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Molecular associations of IFTA

Michael MengelAlberta Transplant Applied Genomics Centre

University of Alberta, Edmonton Canada

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Interstitial fibrosis and tubular atrophy (IFTA) in renal allografts

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IFTA with inflammation

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Issues with doing analysis for the causes / associates of IFTA

• Most studies group comparisons “sick vs. well”, i.e. biopsies with moderate to severe IFTA are compared to normal biopsies without IFTA: starting with the extreme phenotypes

• Cave! The first group generally has a lot of other pathologies while the others are really normal, thus it’s difficult to see whether the findings are related to IFTA or to a co-existing finding, e.g. interstitial inflammation or time post transplant

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Example 1 of a “sick vs. well” studyTitle: “Molecular pathways involved in loss of kidney graft

function with tubular atrophy and interstitial fibrosis.”

Mol Med. 2008 May-Jun;14(5-6):276-85.

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Scoring inflammation in renal allograft biopsies

60% IFTA compartment40% non-scarred compartment

100% Cortex

relative scoring

according to current Banff

rules

25% = Banff i-score 1 “67% i-IFTA”

5% 3% 3% 5%

absolute scoring40% i-IFTA10% i-Banff

nodular

perivascular

subc

ap

sula

r

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Material and Methods

• 129 biopsies for cause classified according to Banff ‘97

• Semi-quantitative assessment as absolute percentages of:– i-Banff = inflammation in non scarred cortex– i-IFTA = inflammation in IFTA– nodular– perivascular– IFTA

• Correlation of extent of histological lesions with gene expression data from microarrays and allograft survival

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0

10

20

30

40

50

60

70

80

90

100

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111 116 121 126

% c

ort

ex in

volv

ed

fibrosis/atrophy

i-Banff

i-IFTA

Infiltrates in biopsies for cause are time dependent

3 months

8 months

27 months

79 months

246 months

129 biopsies ordered by time post TX

Mengel et al. Am J Transplant. 2009 Jan;9(1):169-78.

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Infiltrates and time in BFC

0

5

10

15

20

25

30

<6 months post TX, n=42 >6 months post TX, n=87

me

an

% c

ort

ex

inv

olv

ed

nodular

perivascular

i-Banff

i-IFTA

IFTA

p<0.0001

p<0.0001

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Inflammation in fibrosis/atrophy is associated with worse allograft survival

A

Inclusion criteria: IFTA >5%, i-Banff <25%

graft survival n

≥50% of fibrosis/atrophy show infiltrates 69.6% 46

<50% of fibrosis/atrophy show infiltrates 93.5% 31

censored 77+

uninflamed IFTA

inflamed IFTA

p=0.02

B

biopsies with graft survival n

i-Banff >25%, i-IFTA <25% 69.2% 13 i-IFTA >25%, i-Banff <25% 60.0% 20 both <25% 88.7% 71 censored 104 +

i-Banff

i-IFTA

i <25%

p<0.05

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Gene sets*# i-Banff

t-score i-IFTA IFTA nodular

perivascular

T-cell associated (CATs) 0.534 0.484 0.284 0.246 0.298 ns

γ-Interferon dependent (GRITs) 0.532 0.441 0.258 0.211 0.241 ns

Kidney parenchyma associated (KTs) -0.296 -0.303 -0.199

-0.156 ns ns

Injury and repair associated (IRITs) 0.379 0.355 0.246 0.206 ns ns

Immunoglobulin associated (IGTs) 0.174 ns 0.434 0.398 0.336 ns

B-cell associated (BATs) 0.281 0.279 0.423 0.387 0.355 ns

# given is the highest r-value revealed for one PBT of each particular biological process*Spearman correlation, p<0.001

Table 1: Correlations between Infiltrate types and Pathogenesis Based Transcript sets (PBTs)

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Correlations* between individual genes and histological lesions

0

10

20

30

40

50

60

70

80

90

100

i-Banff Banff t-score i-IFTA fibrosis/atrophy nodular

% c

ontr

ibut

ion

not otherwise annotated

Endothelial activation

B-cell / Ig associated

Injury and repair associated

Kidney parenchyma associated

Macrophage associated

Interferon-γ dependent

T-cell associated

484 probesets 249 probesets 34 probesets172 probesets202 probesets

*r>0.4, p<0.001: no correlations between any genes and perivascular infiltrates at this cut-off

T-c

ell

T-c

ell

T-c

ell

γ-IF

N γ-IF

N

B-c

ell

B-c

ell

B-c

ell

Ma

c

Ma

c

InjuryInjury

Injury

Injury

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Figure 3: Overlap in gene expression between infiltrate types

i-Banff t-score

244

240116 cytotoxic T cell associated

54 not annotated39 γ-interferon dependent

14 Injury and Repair induced17 macrophage associated

9

i-IFTA IFTA

70

13284 not annotated

26 B cell associated15 Injury and Repair induced3 cytotoxic T cell associated

3 Kidney parenchymal1 Endothelial activation 40

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Genes (top 25) correlating with fibrosis/atrophy and i-IFTA

