PCR Array Technology in Biopsy Samples Identifies Up-Regulated mTOR Pathway Genes as Potential Rejection Biomarkers After Kidney Transplantation

Abstract

Background: Antibody-mediated rejection (AMR) is the major cause of kidney transplant rejection. The donor-specific human leukocyte antigen (HLA) antibody (DSA) response to a renal allograft is not fully understood yet. mTOR complex has been described in the accommodation or rejection of transplants and integrates responses from a wide variety of signals. The aim of this study was to analyze the expression of the mTOR pathway genes in a large cohort of kidney transplant patients to determine its possible influence on the transplant outcome. Methods: A total of 269 kidney transplant patients monitored for DSA were studied. The patients were divided into two groups, one with recipients that had transplant rejection (+DSA/+AMR) and a second group of recipients without rejection (+DSA/-AMR and -DSA/-AMR, controls). Total RNA was extracted from kidney biopsies and reverse transcribed to cDNA. Human mTOR-PCR array technology was used to determine the expression of 84 mTOR pathway genes. STRING and REVIGO software were used to simulate gene to gene interaction and to assign a molecular function. Results: The studied groups showed a different expression of the mTOR pathway related genes. Recipients that had transplant rejection showed an over-expressed transcript (≥5-fold) of AKT1S1, DDIT4, EIF4E, HRAS, IGF1, INS, IRS1, PIK3CD, PIK3CG, PRKAG3, PRKCB (>12-fold), PRKCG, RPS6KA2, TELO2, ULK1, and VEGFC, compared with patients that did not have rejection. AKT1S1 transcripts were more expressed in +DSA/-AMR biopsies compared with +DSA/+AMR. The main molecular functions of up-regulated gene products were phosphotransferase activity, insulin-like grown factor receptor and ribonucleoside phosphate binding. The group of patients with transplant rejection also showed an under-expressed transcript (≥5-fold) of VEGFA (>15-fold), RPS6, and RHOA compared with the group without rejection. The molecular function of down-regulated gene products such as protein kinase activity and carbohydrate derivative binding proteins was also analyzed. Conclusions: We have found a higher number of over-expressed mTOR pathway genes than under-expressed ones in biopsies from rejected kidney transplants (+DSA/+AMR) with respect to controls. In addition to this, the molecular function of both types of transcripts (over/under expressed) is different. Therefore, further studies are needed to determine if variations in gene expression profiles can act as predictors of graft loss, and a better understanding of the mechanisms of action of the involved proteins would be necessary.

Keywords: Gene expression; PCR array; antibody-mediated rejection; mTOR; medico-legal autopsy.

Figure 1

Differentially regulated mTOR pathway genes. (A) Up-regulated mTOR pathway genes (B) Down-regulated mTOR pathway genes.

Figure 2

STRING interaction pathway of differentially expressed mTOR pathway genes. Green nodes indicate up-regulated genes and red nodes down-regulated genes comparing +AMR/+DSA and +DSA vs. –AMR/–DSA KT patients. The intensity of the color indicates the degree of regulation of the gene. The blue line shows that the interaction is checked against curated databases and the purple line shows that the interaction is experimentally determined.

Figure 3

STRING interaction pathway of differentially expressed mTOR pathway genes comparing +AMR/+DSA and +DSA vs. –AMR/–DSA KT patients. (A) Green nodes indicate up-regulated genes. (B) Red nodes indicate down-regulated genes. The intensity of the color indicates the degree of regulation of the gene. The blue line shows that the interaction is checked against from curated databases and the purple line shows that the interaction is experimentally determined.

Figure 4

REVIGO TreeMap view of GO terms enriched between up- (upper TreeMaps) or down-regulated (lower TreeMaps) genes in biopsies from +DSA/+AMR patients vs. control recipients (–DSA/–AMR, +DSA/–AMR, and –DSA). Each rectangle is a single cluster representative. The representatives are joined into “superclusters” of loosely related terms that are visualized with distinct colors (indicated by centralized black text). The size of the rectangles reflects the enrichment of the GO term.

Figure 5

KEGG mTOR signaling pathway map comparing +AMR/+DSA and +DSA vs. –AMR/–DSA KT patients. Green color indicates up-regulated genes. Red color indicates down-regulated genes. The intensity of the color indicates the degree of gene regulation.