Development of noninvasive molecular assays to improve disease diagnosis and patient monitoring is a critical need. In renal transplantation, acute rejection (AR) increases the risk for chronic graft injury and failure. Noninvasive diagnostic assays to improve current late and nonspecific diagnosis of rejection are needed. We sought to develop a test using a simple blood gene expression assay to detect patients at high risk for AR. We developed a novel correlation-based algorithm by step-wise analysis of gene expression data in 558 blood samples from 436 renal transplant patients collected across eight transplant centers in the US, Mexico, and Spain between 5 February 2005 and 15 December 2012 in the Assessment of Acute Rejection in Renal Transplantation (AART) study. Gene expression was assessed by quantitative real-time PCR (QPCR) in one center. A 17-gene set—the Kidney Solid Organ Response Test (kSORT)—was selected in 143 samples for AR classification using discriminant analysis (area under the receiver operating characteristic curve [AUC] = 0.94; 95% CI 0.91–0.98), validated in 124 independent samples (AUC = 0.95; 95% CI 0.88–1.0) and evaluated for AR prediction in 191 serial samples, where it predicted AR up to 3 mo prior to detection by the current gold standard (biopsy). A novel reference-based algorithm (using 13 12-gene models) was developed in 100 independent samples to provide a numerical AR risk score, to classify patients as high risk versus low risk for AR. kSORT was able to detect AR in blood independent of age, time post-transplantation, and sample source without additional data normalization; AUC = 0.93 (95% CI 0.86–0.99). Further validation of kSORT is planned in prospective clinical observational and interventional trials. The kSORT blood QPCR assay is a noninvasive tool to detect high risk of AR of renal transplants. Please see later in the article for the Editors' Summary Throughout life, the kidneys filter waste products (from the normal breakdown of tissues and food) and excess water from the blood to make urine. If the kidneys stop working for any reason, the rate at which the blood is filtered decreases, and dangerous amounts of creatinine and other waste products build up in the blood. The kidneys can fail suddenly (acute kidney failure) because of injury or poisoning, but usually failing kidneys stop working gradually over many years (chronic kidney disease). Chronic kidney disease is very common, especially in people who have high blood pressure or diabetes and in elderly people. In the UK, for example, about 20% of people aged 65–74 years have some degree of chronic kidney disease. People whose kidneys fail completely (end-stage kidney disease) need regular dialysis (hemodialysis, in which blood is filtered by an external machine, or peritoneal dialysis, which uses blood vessels in the abdominal lining to do the work of the kidneys) or a renal transplant (the surgical transfer of a healthy kidney from another person into the patient's body) to keep them alive. Our immune system protects us from pathogens (disease-causing organisms) by recognizing specific molecules (antigens) on the invader's surface as foreign and initiating a sequence of events that kills the invader. Unfortunately, the immune system sometimes recognizes kidney transplants as foreign and triggers transplant rejection. The chances of rejection can be minimized by “matching” the antigens on the donated kidney to those on the tissues of the kidney recipient and by giving the recipient immunosuppressive drugs. However, acute rejection (rejection during the first year after transplantation) affects about 20% of kidney transplants. Acute rejection needs to be detected quickly and treated with a short course of more powerful immunosuppressants because it increases the risk of transplant failure. The current “gold standard” method for detecting acute rejection if the level of creatinine in the patient's blood begins to rise is to surgically remove a small piece (biopsy) of the transplanted kidney for analysis. However, other conditions can change creatinine levels, acute rejection can occur without creatinine levels changing (subclinical acute rejection), and biopsies are invasive. Here, the researchers develop a noninvasive test for acute kidney rejection called the Kidney Solid Organ Response Test (kSORT) based on gene expression levels in the blood. For the Assessment of Acute Rejection in Renal Transplantation (AART) study, the researchers used an assay called quantitative polymerase chain reaction (QPCR) to measure the expression of 43 genes whose expression levels change during acute kidney rejection in blood samples collected from patients who had had a kidney transplant. Using a training set of 143 samples and statistical analyses, the researchers identified a 17-gene set (kSORT) that discriminated between patients with and without acute rejection detected by kidney biopsy. The 17-gene set correctly identified 39 of the samples taken from 47 patients with acute rejection as being from patients with acute rejection, and 87 of 96 samples from patients without acute rejection as being from patients without acute rejection. The researchers validated the gene set using 124 independent samples. Then, using 191 serial samples, they showed that the gene set was able to predict acute rejection up to three months before detection by biopsy. Finally, the researchers used 100 blood samples to develop an algorithm (a step-wise calculation) to classify patients as being at high or low risk of acute rejection. These findings describe the early development of a noninvasive tool (kSORT) that might, eventually, help clinicians identify patients at risk of acute rejection after kidney transplantation. kSORT needs to be tested in more patients before being used clinically, however, to validate its predictive ability, particularly given that the current gold standard test against which it was compared (biopsy) is far from perfect. An additional limitation of kSORT is that it did not discriminate between cell-mediated and antibody-mediated immune rejection. These two types of immune rejection are treated in different ways, so clinicians ideally need a test for acute rejection that indicates which form of immune rejection is involved. The authors are conducting a follow-up study to help determine whether kSORT can be used in clinical practice to identify acute rejection and to identify which patients are at greatest risk of transplant rejection and may require biopsy. Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001759.

