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BACKGROUND Rejection is the main cause of kidney allograft failure, and kidney biopsy is the criterion standard method to diagnose it. However, non-invasive techniques to detect kidney transplant rejection are necessary. This study aimed to evaluate urinary chemokines CXCL9 and CXCL10 as potential biomarkers of kidney transplant rejection and to analyze chemokine association with allograft prognosis. MATERIAL AND METHODS We collected 117 urine samples from kidney transplant recipients undergoing allograft biopsy. CXCL9 and CXCL10 levels were measured by ELISA and the ratio to urine creatinine was calculated. Histology and other clinical data were collected from medical records. RESULTS The diagnostic performance of urinary CXCL9/cre in discriminating rejection from all other histological groups showed an ROC AUC value of 0.728 (95% CI 0.632-0.824, p<0.001), and a cut-off value 0.11 ng/mmol had the best sensitivity (76.9%) and specificity (73.1%). The ability of CXCL10/cre to discriminate transplant rejection from all other histological groups had ROC AUC value 0.73 (95% CI 0.63-0.84, P<0.001), the cut-off value 0.42 ng/mmol with best sensitivity (71.4%) and specificity (84.6%). CXCL9 and CXCL10 levels were also increased in patients with polyoma BK virus, recurrent AA amyloidosis, and thrombotic microangiopathy. Patients with higher CXCL9/cre (≥0.11 ng/mmol) and CXCL10/cre (≥0.42 ng/mmol) levels were at increased risk of transplant progression to ESRD (HR 3.25, 95% CI=1.27-8.36, P=0.01), irrespective of serum creatinine at the time of biopsy. CONCLUSIONS Urinary CXCL9/cre and CXCL10/cre were able to distinguished between patients with transplant rejection and those without rejection. High levels of urinary CXCL9/cre and CXCL10/cre were associated with worse allograft survival.

The challenges of long-term graft survival and the side effects of current immunosuppressive therapies in kidney transplantation highlight the need for improved drugs with fewer adverse effects. Biomarkers play a crucial role in quickly detecting post-transplant complications, with new biomarkers showing promise for ongoing monitoring of disease and potentially reducing the need for unnecessary invasive biopsies. The chemokines such as C-X-C motif chemokine ligand 10 (CXCL10), are particularly promising protein biomarkers for acute renal rejection, with urine samples being a desirable source for biomarkers. The aim of this review is to analyze the literature on the potential role of urinary CXCL10 protein in predicting kidney graft injuries. The results of this study demonstrate that evaluating urinary CXCL10 levels is more successful in identifying post-transplant injuries compared to assessing the CXCL10/Cr ratio., (© 2024. The Author(s).)

In kidney transplant recipients, urine CXCL9 and CXCL10 (uCXCL9/10) chemokines have reached a sufficiently high level of evidence to be recommended by the European Society of Organ Transplantation for the monitoring of immune quiescence. To assess the risk of acute rejection (AR), the advantage of uCXCL9/10 is their cost-effectiveness and their high diagnostic performance. Here, we evaluated the feasibility of a next-generation immunoassay for quantifying uCXCL9/10 levels. It demonstrated high efficiency with minimal workflow and a 90-min time to result. Preanalytical studies indicated stability of uCXCL9/10 levels and analytical studies confirmed excellent linearity and precision. In a cohort of 1048 samples collected at biopsy, the results correlated significantly with ELISA quantification and were integrated into a previously validated 8-parameter urine chemokine model. The next generation immunoassay achieved an accuracy of 0.84 for AR diagnosis. This study validates this technology as a robust, locally available and unexpensive platform and marks a significant step towards the widespread implementation of uCXCL9/10, for immune quiescence monitoring. Therefore, we developed an open-access web application using uCXCL9/10 to calculate AR risk and improve clinical decision-making to perform biopsy, ushering in a new era in kidney transplantation, where personalized, data-driven care becomes the norm., (© 2024. The Author(s).)

