Transcript Slide 1

Molecular associations of IFTA
Michael Mengel
Alberta Transplant Applied Genomics Centre
University of Alberta, Edmonton Canada
ATAGC TSI
Interstitial fibrosis and tubular atrophy (IFTA) in
renal allografts
IFTA with inflammation
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
Example 1 of a “sick vs. well” study
Title: “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.
Scoring inflammation in renal allograft biopsies
100% Cortex
nodular
perivascular
subcapsular
10% i-Banff
5%
3%
25% = Banff i-score 1
40% non-scarred
compartment
3%
40% i-IFTA
absolute scoring
“67% i-IFTA”
relative scoring
5%
60% IFTA compartment
according to
current Banff
rules
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
Infiltrates in biopsies for cause are time dependent
100
fibrosis/atrophy
i-Banff
90
i-IFTA
80
% cortex involved
70
60
50
40
30
20
10
0
1
6
11 16
3
months
21 26
31 36
41 46 51
8
months
56 61
66 71
76 81 86
27
months
91 96 101 106 111 116 121 126
79
months
246
months
129 biopsies ordered by time post TX
Mengel et al. Am J Transplant. 2009 Jan;9(1):169-78.
Infiltrates and time in BFC
30
p<0.0001
mean % cortex involved
25
p<0.0001
20
nodular
perivascular
15
i-Banff
i-IFTA
IFTA
10
5
0
<6 months post TX, n=42
>6 months post TX, n=87
Inflammation in fibrosis/atrophy is associated
with worse allograft survival
B
A
uninflamed IFTA
i <25%
inflamed IFTA
i-Banff
p=0.02
i-IFTA
p<0.05
+
Inclusion criteria: IFTA >5%, i-Banff <25%
graft survival
≥50% of fibrosis/atrophy show infiltrates
69.6%
<50% of fibrosis/atrophy show infiltrates
93.5%
censored
biopsies with
n
46
31
77
+
i-Banff >25%, i-IFTA <25%
i-IFTA >25%, i-Banff <25%
both <25%
censored
graft survival
69.2%
60.0%
88.7%
n
13
20
71
104
Table 1: Correlations between Infiltrate types and
Pathogenesis Based Transcript sets (PBTs)
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
Correlations* between individual genes and
histological lesions
484 probesets
249 probesets
202 probesets
172 probesets
34 probesets
100
not otherwise annotated
90
Endothelial activation
80
B-cell / Ig associated
Injury
B-cell
Mac
i-Banff
Banff t-score
i-IFTA
Injury
T-cell
10
Injury
*r>0.4, p<0.001: no correlations between any genes
and perivascular infiltrates at this cut-off
B-cell
20
Macrophage associated
T-cell associated
T-cell
30
Kidney parenchyma associated
Interferon-γ dependent
B-cell
40
γ-IFN
50
γ-IFN
Mac
60
Injury and repair associated
T-cell
% contribution
70
Injury
0
fibrosis/atrophy
nodular
Figure 3: Overlap in gene expression between infiltrate types
i-Banff
244
240
116 cytotoxic T cell associated
54 not annotated
39 γ-interferon dependent
14 Injury and Repair induced
17 macrophage associated
132
i-IFTA
70
84 not annotated
26 B cell associated
15 Injury and Repair induced
3 cytotoxic T cell associated
3 Kidney parenchymal
1 Endothelial activation
t-score
9
IFTA
40
Genes (top 25) correlating with fibrosis/atrophy and i-IFTA
Gene
symbol
CPA3
TPSB2
ABCA8
TPSAB1
GABRP
FCER1A
AGR2
NEGR1
ADAMTSL3
NLGN4X
ENAH
SNAP25
PAPLN
BCMP11
SVEP1
PDE5A
ZFPM2
MS4A2
C7
ROBO1
CHODL
CXCL6
RGS13
PPIF
CABP1
Gene name
mast cell carboxypeptidase A3
mast cell tryptase beta 2
ABC transporters - ATP-binding cassette, sub-family A (ABC1), member 8
mast cell tryptase alpha/beta 1
gamma-aminobutyric acid (GABA) A receptor
receptor for Fc fragment of IgE, high affinity I, expressed predominantely on mast cells
anterior gradient homolog 2
neuronal growth regulator 1
ADAMTS-like 3
neuroligin 4
cytoskeleton regulatory protein hMena
synaptosomal-associated protein
papilin
AGR3 = anterior gradient homolog 3
sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1
phosphodiesterase 5A
zinc finger protein, multitype 2
Fc fragment of IgE, high affinity I, receptor for; beta polypeptide
Complement compnent 7
roundabout, axon guidance receptor, homolog 1
chondrolectin
chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2)
regulator of G-protein signaling 13
peptidylprolyl isomerase F (cyclophilin F, CYP3; Cyp-D)
calcium binding protein 1
Correlation*
r-value
0.619
0.566
0.564
0.555
0.554
0.547
0.523
0.509
0.503
0.499
0.497
0.497
0.486
0.485
0.484
0.479
0.474
0.47
0.468
0.467
0.466
