Evaluation of molecular profiles in calcineurin inhibitor toxicity post-kidney transplant: input to chronic allograft dysfunction

Abstract

The molecular basis of calcineurin inhibitor toxicity (CNIT) in kidney transplantation (KT) and its contribution to chronic allograft dysfunction (CAD) with interstitial fibrosis (IF) and tubular atrophy (TA) were evaluated by: (1) identifying specific CNIT molecular pathways that associate with allograft injury (cross-sectional study) and (2) assessing the contribution of the identified CNIT signature in the progression to CAD with IF/TA (longitudinal study). Kidney biopsies from well-selected transplant recipients with histological diagnosis of CNIT (n = 14), acute rejection (n = 13) and CAD with IF/TA (n = 10) were evaluated. Normal allografts (n = 18) were used as controls. To test CNIT contribution to CAD progression, an independent set of biopsies (n = 122) from 61 KT patients collected at 3 and ~12 months post-KT (range = 9-18) were evaluated. Patients were classified based on 2-year post-KT graft function and histological findings as progressors (n = 30) or nonprogressors to CAD (n = 31). Molecular signatures characterizing CNIT samples were identified. Patients classified as progressors showed an overlap of 7% and 22% with the CNIT signature at 3 and at ~12 months post-KT, respectively, while the overlap was <1% and 1% in nonprogressor patients, showing CNIT at the molecular level as a nonimmunological factor involved in the progression to CAD.

Keywords: Biomarkers; calcineurin inhibitors; kidney transplant; toxicity.

Figure 1. Study design

The study comprise two different phases: Phase I, including a cross-sectional study for discerning pathways involved in CNIT and establishing specific CNIT signatures, and Phase 2, including a longitudinal study for evaluating the contribution of the CNIT signature (from Phase I) in progression to CAD. As specified, gene expression was evaluated in tissue biopsies using microarrays (Training set) and RT-qPCR (Validation set) reactions. In Phase I, samples were classified by a pathologist using Banff criteria. CNIT: calcineurin inhibitor toxicity; AR: acute rejection; IF/TA: interstitial fibrosis and tubular atrophy, NA: normal allograft; RT-qPCR: quantitative real-time PCR.

Figure 2

A. Left, Hierarchical clustering of samples using the differentially expressed genes identified. From top to bottom, CNIT vs. NA, AR vs. NA, and IF/TA vs. NA. Right. Volcano plots for the three comparisons carried out. Differentially expressed genes identified are labeled in red. B. Venn diagram showing overlap between the differentially expressed genes identified under each condition (CNIT, AR and IF/TA vs. NA, respectively). Probe sets names were used as input for these comparisons. NA; normal allograft, CNIT: calcineurin inhibitor toxicity, AR: acute cellular rejection, IF/TA: interstitial fibrosis / tubular atrophy

Figure 3

Network generated by IPA from predicted upstream regulator (Vegf) and their target genes present in the list of CNI toxicity differentially expressed genes. The orange lines indicate predicted activation, while blue lines indicate predicted inhibition. Red boxes stand for over-expressed genes in our analysis, while green boxes indicate down regulation of genes in our data set. A high percentage of congruent predictions are observed.

Figure 4

Volcano plot showing the differentially expressed genes identified labeled in red.

Figure 5

A. Volcano plots generated when comparing 3-month biopsies of non- progressors (top left) and progressors (top right) to NA biopsies. Identified differentially expressed genes are marked in red. The Venn diagrams show overlapping of the differentially expressed genes identified in these comparisons and the CNIT signature. The percentage of CNIT genes identified is indicated under each Venn diagram. B. Same as A, however biopsies collected at 9-months or later post-transplantation were used. Differentially expressed genes are marked in red. The Venn diagram shows overlapping of this gene signature with that of CNIT.