Introduction: Acute rejection (AR) undermines the life-extending benefits of kidney transplantation and is diagnosed using the invasive biopsy procedure. T cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR), or concurrent TCMR + ABMR (Mixed Rejection [MR]) are the three major types of AR. Development of noninvasive biomarkers diagnostic of AR due to any of the three types is a useful addition to the diagnostic armamentarium., Methods: We developed customized RT-qPCR assays and measured urinary cell mRNA copy numbers in 145 biopsy-matched urine samples from 126 kidney allograft recipients. We determined whether the urinary cell three-gene signature diagnostic of TCMR (Suthanthiran et al., 2013) discriminates patients with no rejection biopsies (NR, n = 50) from those with ABMR (n = 28) or MR (n = 20) biopsies., Results: The urinary cell three-gene signature discriminated all three types of rejection biopsies from NR biopsies (P < 0.0001, One-way ANOVA). Dunnett's multiple comparisons test yielded P < 0.0001 for NR vs. TCMR; P < 0.001 for NR vs. ABMR; and P < 0.0001 for NR vs. MR. By bootstrap resampling, optimism-corrected area under the receiver operating characteristic curve (AUC) was 0.749 (bias-corrected 95% confidence interval [CI], 0.638 to 0.840) for NR vs. TCMR (P < 0.0001); 0.780 (95% CI, 0.656 to 0.878) for NR vs. ABMR (P < 0.0001); and 0.857 (95% CI, 0.727 to 0.947) for NR vs. MR (P < 0.0001). All three rejection categories were distinguished from NR biopsies with similar accuracy (all AUC comparisons P > 0.05)., Conclusion: The urinary cell three-gene signature score discriminates AR due to TCMR, ABMR or MR from NR biopsies in human kidney allograft recipients., Competing Interests: Declaration of competing interest M. Suthanthiran has a Consultancy Agreement with CareDx, Inc. Brisbane, CA. D.M. Dadhania D.M.D. has participated in industry sponsored studies organized by AlloVir Inc., CSL Behring, and Memo Therapeutics AG and served as consultant for CareDx, Inc. The other authors of this manuscript declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The kidney allograft has been under continuous attack from diverse injuries since the very beginning of organ procurement, leading to a gradual decline in function, chronic fibrosis, and allograft loss. It is vital to routinely and precisely monitor the risk of injuries after renal transplantation, which is difficult to achieve because the traditional laboratory tests lack sensitivity and specificity, and graft biopsies are invasive with the risk of many complications and time-consuming. Herein, a novel method for the diagnosis of graft injury is demonstrated, using deep learning-assisted surface-enhanced Raman spectroscopy (SERS) of the urine analysis. Specifically, we developed a hybrid SERS substrate composed of gold and silver with high sensitivity to the urine composition under test, eliminating the need for labels, which makes measurements easy to perform and meanwhile results in extremely abundant and complex Raman vibrational bands. Deep learning algorithms were then developed to improve the interpretation of the SERS spectral fingerprints. The deep learning model was trained with SERS signals of urine samples of recipients with different injury types including delayed graft function (DGF), calcineurin-inhibitor toxicity (CNIT), T cell-mediated rejection (TCMR), antibody-mediated rejection (AMR), and BK virus nephropathy (BKVN), which explored the features of these types and achieved the injury differentiation with an overall accuracy of 93.03%. The results highlight the potential of combining label-free SERS spectroscopy with deep learning as a method for liquid biopsy of kidney allograft injuries, which can provide great potential to diagnose and evaluate allograft injuries, and thus extend the life of kidney allografts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Kidney transplantation significantly improves the lives of those with end-stage kidney disease, offering best alternative to dialysis. However, transplant success is threatened by the acute and chronic rejection mechanisms due to complex immune responses against the new organ., Areas Covered: The ongoing research into biomarkers holds promise for revolutionizing the early detection and monitoring of the graft health. Liquid biopsy techniques offer a new avenue, with several diagnostic, predictive, and prognostic biomarkers showing promise in detecting and monitoring kidney diseases and an early and chronic allograft rejection., Expert Opinion: Evaluating the protein composition related to kidney transplant results could lead to identifying biomarkers that provide insights into the graft functionality. Non-invasive proteomic biomarkers can drastically enhance clinical outcomes and change the way how kidney transplants are evaluated for patients and physicians if they succeed in this transition. Hence, the advancement in proteomic technologies, leads toward a significant improvement in understanding of the protein markers and molecular mechanisms linked to the outcomes of kidney transplants. However, the road from discovery to the use of such proteins in clinical practice is long, with a need for continuous validation and beyond the singular research team with comprehensive infrastructure and across research groups collaboration.

Acute renal allograft rejection (ARAR) after kidney transplantation associated with reduced graft survival and eventual graft failure is poorly diagnosed in hospitals. Here, we report the development of Artificial bioMarker Probes (AMPros) for sensitive urinalysis of ARAR in murine models. AMPros spontaneously go to the kidneys after systemic administration, specifically react with the prodromal immune biomarkers to activate their near-infrared fluorescence signals to report cell-mediated rejection, and efficiently undergo renal excretion into urine. Thus, AMPros enable convenient optical urinalysis that detects ARAR prior to histological manifestation of rejection, which is also earlier than current diagnostic methods measuring proinflammatory cytokines and peripheral blood lymphocyte mRNAs. Due to the high kidney specificity, AMPros-based urinalysis discriminates allograft rejection against other non-alloimmune specific diseases, which is unattainable by measurement of serological biomarkers. Such a noninvasive and sensitive urine test holds great promise in continuous monitoring of renal allograft conditions at low resource settings for timely clinical interventions., (© 2023 Wiley-VCH GmbH.)

Kidney transplantation (KT) is the optimal therapeutic strategy for patients with end-stage renal disease. The key to post-transplantation management is careful surveillance of allograft function. Kidney injury may occur from several different causes that require different patient management approaches. However, routine clinical monitoring has several limitations and detects alterations only at a later stage of graft damage. Accurate new noninvasive biomarker molecules are clearly needed for continuous monitoring after KT in the hope that early diagnosis of allograft dysfunction will lead to an improvement in the clinical outcome. The advent of "omics sciences", and in particular of proteomic technologies, has revolutionized medical research. Proteomic technologies allow us to achieve the identification, quantification, and functional characterization of proteins/peptides in biological samples such as urine or blood through supervised or targeted analysis. Many studies have investigated proteomic techniques as potential molecular markers discriminating among or predicting allograft outcomes. Proteomic studies in KT have explored the whole transplant process: donor, organ procurement, preservation, and posttransplant surgery. The current article reviews the most recent findings on proteomic studies in the setting of renal transplantation in order to better understand the effective potential of this new diagnostic approach.

