Gene Expression in Biopsies of Acute Rejection and Interstitial Fibrosis/Tubular Atrophy Reveals Highly Shared Mechanisms That Correlate With Worse Long-Term Outcomes

Abstract

Interstitial fibrosis and tubular atrophy (IFTA) is found in approximately 25% of 1-year biopsies posttransplant. It is known that IFTA correlates with decreased graft survival when histological evidence of inflammation is present. Identifying the mechanistic etiology of IFTA is important to understanding why long-term graft survival has not changed as expected despite improved immunosuppression and dramatically reduced rates of clinical acute rejection (AR) (Services UDoHaH. http://www.ustransplant.org/annual_reports/current/509a_ki.htm). Gene expression profiles of 234 graft biopsy samples were obtained with matching clinical and outcome data. Eighty-one IFTA biopsies were divided into subphenotypes by degree of histological inflammation: IFTA with AR, IFTA with inflammation, and IFTA without inflammation. Samples with AR (n = 54) and normally functioning transplants (TX; n = 99) were used in comparisons. A novel analysis using gene coexpression networks revealed that all IFTA phenotypes were strongly enriched for dysregulated gene pathways and these were shared with the biopsy profiles of AR, including IFTA samples without histological evidence of inflammation. Thus, by molecular profiling we demonstrate that most IFTA samples have ongoing immune-mediated injury or chronic rejection that is more sensitively detected by gene expression profiling. These molecular biopsy profiles correlated with future graft loss in IFTA samples without inflammation.

Keywords: Interstitial fibrosis and tubular atrophy; basic (laboratory) research/science; genomics; graft survival; immunobiology; kidney (allograft) function/dysfunction; kidney transplantation/nephrology; organ transplantation in general; rejection: T cell mediated (TCMR); translational research/science.

Figure 1. Graft survival according to histological phenotype

Interstitial fibrosis and tubular atrophy (IFTA) samples were classified into three subphenotypes according to the degree of inflammation: IFTA plus clinical acute rejection (AR), IFTA with inflammation, and IFTA without inflammation. Biopsies with only AR and normally functioning transplants (TX) were used for survival comparisons. The figure shows graft survival according to these phenotypes in days posttransplant. The insert table shows the number of subjects at key time points by phenotypes.

Figure 2. Differentially expressed genes shared between IFTA and AR

(A) Venn diagram showing differentially expressed genes (DEGs) shared between interstitial fibrosis and tubular atrophy (IFTA) without inflammation and clinical acute rejection (AR). (B) Plots the differential fold changes in gene expression (DEGs) comparing IFTA without inflammation versus AR. A linear regression line and R² statistic demonstrates a highly concordant direction of gene expression between phenotypes; (C) and (D) repeat and validate the analysis using an independent, external dataset. Note 1: Differentially expressed genes and fold changes are calculated in relation to normal transplants (TX) defined by stable function and light histology. Note 2: The subphenotypes of IFTA with and without inflammation were not available for the external data set.

Figure 3. Gene coexpression networks (GCNs)

GCNs were discovered in an unbiased manner using the coexpression of differentially expressed genes for biopsies with clinical acute rejection (AR), interstitial fibrosis and tubular atrophy (IFTA) without AR (i.e. without inflammation), and IFTA with AR (i.e. with inflammation). A number of GCN correlation thresholds (ranging from R² values of 0.6 to 0.9) were tested to examine both loose and tight networks of coexpressed genes. With an increase in the correlation coefficient threshold, a large GCN network split into three smaller and tighter clusters with common biological functions for each. Genes with the most connections (i.e. edges) to other genes in a network are given for each GCN.

Figure 4. Biological functions of clinical acute rejection–gene coexpression network 1 (AR-GCN1) and AR-GCN2 genes

The figure illustrates the biological functions of 107 (56%) of the AR-GCN2 (immune response/inflammation) genes and all 31 of the ARGCN1 (B cell/immunoglobulin production) genes. The genes in the illustration with dashed red border are present in the GCNs. It is important to note that these genes are essentially the same in IFTA-GCN2.

Figure 5. Using the geometric means for each gene coexpression network (GCN) to rank the impact by phenotype

(A) Geometric means of AR-GCN2 transcripts (immune response/inflammation) correlated with the degree of histological inflammation: clinical acute rejection (AR) > interstitial fibrosis and tubular atrophy (IFTA) with AR > IFTA with inflammation (IFTA+i) > IFTA without inflammation > transplants with stable function and normal histology (TX). Note that the geometric mean of AR-GCN2 in IFTA without inflammation was still significantly higher than TX (p = 0.003). (B) In contrast, the geometric means of AR-GCN3 transcripts (metabolism/tissue integrity) were inversely related to inflammation: TX > IFTA without inflammation > AR > IFTA with inflammation > IFTA plus AR. (C and D) Same analyses using the IFTA-GCNs. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.0001.

Figure 6. Correlations between biopsy histology, Banff interstitial fibrosis and tubular atrophy (IFTA) grades, and the geometric means of the three IFTA–coexpression networks (GCNs)

The geometric means (y-axis) are plotted as a function of three interstitial fibrosis and tubular atrophy (IFTA) phenotypes: IFTA with AR, all IFTA biopsies, and IFTA without inflammation (IFTA without i) on the z-axis. In parallel, the geometric means are plotted as a function of Banff IFTA severity grades (x-axis).

Figure 7. Graft survival of subjects with IFTA without inflammation according to expression of our three gene coexpression networks (GCNs)

(A) Interstitial fibrosis and tubular atrophy (IFTA) without inflammation samples clustered into two clusters based on high versus low expression of GCN1 (B cell/immunoglobulin genes). (B) High versus low expression of GCN1 did not demonstrate a difference in graft survival (p = 0.47). (C and D) In contrast, when this analysis is repeated using GCN2 (immune response/inflammatory), graft survival of subjects with IFTA without inflammation correlates with relative expression of GCN2 (p = 0.02). (E and F) Relative expression of GCN3 (metabolism/tissue integrity) also correlates with graft survival (p = 0.03).

Figure 8. Graft survival of subjects with IFTA without inflammation correlates with the expression of 224 differentially expressed “high risk” genes

(A) Interstitial fibrosis and tubular atrophy (IFTA) without inflammation samples clustered into high versus low risk clusters based on expression of 224 differentially expressed transcripts. (B) The high versus low risk sample clusters correlate with graft survival (p = 0.001). DEGs, differentially expressed genes.

Figure 9. Validating the correlation between high risk gene expression and graft survival using an independent external dataset

Interstitial fibrosis and tubular atrophy (IFTA) biopsies from an external dataset (GEO accession number: GSE21374) (16) were clustered into high and low risk subgroups based on expression of the same 224 transcripts that correlated with graft loss. Again, two subject clusters were identified with marked difference in survival curves (p = 0.002). Note that the subphenotypes of IFTA with and without inflammation were not available for this external dataset.

Figure 10

Venn diagram demonstrating the overlap of the 224 differentially expressed genes associated with graft loss to the genes comprising the three interstitial fibrosis and tubular atrophy–gene coexpression networks IFTA-GCNs.