Background. Polyoma virus type BK (BKV) nephritis has emerged as an important cause of renal allograft dysfunction and graft failure. Its diagnosis is contingent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for BKV nephritis could improve clinical outcome. Methods. We obtained 25 urine specimens from 8 renal allograft recipients with biopsy-confirmed BKV nephritis, 31 samples from 28 recipients in whom BKV nephritis was excluded by allograft biopsy, and 74 specimens from 34 patients with stable allograft function. RNA was isolated from the urinary cells and reverse transcribed to complementary DNA. We designed gene-specific oligonucleotide primers and probes for the measurement of messenger RNA (mRNA) encoding BKV VP1 protein and a constitutively expressed 18S ribosomal RNA (rRNA) by real-time quantitative polymerase chain reaction. We explored the hypothesis that BKV VP1 mRNA levels predict BKV nephritis. Results. The levels of BKV VP1 mRNA but not the levels of 18S rRNA predicted BKV nephritis. Analysis involving the receiver operating characteristic curve demonstrated that BKV nephritis can be predicted with a sensitivity of 93.8% and a specificity of 93.9% with the use of a cutoff value of 6.5×10⁵ BKV VP1 mRNA copy number per nanogram of total RNA (P <0.00001). In the receiver operating characteristic curve analysis, the calculated area under the curve was 0.949 (95% confidence interval, 0.912 to 0.987, P 0.2) for 18S rRNA. Conclusions. Measurement of BKV VP1 mRNA in urinary cells offers a noninvasive and accurate means of diagnosing BKV nephritis.

Background. CD103 is displayed on the cell surface of alloreactive CD8 cytotoxic T lymphocytes (CTLs) and is a critical component for the intraepithelial homing of T cells. Because intratubular localization of mononuclear cells is a feature of acute cellular rejection of renal allografts, we explored the hypothesis that CD103 messenger (m)RNA levels in urinary cells predict acute rejection. Methods. We collected 89 urine specimens from 79 recipients of renal allografts. RNA was isolated from the urinary cells, and we measured CD103 mRNA levels and a constitutively expressed 18S ribosomal (r)RNA with the use of real-time quantitative polymerase chain reaction assay. Results. CD103 mRNA levels, but not 18S rRNA levels, were higher in urinary cells from 30 patients with an episode of acute rejection (32 biopsies and 32 urine samples) compared with the levels in 12 patients with other findings on allograft biopsy (12 biopsies and 12 urine samples), 12 patients with biopsy evidence of chronic allograft nephropathy (12 biopsies and 12 urine samples), and 25 patients with stable graft function after renal transplantation (0 biopsies and 33 urine samples) (P = 0.001; one-way analysis of variance). Acute rejection was predicted with a sensitivity of 59% and a specificity of 75% using natural log-transformed value 8.16 CD103 copies per microgram as the cutoff value (P = 0.001). Conclusion. CD103 mRNA levels in urinary cells are diagnostic of acute rejection of renal allografts. Because CD103 is a cell surface marker of intratubular CD8 CTLs, a noninvasive assessment of cellular traffic into the allograft may be feasible by the measurement of CD103 mRNA levels in urinary cells.

Serine proteinase inhibitor (PI)-9 with a reactive center P1 (Glu)-P1′ is a natural antagonist of granzyme B and is expressed in high levels in cytotoxic T lymphocytes (CTL). In view of the role of CTL in acute rejection, we explored the hypothesis that PI-9 would be hyperexpressed during acute rejection. Because PI-9 can protect CTL from its own fatal arsenal and potentially enhance the vitality of CTL, we examined whether PI-9 levels correlate with the severity of rejection as well as predict subsequent graft function. We obtained 95 urine specimens from 87 renal allograft recipients. RNA was isolated from the urinary cells and mRNA encoding PI-9, granzyme B, or perforin and a constitutively expressed 18S rRNA was measured with the use of real-time quantitative polymerase chain reaction assay, and the level of expression was correlated with allograft status. The levels of PI-9 (P =0.001), granzyme B (P <0.0001), and perforin mRNAs (P <0.0001), but not the levels of 18S rRNA (P =0.54), were higher in the urinary cells from the 29 patients with a biopsy-confirmed acute rejection than in the 58 recipients without acute rejection. PI-9 levels were significantly higher in patients with type II or higher acute rejection changes compared with those with less than type II changes (P =0.01). Furthermore, PI-9 levels predicted subsequent graft function (r =0.43, P =0.01). PI-9 mRNA levels in urinary cells are diagnostic of acute rejection, predict renal allograft histology grade, and predict functional outcome following an acute rejection episode.