Background: We developed urinary cell mRNA profiling for noninvasive diagnosis of acute T cell mediated rejection (TCMR) and BK virus nephropathy (BKVN), two significant post-transplant complications. Our profiling protocol for the multicenter Clinical Trial of Transplantation-04 (CTOT-04) study consisted of centrifugation of urine to prepare cell pellets, washes, addition of an RNA preservative, storage at 80 0 C and shipment in cold containers to our Gene Expression Monitoring (GEM) Core for RNA isolation and quantification of mRNA in RT-qPCR assays. To simplify profiling, we developed a filter-based protocol (ZFBP) that eliminated the need for centrifugation, RNA preservative, storage at 80 0 C, and shipment in cold containers for mRNA profiling. Furthermore, we trained kidney allograft recipients to perform the filtration of urine at home using the filter and post the urinary cell lysate containing the RNA at ambient temperature to our GEM Core for profiling. Here, we report our refinement of ZFBP and investigation of its diagnostic performance characteristics., Methods: Total RNA was isolated from kidney allograft biopsy-matched urines using a filter-based protocol complemented by a silica-membrane-based cartridge for mRNA enrichment, the Weill Cornell Hybrid Protocol (WCHP). Absolute copy numbers of CD3ε mRNA, CXCL10 mRNA, and 18S rRNA, components of the CTOT-04 three-gene TCMR diagnostic signature, and urinary cell BKV VP 1 mRNA copy number were measured using RT-qPCR assays. Mann-Whitney test, Fischer exact test, and receiver operating characteristic (ROC) curve analysis were used for data analyses., Results: Urinary cell three-gene TCMR diagnostic signature scores in urines processed using the WCHP discriminated kidney allograft recipients with TCMR (12 TCMR biopsies from 11 patients) from those without TCMR or BKVN (29 No TCMR/No BKVN biopsies from 29 patients). The median (25th and 75th percentiles) score of the CTOT-04 three-gene TCMR diagnostic signature was -0.448 (-1.664, 0.204) in the TCMR group and - 2.542 (-3.267, -1.365) in the No TCMR/ No BKVN group (P = 0.0005, Mann-Whitney test). ROC curve analysis discriminated the TCMR group from the No TCMR/ No BKVN group; the area under the ROC curve (AUROC) was 0.84 (95% Confidence Intervals [CI], 0.69 to 0.98) (P < 0.001), and TCMR was diagnosed with a sensitivity of 67% (95% CI, 35 to 89) at a specificity of 86% (95% CI, 67 to 95) using the CTOT-04 validated cutpoint of -1.213 (P = 0.0016, Fisher exact test). BKV VP1 mRNA copy number in urines processed using the WCHP discriminated patients with BKVN (n = 7) from patients without TCMR or BKVN (n = 29) and the AUROC was 1.0 (95% CI, 1.00 to 1.00) (P < 0.0001) and BKVN was diagnosed with a sensitivity of 86% (95% CI, 42 to 99) at a specificity of 100% (95% CI, 85 to 100) with the previously validated cutpoint of 6.5 × 10 8 BKV-VP1 mRNA copies per microgram of RNA (P < 0.0001, Fisher exact test)., Conclusion: Urine processed using the WCHP predicted TCMR and BKVN in kidney allograft recipients. WCHP represents not only a significant advance toward the portability of urinary cell mRNA profiling but also improved patient management by minimizing their visits for urine collection., (Copyright © 2022. Published by Elsevier B.V.)

Accurate and non-invasive monitoring of allograft posttransplant is essential for early detection of acute cellular rejection and determines the long-term survival of the graft. Clinically, tissue biopsy is the most effective approach for diagnosing transplant rejection. Nonetheless, the procedure is invasive and potentially triggers organ failure. This work aims to design and apply GzmB-responsive nanosensors (GBRNs) that can readily size-change in graft tissues. Subsequently, we investigate the activity of serine protease granzyme B by generating a direct colorimetric urinary readout for non-invasive detection of transplant rejection in under 1 h. In preclinical heart graft mice models of transplant rejection, GBRNs were cleaved by GzmB and excreted by the kidneys via accurate nanometre-size glomerular filtration. By exploiting the catalytic activity of ultrasmall gold nanoclusters, GBRNs urinalysis promotes ultrasensitive surveillance of rejection episodes with a receiver operator characteristic curve area under the curve of 0.896 as well as a 95% confidence interval of about 0.7701-1.000. Besides, the catalytic activity of gold nanoclusters in urine can be detected at point-of-care testing to predict the immunity responses in mice with insufficient immunosuppressive therapy. Therefore, this non-invasive, sensitive, and quantitative method is a robust and informative approach for rapid and routine monitoring of transplant allografts without invasive biopsy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Kidney transplantation is a life-restorative therapy, but immune rejection undermines allograft survival. Urinary cell mRNA profiles offer a noninvasive means of diagnosing kidney allograft rejection, but urine processing protocols have logistical constraints. We aimed to determine whether the centrifugation-based method for urinary cell mRNA profiling could be replaced with a simpler filtration-based method without undermining quality., Methods: We isolated RNA from urine collected from kidney allograft recipients using the Cornell centrifugation-based protocol (CCBP) or the Zymo filter-based protocol (ZFBP) and compared RNA purity and yield using a spectrophotometer or a fluorometer and measured absolute copy number of transcripts using customized real-time quantitative PCR assays. We investigated the performance characteristics of RNA isolated using ZFBP and stored either at -80 °C or at ambient temperature for 2 to 4 days and also when shipped to our Gene Expression Monitoring (GEM) Core at ambient temperature. We examined the feasibility of initial processing of urine samples by kidney allograft recipients trained by the GEM Core staff and the diagnostic utility for acute rejection, of urine processed using the ZFBP., Results: RNA purity (P = 0.0007, Wilcoxon matched paired signed-ranks test) and yield (P < 0.0001) were higher with ZFBP vs. CCBP, and absolute copy number of 18S rRNA was similar (P = 0.79) following normalization of RNA yield by reverse transcribing a constant amount of RNA isolated using either protocol. RNA purity, yield, and absolute copy numbers of 18S rRNA, TGF-β1 mRNA and microRNA-26a were not different (P > 0.05) in the filtrates containing RNA stored either at -80 °C or at ambient temperature for 2 to 4 days or shipped overnight at ambient temperature. RNA purity, yield, and absolute copy numbers of 18S rRNA and TGF-β1 mRNA were also not different (P > 0.05) between home processed and laboratory processed urine filtrates. Urinary cell levels of mRNA for granzyme B (P = 0.01) and perforin (P = 0.0002) in the filtrates were diagnostic of acute rejection in human kidney allografts., Conclusions: Urinary cell mRNA profiling was simplified using the ZFBP without undermining RNA quality or diagnostic utility. Home processing by the kidney allograft recipients, the stability of RNA containing filtrates at ambient temperature, and the elimination of the need for centrifuges and freezers represent some of the advantages of ZFBP over the CCBP for urinary cell mRNA profiling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: It is unknown whether meat intake is beneficial for long-term patient and graft survival in kidney transplant recipients (KTR)., Objectives: We first investigated the association of the previously described meat intake biomarkers 1-methylhistidine and 3-methylhistidine with intake of white and red meat as estimated from a validated food frequency questionnaire (FFQ). Second, we investigated the association of the meat intake biomarkers with long-term outcomes in KTR., Methods: We measured 24-h urinary excretion of 1-methylhistidine and 3-methylhistidine by validated assays in a cohort of 678 clinically stable KTR. Cross-sectional associations were assessed by linear regression. We used Cox regression analyses to prospectively study associations of log2-transformed biomarkers with mortality and graft failure., Results: Urinary 1-methylhistidine and 3-methylhistidine excretion values were median: 282; interquartile range (IQR): 132-598 µmol/24 h and median: 231; IQR: 175-306 µmol/24 h, respectively. Urinary 1-methylhistidine was associated with white meat intake [standardized β (st β): 0.20; 95% CI: 0.12, 0.28; P < 0.001], whereas urinary 3-methylhistidine was associated with red meat intake (st β: 0.30; 95% CI: 0.23, 0.38; P < 0.001). During median follow-up for 5.4 (IQR: 4.9-6.1) y, 145 (21%) died and 83 (12%) developed graft failure. Urinary 3-methylhistidine was inversely associated with mortality independently of potential confounders (HR per doubling: 0.55; 95% CI: 0.42, 0.72; P < 0.001). Both urinary 1-methylhistidine and urinary 3-methylhistidine were inversely associated with graft failure independent of potential confounders (HR per doubling: 0.84; 95% CI: 0.73, 0.96; P = 0.01; and 0.59; 95% CI: 0.41, 0.85; P = 0.004, respectively)., Conclusions: High urinary 3-methylhistidine, reflecting higher red meat intake, is independently associated with lower risk of mortality. High urinary concentrations of both 1- and 3-methylhistidine, of which the former reflects higher white meat intake, are independently associated with lower risk of graft failure in KTR. Future intervention studies are warranted to study the effect of high meat intake on mortality and graft failure in KTR, using these biomarkers., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

