Urinary Cell mRNA Profiles Predictive of Human Kidney Allograft Status.

Immune monitoring of kidney allograft recipients and personalized therapeutics may help reach the aspirational goal of "one transplant for life." The invasive kidney biopsy procedure, the diagnostic tool of choice, has become safer and the biopsy classification more refined. Nevertheless, biopsy-associated complications, interobserver variability in biopsy specimen scoring, and costs continue to be significant concerns. The dynamics of the immune repertoire make frequent assessments of allograft status necessary, but repeat biopsies of the kidney are neither practical nor safe. To address the existing challenges, we developed urinary cell mRNA profiling and investigated the diagnostic, prognostic, and predictive accuracy of absolute levels of a hypothesis-based panel of mRNAs encoding immunoregulatory proteins. Enabled by our refinements of the PCR assay and by investigating mechanistic hypotheses, our single-center studies identified urinary cell mRNAs associated with T cell-mediated rejection, antibody-mediated rejection, interstitial fibrosis and tubular atrophy, and BK virus nephropathy. In the multicenter National Institutes of Health Clinical Trials in Organ Transplantation-04, we discovered and validated a urinary cell three-gene signature of T-cell CD3 ε chain mRNA, interferon gamma inducible protein 10 (IP-10) mRNA, and 18s ribosomal RNA that is diagnostic of subclinical acute cellular rejection and acute cellular rejection and prognostic of acute cellular rejection and graft function. The trajectory of the signature score remained flat and below the diagnostic threshold for acute cellular rejection in the patients with no rejection biopsy specimens, whereas a sharp rise was observed during the weeks before the biopsy specimen that showed acute cellular rejection. Our RNA sequencing and bioinformatics identified kidney allograft biopsy specimen gene signatures of acute rejection to be enriched in urinary cells matched to acute rejection biopsy specimens. The urinary cellular landscape was more diverse and more enriched for immune cell types compared with kidney allograft biopsy specimens. Urinary cell mRNA profile-guided clinical trials are needed to evaluate their value compared with current standard of care.

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

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.

Messenger RNA for FOXP3 in the urine of renal-allograft recipients.

BACKGROUND: The outcome of renal transplantation after an episode of acute rejection is difficult to predict, even with an allograft biopsy. METHODS: We studied urine specimens from 36 subjects with acute rejection, 18 subjects with chronic allograft nephropathy, and 29 subjects with normal biopsy results. Levels of messenger RNA (mRNA) for FOXP3, a specification and functional factor for regulatory T lymphocytes, and mRNA for CD25, CD3epsilon, perforin, and 18S ribosomal RNA (rRNA) were measured with a kinetic, quantitative polymerase-chain-reaction assay. We examined associations of mRNA levels with acute rejection, rejection reversal, and graft failure. RESULTS: The log-transformed mean (+/-SE) ratio of FOXP3 mRNA copies to 18S ribosomal RNA copies was higher in urine from the group with acute rejection (3.8+/-0.5) than in the group with chronic allograft nephropathy (1.3+/-0.7) or the group with normal biopsy results (1.6+/-0.4) (P<0.001 by the Kruskal-Wallis test). FOXP3 mRNA levels were inversely correlated with serum creatinine levels measured at the time of biopsy in the acute-rejection group (Spearman's correlation coefficient = -0.38, P=0.02) but not in the group with chronic allograft nephropathy or the group with normal biopsy results. Analyses of receiver-operating-characteristic curves demonstrated that reversal of acute rejection can be predicted with 90 percent sensitivity and 73 percent specificity with use of the optimal identified cutoff for FOXP3 mRNA of 3.46 (P=0.001). FOXP3 mRNA levels identified subjects at risk for graft failure within six months after the incident episode of acute rejection (relative risk for the lowest third of FOXP3 mRNA levels, 6; P=0.02). None of the other mRNA levels were predictive of reversal of acute rejection or graft failure. CONCLUSIONS: Measurement of FOXP3 mRNA in urine may offer a noninvasive means of improving the prediction of outcome of acute rejection of renal transplants.

Matching T-cell receptors identified in renal biopsies and urine at the time of acute rejection in pediatric renal transplant patients.

