Progressive histological damage in renal allografts is associated with expression of innate and adaptive immunity genes

Abstract

The degree of progressive chronic histological damage is associated with long-term renal allograft survival. In order to identify promising molecular targets for timely intervention, we examined renal allograft protocol and indication biopsies from 120 low-risk pediatric and adolescent recipients by whole-genome microarray expression profiling. In data-driven analysis, we found a highly regulated pattern of adaptive and innate immune gene expression that correlated with established or ongoing histological chronic injury, and also with development of future chronic histological damage, even in histologically pristine kidneys. Hence, histologically unrecognized immunological injury at a molecular level sets the stage for the development of chronic tissue injury, while the same molecular response is accentuated during established and worsening chronic allograft damage. Irrespective of the hypothesized immune or nonimmune trigger for chronic allograft injury, a highly orchestrated regulation of innate and adaptive immune responses was found in the graft at the molecular level. This occurred months before histologic lesions appear, and quantitatively below the diagnostic threshold of classic T-cell or antibody-mediated rejection. Thus, measurement of specific immune gene expression in protocol biopsies may be warranted to predict the development of subsequent chronic injury in histologically quiescent grafts and as a means to titrate immunosuppressive therapy.

Figure 1. Classification of the 120 renal allograft biopsy samples included in the current study

*Compilation of discovery sets and validation sets from Project 1. **Patients different from those in Projects 1–3. CADI, Chronic Allograft Damage Index; mo, months; TX, transplantation. ^$The same data set as the low CADI group of the validation set of Project 1. ^#The same data set as the high CADI group of the validation set of Project 1.

Figure 2. Gene expression and CADI score (Project 1)

After significance analysis of microarrays, comparing gene expression of biopsies with high versus low CADI score, the probe set lists were analyzed using the Ingenuity Pathway Analysis program (Ingenuity Systems) and demonstrated highly significant enrichment of immune genes. The immune gene set scores represented in the graphs were calculated as the geometric mean of fold changes (unlogged values) across all probe sets (not only the significant probe sets) within the Ingenuity Pathways Knowledge Base (IPKB) lists for the different immune cell functions (Supplementary Table S5 online). (a) Comparison of the immune gene set scores between biopsies with high versus low CADI scores in a discovery set (n=24) and (b) in a validation set (n=24). P-values represented in the graphs were calculated using Mann–Whitney tests. The error bars represent mean±standard error of the mean. CADI, Chronic Allograft Damage Index; NK, natural killer; TX, transplantation.

Figure 3. Gene expression dynamics and CADI score over time after transplantation (Project 2)

Enrichment analysis of the probe set list that was generated from unpaired time course analysis (signed area test on 72 serial protocol biopsies at 0, 6, and 24 months after transplantation) comparing progressors (N=12 patients) with nonprogressors (N=12 patients) showed highly significant enrichment of immune genes involved in both innate and adaptive immunity. The immune gene set scores represented in the graphs were calculated as the geometric mean of fold changes (unlogged values) across all probe sets (not only the significant probe sets) within the Ingenuity Pathways Knowledge Base lists for the different immune cell functions (Supplementary Table S5 online). P-values represented in the graphs were calculated using repeated measures analysis of variance. The error bars represent mean±standard error of the mean. The significant P-values of the enrichment analysis from the comparison between progressors and non-progressors are given in Table 2 (Project 2). CADI, Chronic Allograft Damage Index; NK, natural killer; NS, nonsignificant.

Figure 4. Association of 6 months gene expression with CADI score at 24 months (Project 3)

(a) After significance analysis of microarrays, comparing gene expression of 6-month biopsies of progressors versus nonprogressors, the probe set list was analyzed using the Ingenuity Pathway Analysis program (Ingenuity Systems) and demonstrated significant enrichment of immune genes. The immune gene set scores represented in the graphs were calculated as the geometric mean of fold changes (unlogged values) across all probe sets (not only the significant probe sets) within the Ingenuity Pathways Knowledge Base lists for the different immune cell functions (Supplementary Table S5 online). P-values represented in the graphs were calculated using Mann–Whitney tests. The error bars represent mean±standard error of the mean. (b) Receiver operating characteristic-curve analysis demonstrates good accuracy of the 6-month immune gene set scores for prediction of subsequent damage. AUC, area under the curve; CADI, Chronic Allograft Damage Index; NK, natural killer.

Figure 5. Immune gene set scores as quantitative marker for inflammation (Project 4)

(a) There was a gradual association between the CADI score/T-cell-mediated rejection and the different immune gene set scores, as calculated from the Ingenuity Pathways Knowledge Base. Therefore, these immune gene set scores not only represent a good marker for ongoing and future chronic histological damage, but also offer a quantifiable measure for other types of inflammation in the graft. P-values represented in the graphs were calculated using Kruskall–Wallis tests. The error bars represent mean±standard error of the mean. (b) Spearman correlation analysis between histological features and gene set scores in the 48 rejection-free biopsies (left panel) and the 72 biopsies (48 rejection-free biopsies + 24 biopsies with T-cell-mediated rejection) included in Project 4. The numbers given in the boxes represent correlation coefficients; the filled colors correspond to the statistical significance of the correlation. Even in the absence of rejection (left panel), the correlation between the scores of histological inflammation and inflammatory gene set scores was high (especially for inflammation in atrophic areas and the total i-score), which demonstrates that the gene sets capture subtle inflammation. When rejection biopsies were included in the correlation analysis (right panel), interstitial inflammation and tubulitis correlated highly significantly with the gene set scores. The gene set scores therefore represent a global assessment of the inflammatory burden (without information of the exact anatomical localization of the cells that express the inflammatory genes) and a quantitative marker for ongoing inflammation, from moderate subclinical inflammation associated with chronic histological damage to marked inflammation of Banff-grade acute T-cell-mediated rejection. CADI, Chronic Allograft Damage Index; IF/TA, interstitial fibrosis/tubular atrophy; NK, natural killer; NS, nonsignificant.