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., (Copyright © 2021 by the American Society of Nephrology.)

Extracellular vesicles (EVs) are nanoparticles that transmit molecules from releasing cells to target cells. Recent studies link urinary EVs (uEV) to diverse processes such as infection and rejection after kidney transplantation. This, and the unmet need for biomarkers diagnosing kidney transplant dysfunction, has led to the current high level of interest in uEV. uEV provide non-intrusive access to local protein, DNA, and RNA analytics without invasive biopsy. To determine the added value of uEV measurements for detecting allograft dysfunction after kidney transplantation, we systematically included all related literature containing directly relevant information, with the addition of indirect evidence regarding urine or kidney injury without transplantation. According to their varying characteristics, uEV markers after transplantation could be categorized into kidney-specific, donor-specific, and immune response-related (IR-) markers. A few convincing studies have shown that kidney-specific markers (PODXL, ion cotransporters, SYT17, NGAL, and CD133) and IR-markers (CD3, multi-mRNA signatures, and viral miRNA) could diagnose rejection, BK virus-associated nephropathy, and calcineurin inhibitor nephrotoxicity after kidney transplantation. In addition, some indirect proof regarding donor-specific markers (donor-derived cell-free DNA) in urine has been demonstrated. Together, this literature review provides directions for exploring novel uEV markers' profiling complications after kidney transplantation.

We investigated the clinical relevance of urinary cytokines/chemokines reflecting intrarenal immunologic micromilieu as prognostic markers and the optimal measurement timing after living donor kidney transplantation (LDKT). This prospective cohort study included 77 LDKT patients who were followed for ≥ 5 years. Patients were divided into control (n = 42) or acute rejection (AR, n = 35) group. Early AR was defined as AR occurring within 3 months. Serum and urine cytokines/chemokines were measured serially as follows: intraoperative, 8/24/72 h, 1 week, 3 months, and 1 year after LDKT. Intrarenal total leukocytes, T cells, and B cells were analyzed with immunohistochemistry followed by tissueFAXS. Urinary MCP-1 and fractalkine were also analyzed in a validation cohort. Urinary MCP-1 after one week was higher in the AR group. Urinary MCP-1, fractalkine, TNF-α, RANTES, and IL-6 after one week were significantly higher in the early AR group. Intrarenal total leukocytes and T cells were elevated in the AR group compared with the control group. Urinary fractalkine, MCP-1, and IL-10 showed positive correlation with intrarenal leukocyte infiltration. Post-KT 1 week urinary MCP-1 showed predictive value in the validation cohort. One-week post-KT urinary MCP-1 may be used as a noninvasive diagnostic marker for predicting AR after LDKT., (© 2021. The Author(s).)

Background: In kidney transplantation, fibrosis represents the final and irreversible consequence of the pathogenic mechanisms that lead to graft failure, and in the late stages it irremediably precedes the loss of renal function. The invasiveness of kidney biopsy prevents this condition from being frequently monitored, while clinical data are rather unspecific. The objective of this study was to find noninvasive biomarkers of kidney rejection., Methods: We carried out proteomic analysis of the urinary Extracellular Vesicles (uEVs) from a cohort of kidney transplant recipients (n = 23) classified according to their biopsy-based diagnosis and clinical parameters as interstitial fibrosis and tubular atrophy (IFTA), acute cellular rejection (ACR), calcineurin inhibitors toxicity (CNIT) and normal kidney function (NKF)., Results: Shotgun mass spectrometry of uEV-proteins identified differential expression of several proteins among these different groups. Up to 23 of these proteins were re-evaluated using targeted proteomics in a new independent cohort of patients (n = 41) classified in the same diagnostic groups. Among other results, we found a differential expression of vitronectin (VTN) in patients displaying chronic interstitial and tubular lesions (ci and ct mean > 2 according to Banff criteria). These results were further confirmed by a pilot study using enzyme-linked immunosorbent assay (ELISA)., Conclusion: Urinary vitronectin levels are a potential stand-alone biomarker to monitor fibrotic changes in kidney transplant recipients in a non-invasive fashion.