Urinary monitoring of kidney allograft function has been used for many years. More recently, molecular identification of cytotoxic T-cell products has been used as a diagnostic tool in acute rejection. Monitoring of T-cell infiltrates by analysis of the T-cell receptor (TcR) gene usage has been performed on biopsies with acute and chronic rejection, but not on urine samples. The aim of this study was to identify and compare TRBV gene usage assessing the CDR3 (Complementarity Determining Region 3) length distribution and sequence in urine and biopsies of pediatric renal allograft patients at the time of acute rejection and compare them with peripheral blood. We studied four pediatric renal transplant recipients with acute cellular rejection. We identified restricted and matched TRBV CDR3 spectratypes with overexpressed TRBV families and show identical, clonally expanded TRBV CDR3 sequences in all four patients present in the urine and renal allograft. We demonstrate that urinary monitoring can detect graft-infiltrating lymphocytes in acute rejection and may have a role in the monitoring of renal transplants.

Immunocytological determination of lymphocytes and monocytes/macrophages in urinary sediments of renal allograft recipients.

BACKGROUND: Urinary studies using Papanicolaou staining following kidney transplantation led to the conjecture that acute allograft rejection might be accompanied by an increased lymphocyturia. However, it is difficult to distinguish lymphoid cells from other urinary cells using conventional stains. METHODS: Staining of urinary lymphocytes using FITC-labelled antibodies is complicated by a high unspecific fluorescence that limits the evaluation. Therefore, we developed a method to stain urinary lymphocytes using enzyme-linked antibodies. The cells were cytocentrifuged onto microscope slides and were fixed. RESULTS: By means of a combined evaluation of Papanicolaou and immunocytochemical staining, CD3-positive pan T cells, CD4-positive T-helper cells, CD8-positive cytotoxic/suppressor cells, and CD14-positive monocytes/macrophages of urinary sediments were determined in 41 kidney graft recipients following renal transplantation. During periods of normal graft function, neither positive lymphocytes nor positive monocytes/macrophages were found in the urinary sediments. However, in the course of acute allograft rejection a significant increase in positive lymphocytes and positive monocytes/macrophages could be observed. Interestingly, in cases of acute allograft rejection the distribution of urinary lymphocytes and monocytes was comparable to the distribution of infiltrating immunocompetent cells in renal allograft biopsies. CONCLUSION: The present study demonstrates that immunocytochemical staining via enzyme-conjugated antibodies is a reliable method to visualize T lymphocytes and monocytes/macrophages in the urinary sediment, and that this technique may be of special diagnostic value in the diagnosis of acute allograft rejection.

Urine cytology and urine flow cytometry in renal transplantation--a prospective double blind study.

Urine cytology (UC) has proved to correlate well with core and fine-needle aspiration kidney biopsies of renal allograft recipients undergoing acute rejection (AR). This study was undertaken to compare the relative usefulness of urine flow immunocytometry (UFC) (using fluorescinated antibodies anti-HLA-DR, anti-CD3 and antirenal epithelial cells) with UC in its ability to diagnose AR by analyzing 200 urine specimens during a prospective double-blind study of 40 renal transplant recipients. Clinical diagnosis was retrospectively assigned to one of the following categories: group I--AR, 15; group II--ischemic injury period (first 5 days postop.), 12; group III, 173 (including 168 stable grafts, 1 pyelonephritis and 4 cyclosporine toxicity), by investigators blinded to the urine results. Both tests were highly sensitive for the diagnosis of AR (UC = 86.6% vs. UFC = 100%; P = NS) with a specificity after the ischemic injury period of 78% by UC and 87.9% by UFC. Samples obtained during AR revealed higher levels of expression of HLA-DR as well as higher numbers of CD3-positive cells. These tests had specificity values of 95.3% and 97.6%, respectively, for the diagnosis of AR. The degree of immune activation (established by numbers of lymphocytes/lymphoblasts seen by UC) correlated with the severity of biopsy-proven ARs and with response to antirejection therapy. In conclusion, both test are highly accurate in diagnosing AR. The highest specificity value was obtained when both UC and UFC were utilized together (93%). We suggest that the routine use of these tests can provide an important adjunct to the evaluation of renal transplant recipients.