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.
Highlights
Kidney-derived cells exist in the urine of both healthy individual and kidney transplant patient, and these cells contain various molecules associated with ongoing kidney injury or allograft status
In 2001, Suthanthiran et al [1] first reported that mRNA levels of granzyme B and perforin were increased in the urinary cells of patients diagnosed with acute rejection (AR) by biopsy, and it was suggested that measurement of these mRNA levels in urine could be a potential noninvasive AR diagnostic tool
To validate mRNA levels in urinary cells, 90 urine samples were collected from 88 kidney transplant patients
Summary
Kidney-derived cells exist in the urine of both healthy individual and kidney transplant patient, and these cells contain various molecules associated with ongoing kidney injury or allograft status. Normalized urinary mRNAs by the total amount of RNA developed by Suthanthiran et al [2] discriminated patients with AR from those with no AR, but normalization of target mRNA using 18S rRNA or other housekeeping genes is still an issue of debate [4,5,6,7,8,9]. Another method of analyzing real-time PCR data is the relative quantification known as the 2-ΔΔC method, which is more convenient and widely using method in biologic experiments [10, 11]
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