Objective: To evaluate the clinical and laboratory characteristics in pregnancy that differentiate preeclampsia from acute renal allograft rejection and to investigate the maternal, neonatal, and graft sequelae of these diagnoses., Methods: We conducted a retrospective case-controlled registry study of data abstracted from Transplant Pregnancy Registry International deliveries between 1968 and 2019. All adult kidney transplant recipients with singleton pregnancies of at least 20 weeks of gestation were included. Acute rejection was biopsy proven and preeclampsia was diagnosed based on contemporary criteria. Variables were compared using χ2, Fisher exact, and Wilcoxon rank sum tests as appropriate. Multivariable linear regression was used to analyze preterm birth. Kaplan-Meier curves with log-rank test and Cox proportional hazards model were used to compare graft loss over time., Results: There were 26 pregnant women with biopsy-confirmed acute rejection who were matched by the year they conceived to 78 pregnant women with preeclampsia. Recipients with acute rejection had elevated peripartum serum creatinine levels (73% vs 14%, P<.001), with median intrapartum creatinine of 3.90 compared with 1.15 mg/dL (P<.001). Conversely, only patients with preeclampsia had a significant increase in proteinuria from baseline. Although there were no significant differences in maternal outcomes, graft loss within 2 years postpartum (42% vs 10%) and long-term graft loss (73% vs 35%) were significantly increased in recipients who experienced acute rejection (P<.001 for both). The frequency of delivery before 32 weeks of gestation was 53% with acute rejection and 20% with preeclampsia. After controlling for hypertension and immunosuppressant use, acute rejection was associated with higher frequency of delivery at less than 32 weeks of gestation (adjusted odds ratio 4.04, 95% CI 1.10-15.2)., Conclusion: In pregnancy, acute rejection is associated with higher creatinine levels, and preeclampsia is associated with increased proteinuria. Acute rejection in pregnancy carries a risk of prematurity and graft loss beyond that of preeclampsia for kidney transplant recipients., Funding Source: The Transplant Pregnancy Registry International is supported in part by an educational grant from Veloxis Pharmaceuticals., Competing Interests: Financial Disclosure Lisa Coscia reports that money was paid to her institution from Veloxis Pharmaceuticals. The other authors did not report any potential conflicts of interest., (Copyright © 2021 by the American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Background: Children are at high risk for subclinical rejection, and kidney biopsy is currently used for surveillance. Our objective was to test how novel rejection biomarkers such as urinary CXCL10 may influence clinical decision-making to indicate need for a biopsy., Methods: A minimum dataset for standard decision-making to indicate a biopsy was established by an expert panel and used to design clinical vignettes for use in a survey. Pediatric nephrologists were recruited to review the vignettes and A) estimate rejection risk and B) decide whether to biopsy; first without and then with urinary CXCL10/Cr level. Accuracy of biopsy decisions was then tested against the biopsy results. IRA was assessed by Fleiss Kappa (κ) for binary choice and ICC for probabilities., Results: Eleven pediatric nephrologists reviewed 15 vignettes each. ICC of probability assessment for rejection improved from poor (0.28, P < .01) to fair (0.48, P < .01) with addition of CXCL10/Cr data. It did not, however, improve the IRA for decision to biopsy (K = 0.48 and K = 0.43, for the comparison). Change in clinician estimated probability of rejection with additional CXCL10/Cr data was correlated with CXCL10/Cr level (r 2  = 0.7756, P < .0001). Decision accuracy went from 8/15 (53.3%) cases to 11/15 (73.3%) with CXCL10/Cr, although improvement did not achieve statistical significance. Using CXCL10/Cr alone would have been accurate in 12/15 cases (80%)., Conclusion: There is high variability in decision-making on biopsy indication. Urinary CXCL10/Cr improves probability estimates for risk of rejection. Training may be needed to assist nephrologists in better integrate biomarker information into clinical decision-making., (© 2020 Wiley Periodicals LLC.)

Background: Early identification and treatment of kidney transplant rejection episodes is vital to limit loss of function and prolong the life of the transplanted kidney and recipient. Current practice depends on detecting a creatinine rise. A biomarker to diagnose transplant rejection at an earlier time point than current practice, or to inform earlier decision making to biopsy, could be transformative. It has previously been shown that urinary nitrate concentration is elevated in renal transplant rejection. Nitrate is a nitric oxide (NO) oxidation product. Transplant rejection upregulates NO synthesis via inducible nitric oxide synthase leading to elevations in urinary nitrate concentration. We have recently validated a urinary nitrate concentration assay which could provide results in a clinically relevant timeframe. Our aim was to determine whether urinary nitrate concentration is a useful tool to predict renal transplant rejection in the context of contemporary clinical practice., Methods: We conducted a prospective observational study, recruiting renal transplant participants over an 18-month period. We made no alterations to the patients' clinical care including medications, immunosuppression, diet and frequency of visits. We collected urine samples from every clinical attendance. We assessed the urinary nitrate to creatinine ratio (uNCR) between patient groups: routine attendances, biopsy proven rejection, biopsy proven no rejection and other call backs. uNCR was examined over time for those with biopsy proven transplant rejection. These four groups were compared using an ANOVA test., Results: A total of 2656 samples were collected. uNCR during biopsy proven rejection, n = 15 (median 49 μmol/mmol, IQR 23-61) was not significantly different from that of routine samples, n = 164 (median 55 μmol/mmol, IQR 37-82) (p = 0.55), or biopsy proven no rejection, n = 12 (median 39 μmol/mmol, IQR 21-89) (P = 0.77). Overall uNCR was highly variable with no diagnostic threshold for kidney transplant rejection. Furthermore, within-patient uNCR was highly variable over time, and thus it was not possible to produce individualised patient thresholds to identify rejection. The total taking Tacrolimus was 204 patients, with no statistical difference between the uNCR of all those on Tacrolimus, against those not, p = 0.18., Conclusion: The urinary nitrate to creatinine ratio is not a useful biomarker for renal transplant rejection.