Affymetrix ID Gene symbol

Gene name Correlation* r-value

205624_at CPA3 mast cell carboxypeptidase A3 0.619207134_x_at TPSB2 mast cell tryptase beta 2 0.566204719_at ABCA8 ABC transporters - ATP-binding cassette, sub-family A (ABC1), member 8 0.564205683_x_at TPSAB1 mast cell tryptase alpha/beta 1 0.555205044_at GABRP gamma-aminobutyric acid (GABA) A receptor 0.554211734_s_at FCER1A receptor for Fc fragment of IgE, high affinity I, expressed predominantely on mast cells 0.547209173_at AGR2 anterior gradient homolog 2 0.523229461_x_at NEGR1 neuronal growth regulator 1 0.509213974_at ADAMTSL3 ADAMTS-like 3 0.503221933_at NLGN4X neuroligin 4 0.499228310_at ENAH cytoskeleton regulatory protein hMena 0.497202508_s_at SNAP25 synaptosomal-associated protein 0.497226435_at PAPLN papilin 0.486228241_at BCMP11 AGR3 = anterior gradient homolog 3 0.485219552_at SVEP1 sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 0.484227088_at PDE5A phosphodiesterase 5A 0.479219778_at ZFPM2 zinc finger protein, multitype 2 0.474207496_at MS4A2 Fc fragment of IgE, high affinity I, receptor for; beta polypeptide 0.47202992_at C7 Complement compnent 7 0.4681558714_at ROBO1 roundabout, axon guidance receptor, homolog 1 0.467219867_at CHODL chondrolectin 0.466206336_at CXCL6 chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) 0.466210258_at RGS13 regulator of G-protein signaling 13 0.462201489_at PPIF peptidylprolyl isomerase F (cyclophilin F, CYP3; Cyp-D) -0.47208321_s_at CABP1 calcium binding protein 1 -0.49

*Spearman correlation with extent of fibrosis/atrophy or i-IFTA, p<0.001

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0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

% T cells (CD3+) % Macropahges(CD68+)

% B cells (CD20+) % plasma cells(CD138+)

% IgG4 plasma cells(IgG4+)

% mast cells(tryptase+)

%

i-non-IFTA

i-IFTA

Confirmation by immunohistochemistry

p 0.006

p 0.02

p 0.006

p 0.05

p 0.0004

i-Banff

i-IFTA

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Mast cell associate transcript set (MACAT)

4 mast cell associated transcripts– CPA3 (Carboxypeptidase 3)– TPSB2 (Tryptase beta 2) – TPSAB1 ( Tryptase alpha-beta 1)– FCER1A (Fc fragment of IgE, high affinity I, receptor)

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0

0.5

1

1.5

2

2.5

i-non-IFTA<25%, n=109

i-non-IFTA>25%, n=20

i-IFTA <25%,n=102

i-IFTA >25%,n=27

IFTA <25%,n=87

IFTA >25%,n=42

Mas

t cel

l PBT

sco

re

p<0.0001

i-Banff<25%, n=109

i-Banff>25%, n=20

Mast cell associated transcripts are a molecular correlate of IFTA

Mast cell transcripts correlate with

time post TX: r=0.55, p <0.01

i-IFTA: r=0.63, p <0.01

IFTA: r=0.61, p <0.01

delta GFR: r= -0.35, p <0.0001

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In biopsies with IFTA increased expression of Mast cell associated transcript is associated with worse allograft survival

Inclusion criteria: at least IFTA grade I

graft survival n

high* MACAT score 71.2% 29

low* MACATscore 96.6% 59

censored 88

*low = lowest tertile of MACAT score of the included 88 biopsies*high = intermediate and highest tertile of MACAT score of the included 88 biopsies

+

high mast cell scoresp=0.01

low mast cell scores

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Summary IFTA and infiltrates

• There are two inflammatory compartments in renal allografts:

– i-Banff (non-scarred): • time-independent, T-cell, γ-interferon, macrophage associated,

prognostic relevant

– i-IFTA (scarred): • time-dependent, T-cell, γ-interferon, macrophage + B-cell, mast

cell associated, prognostic relevant

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Relationship between IFTA and function in native kidneys

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Relationship between IFTA and function in renal allografts (biopsies for cause)

Kasiske et al. Kidney Int. 1991;40:514-524

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IMATs (Mactrophage activation)

IGTs (Plasma cell infiltration)

CISTs (severe injury)

GSTs (severe injury)

BATs (B cell infiltration)

