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

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 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”

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 3 months 8 27 79 246 129 biopsies ordered by time post TX Mengel et al. Am J Transplant. 2009 Jan;9(1):169-78.

Infiltrates and time in BFC p<0.0001 p<0.0001

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 69.6% 46 <50% of fibrosis/atrophy show infiltrates 93.5% 31 censored 77 + 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 uninflamed IFTA inflamed IFTA p=0.02

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

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

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

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

Confirmation by immunohistochemistry p 0.006 i-Banff i-IFTA p 0.02 p 0.006 p 0.0004 p 0.05

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 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

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

(% 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) KT1 (Parenchymal transcripts) IRITD5 (Injury) IRITD3 (Injury) IRITD1 (Injury) CMATs (Macrophage infiltration) CATs (T cell infiltration) 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 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

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 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

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

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, 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