Both absolute and relative quantification of urinary mRNA are useful for non-invasive diagnosis of acute kidney allograft rejection

Abstract

Urinary mRNA analysis with three-gene set (18S rRNA, CD3ε, and IP-10) has been suggested as a non-invasive biomarker of acute rejection (AR) in kidney transplant recipients using quantitative real-time PCR (qPCR). Application of droplet digital PCR (ddPCR), which has been suggested to provide higher sensitivity, accuracy, and absolute quantification without standard curves, could be a useful method for the quantifying low concentration of urinary mRNA. We investigated the urinary expression of these three genes in Korean patients with kidney transplantation and also evaluated the usefulness of ddPCR. 90 urine samples were collected at time of allograft biopsy in kidney recipients (n = 67) and from patients with stable renal function more than 10 years (n = 23). Absolute quantification with both PCR system showed significant higher mRNA levels of CD3ε and IP-10 in AR patients compared with stable transplants (STA), but there was no difference in 18S rRNA expression across the patient groups. To evaluate discrimination between AR and STA, ROC curve analyses of CTOT-4 formula yielded area under the curve values of 0.72 (95% CI 0.60–0.83) and 0.77 (95% CI 0.66–0.88) for qPCR and ddPCR, respectively. However, 18S normalization of absolute quantification and relative quantification with 18S showed better discrimination of AR from STA than those of the absolute method. Our data indicate that ddPCR system without standard curve would be useful to determine the absolute quantification of urinary mRNA from kidney transplant recipients. However, comparative method also could be useful and convenient in both qPCR and ddPCR analysis.

Clinical significance of CD161+CD4+ T cells in the development of chronic antibody-mediated rejection in kidney transplant recipients

“…Therefore, in this study we divided the patients into cAMR and IF/TA according to Banff classification [ 26 ]. As a control group, we included the LTGS group from multiple transplant centers; these patients were at least 10 years post-transplantation and showed MDRD eGFR > 50 mL/min/1.73 m 2 [ 27 ].…”

Phenotype and molecular signature of CD8+ T cell subsets in T cell- mediated rejections after kidney transplantation

“…In an ex vivo study to compare CD8 + T cell subsets among clinical groups, peripheral-blood mononuclear-cell (PBMC) samples were chosen from the ARTKT-1 (assessment of immunologic risk and tolerance in kidney transplantation) study, a cross-sectional sample collection study of KTRs who had received kidney allograft biopsy or who had long-term allograft survival (LTGS) with stable allograft function (MDRD eGFR � 50 mL/min/1.73 m 2 ) over ten years at four different transplant centers (Kyoung Hee University Hospital at Gangdong, Kyung Hee University Hospital, Kyungpook National University Hospital, Seoul St. Mary's Hospital of Catholic University of Korea) from August 2013 to July 2015. [17][18][19][20] ARTKT-1 was used only to identify participants and access kidney tissue. Among the PBMC samples collected for the ARTKT-1 study, we used a total of 121 samples from 32 patients with normal biopsy without any evidence of rejection (NC group) and 50 patients who showed T cell-mediated rejection (TCMR) on allograft biopsy with Banff classification assessed by a single pathologist (TCMR group) [21] and 39 patients with LTGS for this study.…”

Urinary Biomarkers for Diagnosis and Prediction of Acute Kidney Allograft Rejection: A Systematic Review

“…In accordance with previous studies, the most extensively assessed urinary biomarkers were C-X-C motif chemokine ligand 9 (CXCL9) and 10 (CXCL10), usually adjusted for urinary creatinine concentration. In detail, 12/38 (32%) studies either addressed CXCL9 and CXCL10 alone, in combination, or in the context of particular scores or formulas [16,17,24,26,[34][35][36]44,47,[50][51][52]. Other directly targeted cytokines and interleukins were chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 [22,31,43], CXCL13 [29], interleukin 10 (IL10) with interferon gamma (IFNγ) [46] and tumor necrosis factor alpha (TNFα) [32].…”

“…Two studies [35,44] investigated the diagnostic accuracy of the CTOT4 formula (CXCL10 mRNA, CD3ε mRNA, 18S rRNA), previously validated by the CTOT-4 (clinical trials in organ transplantation-4) multicenter study group [54]. CXCL10 was also included in newly derived scores such as the Q score, composed of six DNA, protein, and metabolite urinary biomarkers (cell-free DNA, methylated cell-free DNA, clusterin, total protein, creatinine, and CXCL10) [17], while CXCL9 and CXCL10 genes were included in the uCRM score [24].…”