Noninvasive tools for diagnosis or prediction of acute kidney allograft rejection have been extensively investigated in recent years. Biochemical and molecular analyses of blood and urine provide a liquid biopsy that could offer new possibilities for rejection prevention, monitoring, and therefore, treatment. Nevertheless, these tools are not yet available for routine use in clinical practice. In this systematic review, MEDLINE was searched for articles assessing urinary biomarkers for diagnosis or prediction of kidney allograft acute rejection published in the last five years (from January 1, 2015 to May 31, 2020). This review follows the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines. Articles providing targeted or unbiased urine sample analysis for the diagnosis or prediction of both acute cellular and antibody-mediated kidney allograft rejection were included, analyzed, and graded for methodological quality with a particular focus on study design and diagnostic test accuracy measures. Urinary C-X-C motif chemokine ligands were the most promising and frequently studied biomarkers. The combination of precise diagnostic reference in training sets with accurate validation in real-life cohorts provided the most relevant results and exciting groundwork for future studies.

Background: Immunosuppressive drugs (ISD) are an essential tool in the treatment of transplant rejection and immune-mediated diseases. Therapeutic drug monitoring (TDM) for determination of ISD concentrations in biological samples is an important instrument for dose personalization for improving efficacy while reducing side effects. While currently ISD concentration measurements are performed at specialized, centralized facilities, making the process complex and laborious for the patient, various innovative technical solutions have recently been proposed for bringing TDM to the point-of-care (POC)., Content: In this review, we evaluate current ISD-TDM and its value, limitations, and proposed implementations. Then, we discuss the potential of POC-TDM in the era of personalized medicine, and provide an updated review on the unmet needs and available technological solutions for the development of POC-TDM devices for ISD monitoring. Finally, we provide concrete suggestions for the generation of a meaningful and more patient-centric process for ISD monitoring., Summary: POC-based ISD monitoring may improve clinical care by reducing turnaround time, by enabling more frequent measurements in order to obtain meaningful pharmacokinetic data (i.e., area under the curve) faster reaction in case of problems and by increasing patient convenience and compliance. The analysis of the ISD-TDM field prompts the evolution of POC testing toward the development of fully integrated platforms able to support clinical decision-making. We identify 4 major areas requiring careful combined implementation: patient usability, data meaningfulness, clinicians' acceptance, and cost-effectiveness., (© American Association for Clinical Chemistry 2020.)

The main complication associated with renal graft loss is immune rejection. The gold standard for the diagnosis of renal graft rejection is percutaneous renal biopsy, which is expensive and can lead to complications. Inflammation is one of the main pathogenic pathways in allograft rejection, and urine samples seem to be efficient windows to explore the allograft condition with a high cost-benefit ratio. This study aimed to evaluate the messenger ribonucleic acid (mRNA) profile expression pattern for interleukin (IL) 2, IL-4, IL-6, IL-8, and IL-10; tumor necrosis factor alfa; gamma interferon; and transforming growth factor β1 in the urine renal cells of patients with a diagnosis of humoral rejection and patients with a diagnosis of normal biopsy. METHODS: An observational, cross-sectional analytical study was performed. All kidney transplants were performed at the Organ Transplant Department between 2018 and 2019. Also, a healthy control with a normal blood test and no apparent infection was included. mRNA from urine samples and biopsies was isolated, and the expression of interleukins was analyzed in PCR real time. Data were analyzed by Shapiro-Wilk and Kruskal-Wallis tests. RESULTS: The proinflammatory IL expression pattern in urine samples of kidney rejection group showed overexpression for IL-8 (P = .0001). No differences were observed in the rest of the interleukins analyzed. When we compared the results in the rejected versus not rejected transplanted patients with a group of apparently healthy subjects, the difference remains consistent. Thus, mRNA of IL-8 could function as a diagnostic tool in cases of chronic damage secondary to fibrosis., (Copyright © 2020. Published by Elsevier Inc.)

Acute T cell-mediated rejection (TCMR) is a major complication after renal transplantation. TCMR diagnosis is very challenging and currently depends on invasive renal biopsy and nonspecific markers such as serum creatinine. A noninvasive metabolomics panel could allow early diagnosis and improved accuracy and specificity. We report, in this study, on urine metabolome changes in renal transplant recipients diagnosed with TCMR, with a view to future metabolomics-based diagnostics in transplant medicine. We performed urine metabolomic analyses in three study groups: (1) 7 kidney transplant recipients with acute TCMR, (2) 15 kidney transplant recipients without rejection but with impaired kidney function, and (3) 6 kidney transplant recipients with stable renal function, using 1 H-nuclear magnetic resonance. Multivariate modeling of metabolites suggested a diagnostic panel where the diagnostic accuracy of each metabolite was calculated by receiver operating characteristic curve analysis. The impaired metabolic pathways associated with TCMR were identified by pathway analysis. In all, a panel of nine differential metabolites encompassing nicotinamide adenine dinucleotide, 1-methylnicotinamide, cholesterol sulfate, gamma-aminobutyric acid (GABA), nicotinic acid, nicotinamide adenine dinucleotide phosphate, proline, spermidine, and alpha-hydroxyhippuric acid were identified as novel potential metabolite biomarkers of TCMR. Proline, spermidine, and GABA had the highest area under the curve (>0.7) and were overrepresented in the TCMR group. Nicotinate and nicotinamide metabolism was the most important pathway in TCMR. These findings call for clinical validation in larger study samples and suggest that urinary metabolomics warrants future consideration as a noninvasive research tool for TCMR diagnostic innovation.