GRIT1 (Ifng effects)

GRIT2 (Ifng effects)

KT1 (Parenchymal transcripts)

IRITD5 (Injury)

IRITD3 (Injury)

IRITD1 (Injury)

CMATs (Macrophage infiltration)

CATs (T cell infiltration)

An

no

tati

on

of

pro

bes

ets

(% o

f to

tal o

f co

rrel

atin

g p

rob

eset

s)

0

5

10

15

20

25

30

35

40

45

Negative Correlation Positive Correlation

n = 144

n = 224

GFR transcript set

Molecular correlates of eGFR at the time of biopsy

Bunnang and Einecke et al. J Am Soc Nephrol. 2009 (5):1149-60

GFRT Negative

GFRT Positive

Histologic lesions (Banff scores)*

g 0.09 -0.10

cg 0.03 0.03

i 0.33** -0.32**

ci 0.29** -0.20**

t 0.16 -0.19*

ct 0.29** -0.20*

v -0.02 -0.02

cv -0.09 0.17*

ah -0.17* 0.21*

mm 0.17 0.05

PTC 0.07 -0.08

Renal function

eGFR at Biopsy -0.54** 0.50**

Functional deterioration from baseline (delta eGFR) -0.28** -0.30**

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Significant overlap between injury / repair / and IFTA associated transcripts

Am J Transplant. 2007 Nov;7(11):2483-95.

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Numerous “fibrosis genes” are associated with injury and repair

Early IRITs Intermediate IRITs Late IRITs

Category % of genes in the list % of genes in the list % of genes in the list

Response to stress (832) 4.8 7.4 9.1

Morphogenesis (n=951) 5.2 8.0 6.9

Organ development (991) 3.8 8.9 8.7

Embryonic kidney (n=904) 7.1 10.6 6.1

Ureteric bud vs mesenchyme (n=933) 6.7 12.6 7.4

Embryonic mesenchyme (n=951) 2.9 9.7 11.3

Mesenchyme vs ureteric bud (n=751) 4.8 8.0 19.9

Cell cycle and cell proliferation (n=1008) 2.4 8.3 10.4

Tgfb1/fibrogenesis (n=48) 1.9 3.2 6.1

Collagen (n=36) 0.5 1.1 4.3

ECM (n=431) 1.9 4.0 12.1

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Molecular changes in protocol biopsiesBackground and Hypothesis

early protocol biopsy

subclinical pathology = harbinger of more severe, clinical overt, irreversible pathology

therapeutic intervention prevention of irreversible chronic allograft damage

assessment of the

subclinical molecular phenotype of an allograft to further corroborate this hypothesis

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No molecular evidence for EMT in the onset of IFTA in early protocol biopsies

J Am Soc Nephrol. 2008 Aug;19(8):1571-83.

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Molecular associates of subclinical IFTA at 12-months in clinically uncomplicated living donor kidney transplants

Transplantation. 2007 Jun 15;83(11):1466-76

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PBT-annotation of top 100 transcripts correlating* in 6-week protocol biopsies with future onset of

IFTA in 6-month protocol biopsies

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

correlated with onset of IFTA at 6-months post TX

correlated with delta clearance 6-weeks to 12-months

correlated with prevalence of acute rejection between 6-weeks and 12-months

after transplantation

Endothel associated transcripts

TGF-β related transcripts

kidney parenchymal transcripts

injury induced transcripts

interferon-γ inducible transcripts

macrophage associated transcripts

B cell associated transcripts

NK cell associated transcripts

T cell associated transcripts

*Spearman correlation, p<0.001

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PBT-annotation of top 100 transcripts correlating* with future end points in PB

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

correlated with onset of IFTA at 6-months post TX

correlated with delta clearance 6-weeks to 12-months

correlated with prevalence of acute rejection between 6-weeks and 12-months

after transplantation

Endothel associated transcripts

TGF-β related transcripts

kidney parenchymal transcripts

injury induced transcripts

interferon-γ inducible transcripts

macrophage associated transcripts

B cell associated transcripts

NK cell associated transcripts

T cell associated transcripts

*Spearman correlation, p<0.001

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Conclusions: Molecules and IFTA

• IFTA by histology is associated with inflammation and injury / repair molecules = sign of active/progressive IFTA or attempt to recovery?

• Conclusion about cause for IFTA is yet not possible• Some molecular correlates (e.g. mast cells) might

be used as a measurement for IFTA• More detailed resolution of the overall molecular

disturbance might provide potential therapeutic targets for a non-cause specific anti IFTA treatment

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Outlook

• Test reproducibility for IF, TA, i-Banff, i-IFTA, and total i-score: – if feasible, reporting of the different

inflammatory and morphological compartments might allow to design new clinical trials

– i-Banff and i-IFTA might be amenable to different therapies