-2

TS

H6

0

TI4

7

MJ

72

Renal transcripts

CTL transcripts

Combined analysis of renal genes and CATs – Combined analysis of renal genes and CATs – Principal Component AnalysisPrincipal Component Analysis

(published Database)(published Database)

Well functioning transplants

Dysfunction but no rejection

Acute Rejection

Controls Rejection No Rejection

Correlation of alterations in expression of CATs and Correlation of alterations in expression of CATs and Renal genesRenal genes

(Human Kidney Biopsies – published Database)(Human Kidney Biopsies – published Database)

Summary – Renal genesSummary – Renal genes

• Mouse isografts show some alterations in expression of renal genes.

• In allografts, decreased expression of Renal genes is much more profound and sustained.

• Decreased expression of many Renal genes occurs before tubulitis develops.

ConclusionsConclusions

• Pathogenesis-based transcript sets may be the basis for a secure and robust diagnosis of T cell mediated rejection but require further refinement

• The combined analysis of several transcript sets plus further refinements may increase sensitivity and specificity.

Where to go from hereWhere to go from here

• Further refine the sets of CTL associated transcripts and

renal genes

• Develop additional signatures

Improve understanding of mechanisms of diseaseImprove understanding of mechanisms of disease

(targets for drug discovery)(targets for drug discovery)

• Develop diagnostic and monitoring toolsDevelop diagnostic and monitoring tools

• Adaptation as end points for clinical trialsAdaptation as end points for clinical trials

• Application to other transplants Application to other transplants

• Application to other inflammatory processesApplication to other inflammatory processes