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.

Evaluating the Link between BAFF System Gene Expression and Acute Rejection Development in Kidney Transplantation

“…Elevated BAFF levels have been associated with AMR. However, it must be taken into account that most studies have been carried out on soluble and non-transcribed BAFF [ 14 , 27 ], so the results may not correspond precisely to the same evaluated reality. The study by Thibault-Espitia et al, shows that high levels of R-BAFF transcripts and low levels of BAFF transcripts have an increased risk of long-term graft dysfunction [ 9 ].…”

“…The Kruskal–Wallis test and Dunn’s post hoc test with Bonferroni adjustment for multiple comparisons were employed to compare three or more groups. Correlation analyses were carried out using the Spearman index, as previously described [ 27 , 28 ].…”

Computational Prediction of Biomarkers, Pathways, and New Target Drugs in the Pathogenesis of Immune-Based Diseases Regarding Kidney Transplantation Rejection

“…The Kruskal Wallis test and Dunn’s post hoc test with Bonferroni correction for multiple comparisons compared three or more groups. Correlation analyzes were carried out using the Spearman index, as previously published ( 5 , 21 , 22 ). For the longitudinal comparison of two related groups, the Wilcoxon non-parametric test for related samples was used.…”

“…The severity and occurrence of rejection in kidney transplant (KT) patients depend on numerous variables that can affect the magnitude and nature of immune responses. Understanding how genetic and molecular factors affect the effector functions of immune cells and donor-specific antibodies (DSA) can better renal stratification receptors based on their immunological risk and thus help the clinician make better decisions to anticipate adverse events (1)(2)(3)(4)(5).…”

Monitoring of Serological, Cellular and Genomic Biomarkers in Transplantation, Computational Prediction Models and Role of Cell-Free DNA in Transplant Outcome

“…The application of interactions promises to be ideal for identifying rejection with these new potential biomarkers or drug targets and allow us to discard the hackneyed hypothesis of a “gene interaction” disease [ 26 , 66 ]. Finally, we must work on undefined bioinformatic processes such as analyzing interest interactions, obtaining problem genes of a specific pathology using online databases and previous bibliography, defining modules, enriching hundreds of pathways and molecular mechanisms and clinical validation, estimation and prediction of the obtained results [ 92 , 93 ]. The STRING database [ 94 ] and DisGeNET, which contain genes associated with human pathologies, can also integrate known and expected physical/functional interactions between proteins.…”