0.466
0.462
-0.47
-0.49
Confirmation by immunohistochemistry
70
p 0.006
65
60
55
i-Banff
i-non-IFTA
50
45
i-IFTA
i-IFTA
p 0.02
%
40
35
30
25
20
p 0.006
15
p 0.0004
10
p 0.05
5
0
% T cells (CD3+)
% Macropahges
(CD68+)
% B cells (CD20+)
% plasma cells
(CD138+)
% IgG4 plasma cells
(IgG4+)
% mast cells
(tryptase+)
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)
Mast cell associated transcripts are a molecular
correlate of IFTA
Mast cell PBT score
2.5
p<0.0001
2
Mast cell transcripts correlate with
1.5
time post TX: r=0.55, p <0.01
1
0.5
i-IFTA:
r=0.63, p <0.01
IFTA:
r=0.61, p <0.01
delta GFR:
r= -0.35, p <0.0001
0
i-Banff
i-non-IFTA
<25%,n=109
n=109
<25%,
i-Banff
i-non-IFTA
>25%,n=20
n=20
>25%,
i-IFTA <25%,
n=102
i-IFTA >25%,
n=27
IFTA <25%,
n=87
IFTA >25%,
n=42
In biopsies with IFTA increased expression of Mast cell associated
transcript is associated with worse allograft survival
low mast cell scores
p=0.01
high mast cell scores
+
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
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
Relationship between IFTA and function in native
kidneys
Relationship between IFTA and function in renal allografts
(biopsies for cause)
Kasiske et al. Kidney Int. 1991;40:514-524
Molecular correlates of eGFR at the time of biopsy
GFR transcript set
Annotation of probesets
(% of total of correlating probesets)
45
n = 144
GFRT
Negative
GFRT
Positive
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*
10
ct
0.29**
-0.20*
5
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
eGFR at Biopsy
-0.54**
0.50**
Functional deterioration from
baseline (delta eGFR)
-0.28**
-0.30**
40
35
n = 224
30
25
Histologic lesions (Banff scores)*
20
15
0
Negative Correlation
IMATs
IGTs
CISTs
GSTs
BATs
GRIT1
GRIT2
KT1
IRITD5
IRITD3
IRITD1
CMATs
CATs
Positive Correlation
(Mactrophage activation)
(Plasma cell infiltration)
(severe injury)
(severe injury)
(B cell infiltration)
(Ifng effects)
(Ifng effects)
(Parenchymal transcripts)
(Injury)
(Injury)
(Injury)
(Macrophage infiltration)
(T cell infiltration)
Renal function
Bunnang and Einecke et al. J Am Soc Nephrol. 2009 (5):1149-60
Significant overlap between injury / repair /
and IFTA associated transcripts
Am J Transplant. 2007 Nov;7(11):2483-95.
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
Molecular changes in protocol biopsies
Background 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
No molecular evidence for EMT in the onset of IFTA in
early protocol biopsies
J Am Soc Nephrol. 2008 Aug;19(8):1571-83.
Molecular associates of subclinical IFTA at 12-months in
clinically uncomplicated living donor kidney transplants
Transplantation. 2007 Jun 15;83(11):1466-76
PBT-annotation of top 100 transcripts correlating*
in 6-week protocol biopsies with future onset of
IFTA in 6-month protocol biopsies
100%
90%
80%
Endothel associated transcripts
70%
TGF-β related transcripts
60%
kidney parenchymal transcripts
50%
injury induced transcripts
40%
interferon-γ inducible transcripts
30%
macrophage associated transcripts
B cell associated transcripts
20%
NK cell associated transcripts
10%
T cell associated transcripts
0%
correlated with onset of IFTA at 6-months correlated with delta clearance 6-weeks to correlated with prevalence of acute
post TX
12-months
rejection between 6-weeks and 12-months
after transplantation
*Spearman correlation, p<0.001
PBT-annotation of top 100 transcripts correlating*
with future end points in PB
100%
90%
80%
Endothel associated transcripts
70%
TGF-β related transcripts
60%
kidney parenchymal transcripts
50%
injury induced transcripts
40%
interferon-γ inducible transcripts
30%
macrophage associated transcripts
B cell associated transcripts
20%
NK cell associated transcripts
10%
T cell associated transcripts
0%
correlated with onset of IFTA at 6-months correlated with delta clearance 6-weeks to correlated with prevalence of acute
post TX
12-months
rejection between 6-weeks and 12-months
after transplantation
*Spearman correlation, p<0.001
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
Outlook
• Test reproducibility for IF, TA, i-Banff, iIFTA, 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