The aim of this study was to identify potential proteomic biomarkers for chronic active antibody-mediated rejection (CAMR) in kidney transplant recipients (KTRs). Among 385 KTRs enrolled in a cross-sectional multicenter study, 26 KTRs with biopsy-proven CAMR, 57 KTRs with long-term graft survival (LGS), and 10 rejection-free matched KTRs were included. A proteomic approach was employed to measure urinary extracellular vesicle (EV) changes in the KTRs. The urinary EVs were trypsin-digested using a gel-assisted protocol and quantified by label-free liquid chromatography with tandem mass spectrometry, using a data-dependent acquisition (DDA) mode. Western blot analysis was performed to confirm the protein levels for each candidate biomarker. Analysis of the isolated EV proteins revealed 93 and 97 proteins in the CAMR and LGS patients, respectively. Proteins that were identical in both groups were excluded and only high-significance proteins with a fold change of at least 1.5 were selected as candidate biomarkers. Six proteins (APOA1, TTR, PIGR, HPX, AZGP1, and CP) that were distinguishable between CAMR and LGS were selected. The proteins were confirmed by immunoblot analyses using independently acquired urinary EV samples. AZGP1 in particular was found to be a CAMR-specific proteomic biomarker that was distinguishable from the rejection-free control group with matching kidney function, duration of transplantation, and age. We identified and validated six proteomic biomarkers for CAMR and clarified one CAMR-specific proteomic biomarker in KTRs. Further clinical trials are needed before these rejection-specific biomarkers can be applied for the early prediction, diagnosis, and monitoring of the clinical response of KTRs to the treatment of CAMR., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Creatinine and proteinuria are used to monitor kidney transplant patients. However, renal biopsies are needed to diagnose renal graft rejection. Here, we assessed whether the quantification of different urinary cells would allow non-invasive detection of rejection. Urinary cell numbers of CD4 + and CD8 + T cells, monocytes/macrophages, tubular epithelial cells (TEC), and podocalyxin(PDX)-positive cells were determined using flow cytometry and were compared to biopsy results. Urine samples of 63 renal transplant patients were analyzed. Patients with transplant rejection had higher amounts of urinary T cells than controls; however, patients who showed worsening graft function without rejection had similar numbers of T cells. T cells correlated with histological findings (interstitial inflammation p = 0.0005, r = 0.70; tubulitis p = 0.006, r = 0.58). Combining the amount of urinary T cells and TEC, or T cells and PDX + cells, yielded a significant segregation of patients with rejection from patients without rejection (all p < 0.01, area under the curve 0.89-0.91). Urinary cell populations analyzed by flow cytometry have the potential to introduce new monitoring methods for kidney transplant patients. The combination of urinary T cells, TEC, and PDX-positive cells may allow non-invasive detection of transplant rejection.

Monitoring allograft function after kidney transplant has routinely relied on the use of nonspecific markers, such as serum creatinine, glomerular filtration rate, proteinuria, and donor-specific antibodies. These traditional markers have low sensitivity and fail to detect subclinical changes. Diagnosis of renal allograft dysfunction still requires an allograft biopsy, as it remains the criterion standard for assessment of graft status. However, renal biopsy is an invasive procedure, and sampling errors may result in misdiagnosis, perhaps causing graft failure. New biomarkers have been developed to monitor allograft function, although many are not yet routinely used. Other shortcomings, such as lack of standardization and high cost, should be solved before their widespread application in the clinic. A recipient's immune status could be monitored by use of urine or blood samples. These include functional cell-based assays and the evaluation of molecular expression at the messenger RNA or protein levels. Molecular technologies, including molecular microscope diagnostic systems, have been recently developed to improve the yield of histologic evaluation of the allograft biopsy. Prospective, interventional trials are required to demonstrate whether these new biomarkers improve patient or transplant outcomes. Implementation of these technologies into standard clinical practice remains challenging until their generalizability, cost, ease of interpretation, and the identification of patients who may benefit from more than standard-of-care surveillance can be determined. These biomarkers could allow immunosuppressive therapy to be individualized for patients.

Host-microbiota interactions can modulate the immune system both at local and systemic levels, with potential consequences for organ transplantation outcomes. In this study, we hypothesized that differences in the urinary microbiome following kidney transplantation would be associated with posttransplantation status: stable, minimally immunosuppressed, or tolerant. One hundred thirteen urine samples from stable (n = 51), minimally immunosuppressed (n = 19), and spontaneously tolerant (n = 16) patients, paired with age-matched controls (n = 27) were profiled and compared to each other at a taxonomic level with special interest in the immunosuppressive regimen. All comparisons and correlations were adjusted on sex and time posttransplantation. Our results highlighted a unique and specific urinary microbiota associated with spontaneous tolerance characterized by a high diversity and a clear Proteobacteria profile. Finally, we report that this profile is (1) impacted by gender, (2) inversely correlated with immunosuppressive drugs (calcineurin inhibitors and mammalian target of rapamycin inhibitors), and (3) stable in time., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The pathophysiology of late kidney-allograft failure remains complex and poorly understood. Activation of filtered or locally produced complement may contribute to the progression of renal failure through tubular C5b-9 formation. This study aimed to determine urinary properdin and sC5b-9 excretion and assess their association with long-term outcome in renal transplant recipients (RTR). Methods: We measured urinary properdin and soluble C5b-9 in a well-defined cross-sectional cohort of RTR. Urinary specimens were taken from a morning urine portion, and properdin and sC5b-9 were measured using an enzyme-linked-immunosorbent assay (ELISA). Cox proportional hazard regression analyses were used to investigate prospective associations with death-censored graft failure. Results: We included 639 stable RTR at a median [interquartile range] 5.3 (1.8-12.2) years after transplantation. Urinary properdin and sC5b-9 excretion were detectable in 161 (27%) and 102 (17%) RTR, respectively, with a median properdin level of 27.6 (8.6-68.1) ng/mL and a median sC5b-9 level of 5.1 (2.8-12.8) ng/mL. In multivariable-adjusted Cox regression analyses, including adjustment for proteinuria, urinary properdin (HR, 1.12; 95% CI 1.02-1.28; P = 0.008) and sC5b-9 excretion (HR, 1.34; 95% CI 1.10-1.63; P = 0.003) were associated with an increased risk of graft failure. If both urinary properdin and sC5b-9 were detectable, the risk of graft failure was further increased (HR, 3.12; 95% CI 1.69-5.77; P < 0.001). Conclusions: Our findings point toward a potential role for urinary complement activation in the pathogenesis of chronic allograft failure. Urinary properdin and sC5b-9 might be useful biomarkers for complement activation and chronic kidney allograft deterioration, suggesting a potential role for an alternative pathway blockade in RTR., (Copyright © 2019 Lammerts, Eisenga, Alyami, Daha, Seelen, Pol, van den Born, Sanders, Bakker and Berger.)

