1. Molecular associations of IFTA
Michael Mengel
Alberta Transplant Applied Genomics Centre
University of Alberta, Edmonton Canada

2. Interstitial fibrosis and tubular atrophy (IFTA) in renal allografts

3. IFTA with inflammation

4. 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

5. 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 May-Jun;14(5-6):

6. Scoring inflammation in renal allograft biopsies
100% Cortex 5% 3%
absolute scoring
40% i-IFTA
10% i-Banff
nodular
perivascular
subcapsular
60% IFTA compartment
40% non-scarred
compartment
relative scoring
according to current Banff rules
25% = Banff i-score 1
“67% i-IFTA”

7. 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

8. Infiltrates in biopsies for cause are time dependent3
months 8 27 79
246
129 biopsies ordered by time post TX
Mengel et al. Am J Transplant Jan;9(1):

9. Infiltrates and time in BFC
p<0.0001
p<0.0001

10. Inflammation in fibrosis/atrophy is associated with worse allograft survival
Inclusion criteria: IFTA >5%, i-Banff <25%
graft survival n
≥50% of fibrosis/atrophy show infiltrates %
<50% of fibrosis/atrophy show infiltrates %
censored + B
biopsies with graft survival n
i-Banff >25%, i-IFTA <25% %
i-IFTA >25%, i-Banff <25% %
both <25% %
censored +
i-Banff
i-IFTA
i <25%
p<0.05
uninflamed IFTA
inflamed IFTA
p=0.02

11. 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
Kidney parenchyma associated (KTs)
-0.296
-0.303
-0.199
-0.156
Injury and repair associated (IRITs)
0.379
0.355
0.206
Immunoglobulin associated (IGTs)
0.174
0.434
0.398
0.336
B-cell associated (BATs)
0.281
0.279
0.423
0.387
# given is the highest r-value revealed for one PBT of each particular biological process
*Spearman correlation, p<0.001

12. Correlations* between individual genes and histological lesions
484 probesets
249 probesets
202 probesets
172 probesets
34 probesets
Injury
Injury
Mac
Mac
B-cell
γ-IFN
γ-IFN
*r>0.4, p<0.001: no correlations between any genes and perivascular infiltrates at this cut-off
B-cell
B-cell
T-cell
T-cell
Injury
T-cell
Injury

13. Figure 3: Overlap in gene expression between infiltrate types
i-Banff
240
116 cytotoxic T cell associated
54 not annotated
39 γ-interferon dependent
14 Injury and Repair induced
17 macrophage associated
t-score
244 9
i-IFTA
IFTA 70
132
84 not annotated
26 B cell associated
15 Injury and Repair induced
3 cytotoxic T cell associated
3 Kidney parenchymal
1 Endothelial activation 40

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

15. Confirmation by immunohistochemistry
p 0.006
i-Banff
i-IFTA
p 0.02
p 0.006 p
p 0.05

16. 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)

17. Mast cell associated transcripts are a molecular correlate of IFTA
i-Banff
<25%, n=109
>25%, n=20
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

18. 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 %
low* MACATscore %
censored
*low = lowest tertile of MACAT score of the included 88 biopsies
*high = intermediate and highest tertile of MACAT score of the included 88 biopsies +

19. 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

20. Relationship between IFTA and function in native kidneys

21. Relationship between IFTA and function in renal allografts (biopsies for cause)
Kasiske et al. Kidney Int. 1991;40:

22. (% of total of correlating probesets) Annotation of probesets
Molecular correlates of eGFR at the time of biopsy
GFR transcript set
(% of total of correlating probesets)
Annotation of probesets 5 10 15 20 25 30 35 40 45
Negative Correlation
Positive Correlation
n = 144
n = 224
GFRT Negative
GFRT Positive
Histologic lesions (Banff scores)* g
0.09
-0.10 cg
0.03 i
0.33**
-0.32** ci
0.29**
-0.20** t
0.16
-0.19* ct
-0.20* v
-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**
IMATs (Mactrophage activation)
IGTs (Plasma cell infiltration)
CISTs (severe injury)
GSTs (severe injury)
BATs (B cell infiltration)
GRIT1 (Ifng effects)
GRIT2 (Ifng effects)
KT (Parenchymal transcripts)
IRITD5 (Injury)
IRITD3 (Injury)
IRITD1 (Injury)
CMATs (Macrophage infiltration)
CATs (T cell infiltration)
Bunnang and Einecke et al. J Am Soc Nephrol (5):

23. Significant overlap between injury / repair / and IFTA associated transcripts
Am J Transplant Nov;7(11):

24. Numerous “fibrosis genes” are associated with injury and repair
Early IRITs
Intermediate IRITs
Late IRITs
Category
% 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
Embryonic mesenchyme (n=951)
2.9
9.7
11.3
Mesenchyme vs ureteric bud (n=751)
19.9
Cell cycle and cell proliferation (n=1008)
2.4
8.3
10.4
Tgfb1/fibrogenesis (n=48)
1.9
3.2
Collagen (n=36)
0.5
1.1
4.3
ECM (n=431)
4.0
12.1

25. 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

26. No molecular evidence for EMT in the onset of IFTA in early protocol biopsies
J Am Soc Nephrol Aug;19(8):

27. Molecular associates of subclinical IFTA at 12-months in clinically uncomplicated living donor kidney transplants
Transplantation Jun 15;83(11):

28. PBT-annotation of top 100 transcripts correlating
PBT-annotation of top 100 transcripts correlating* in 6-week protocol biopsies with future onset of IFTA in 6-month protocol biopsies
*Spearman correlation, p<0.001

29. PBT-annotation of top 100 transcripts correlating
PBT-annotation of top 100 transcripts correlating* with future end points in PB
*Spearman correlation, p<0.001

30. 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

31. 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