Molecular pathways involved in loss of kidney graft function with tubular atrophy and interstitial fibrosis

Abstract

Loss of kidney graft function with tubular atrophy (TA) and interstitial fibrosis (IF) causes most kidney allograft losses. We aimed to identify the molecular pathways involved in IF/TA progression. Kidney biopsies from normal kidneys (n = 24), normal allografts (n = 6), and allografts with IF/TA (n = 17) were analyzed using high-density oligonucleotide microarray. Probe set level tests of hypotheses tests were conducted to identify genes with a significant trend in gene expression across the three groups using Jonckheere-Terpstra test for trend. Interaction networks and functional analysis were used. An unsupervised hierarchical clustering analysis showed that all the IF/TA samples were associated with high correlation. Gene ontology classified the differentially expressed genes as related to immune response, inflammation, and matrix deposition. Chemokines (CX), CX receptor (for example, CCL5 and CXCR4), interleukin, and interleukin receptor (for example, IL-8 and IL10RA) genes were overexpressed in IF/TA samples compared with normal allografts and normal kidneys. Genes involved in apoptosis (for example, CASP4 and CASP5) were importantly overexpressed in IF/TA. Genes related to angiogenesis (for example, ANGPTL3, ANGPT2, and VEGF) were downregulated in IF/TA. Genes related to matrix production-deposition were upregulated in IF/TA. A distinctive gene expression pattern was observed in IF/TA samples compared with normal allografts and normal kidneys. We were able to establish a trend in gene expression for genes involved in different pathways among the studied groups. The top-scored networks were related to immune response, inflammation, and cell-to-cell interaction, showing the importance of chronic inflammation in progressive graft deterioration.

Figure 1

(A) Plot of two-dimensional multidimensional scaling coordinates derived from quality assessment data for the included microarrays. The plot revealed similar distributions, as desired. (B) Pairwise scatterplots and Pearson correlation for the three independent isolations from one kidney sample.

Figure 2

Unsupervised hierarchical clustering analysis including all the study kidney sample tissues. The dendrogram shows that the IF/TA tissue samples clustered with high correlation (black boxes, IF/TA samples).

Figure 3

Two-dimensional hierarchical clustering results for differentially expressed genes among kidney samples. This set was created using Pearson (centered) correlation cluster analysis, using BRB-ArrayTools version 3.0.2. Each vertical column represents an independent experiment. The fold changes in mRNA levels in the samples are represented by green and red squares, showing decreased and increased levels between the RNA samples, respectively. The color scale indicates the magnitude of fold changes. Dark blue boxes, normal kidney allograft samples; light blue boxes, IF/TA kidney samples; pink boxes, normal kidney samples.

Figure 4

The top-two scoring network of interactions among the differentially expressed genes between normal, normal allograft, and IF/TA kidney tissues. Interconnections of significant functional networks in IF/TA are indicated. The meaning of the node shapes and interaction edges is also indicated.

Figure 5

Canonical pathway analysis. Leukocyte transvasation signaling: genes differentially expressed in IF/TA samples. Red, overexpressed genes; green, underexpressed genes.