Background: Circular RNAs (circRNAs) have recently been described as novel noncoding regulators of gene expression. They are detectable in the blood of patients with acute kidney injury. We tested whether circRNAs were present in urine and could serve as new predictors of outcome in renal transplant patients with acute rejection., Methods: A global circRNA expression analysis using RNA from urine of patients with acute T cell-mediated renal allograft rejection and control transplant patients was performed. Dysregulated circRNAs were confirmed in a cohort of 62 patients with acute rejection, 10 patients after successful antirejection therapy, 18 control transplant patients without rejection, and 13 stable transplant patients with urinary tract infection., Results: A global screen revealed several circRNAs to be altered in urine of patients with acute rejection. Concentrations of 2 circRNAs including hsa&#95;circ&#95;0001334 and hsa&#95;circ&#95;0071475 were significantly increased. These were validated in the whole cohort of patients. hsa&#95;circ&#95;0001334 was upregulated in patients with acute rejection compared with controls. Concentrations of hsa&#95;circ&#95;0001334 normalized in patients with acute rejection following successful antirejection therapy. hsa&#95;circ&#95;0001334 was associated with higher decline in glomerular filtration rate 1 year after transplantation., Conclusions: CircRNA concentrations are significantly dysregulated in patients with acute rejection at subclinical time points. Urinary hsa&#95;circ&#95;0001334 is a novel biomarker of acute kidney rejection, identifying patients with acute rejection and predicting loss of kidney function., (© 2019 American Association for Clinical Chemistry.)

Background: To validate a novel method for post-transplant surveillance to detect kidney allograft rejection via a characteristic constellation of the urine metabolites alanine, citrate, lactate, and urea investigated by nuclear magnetic resonance (NMR) spectroscopy a first prospective, observational study was performed., Methods: Within the UMBRELLA study 986 urine specimens were collected from 109 consecutively enrolled renal transplant recipients, and metabolite constellations were analyzed. A metabolite rejection score was calculated and compared to histopathological results of corresponding indication and protocol allograft biopsies (n = 206)., Findings: The metabolite constellation was found to be a useful biomarker to non-invasively detect acute allograft rejection (AUC = 0.75; 95% confidence interval (CI) 0.68-0.83; based on 46 cases and 520 control samples). Combined analysis of the metabolite rejection score and the estimated glomerular filtration rate (eGFR) at the time of urine sampling further improved the overall test performance significantly (AUC = 0.84; 95% CI 0.76-0.91; based on 42 cases and 468 controls). Regarding the time course analysis in patients without rejection episodes the test results remained well below a diagnostic threshold associated with high risk of acute rejection. In other cases, a marked increase above this threshold indicated acute allograft rejection already six to ten days before diagnostic renal biopsies were performed., Interpretation: A combination of an NMR-based urine metabolite analysis and eGFR is promising as a non-invasive test for post-transplant surveillance and to support decision making whether renal allografts need histopathological evaluation., (Copyright © 2019. Published by Elsevier B.V.)

Introduction: Early prognostic markers that identify high‑risk kidney transplant recipients may lead to optimization of immunosuppressive therapy and improved long‑term outcomes., Objectives: The aim of this study was to assess whether the measurement of urinary concentrations of CCL2 and CXCL10 chemokines can be a valuable noninvasive tool for identifying ongoing pathological processes in a kidney allograft., Patients and Methods: The study included 40 patients who underwent a protocol biopsy within 1‑year post kidney transplant. The urinary concentrations of CCL2 and CXCL10 with reference to creatinine in urine were assayed in all patients. On the basis of biopsy results, a study group was selected (n = 25), including patients with a diagnosis of interstitial fibrosis and tubular atrophy grades II to III (n = 16), BK virus (BKV) nephropathy (n = 4), or mild inflammatory lesions fulfilling the criteria for mild rejection processes or borderline lesions (n = 11). Patients with normal biopsy results were included in a control group (n = 15)., Results: The ratio of CCL2 to creatinine (CCL2:Cr) was a significant independent predictor of BKV ephropathy (odds ratio, 1.1; 95% CI, 1.0-1.2; P = 0.04). The CXCL10:Cr ratio was not found to be an independent predictor of BKV nephropathy (odds ratio, 1.3; 95% CI, 0.99-1.71; P = 0.06)., Conclusions: The CCL2:Cr and CXCL10:Cr ratios may predict BKV nephropathy. The diagnostic value of CCL2 and CXCL10 in BKV infection should be further evaluated.

Kidney transplantation is the treatment of choice for patients with advanced chronic kidney disease (CKD) and end stage renal disease (ESRD). However, acute rejection (AR) is a common complication in kidney transplantation and is associated with reduced graft survival. Current diagnosis of AR relies mainly on clinical monitoring including serum creatinine, proteinuria, and confirmation by histopathologic assessment in the biopsy specimen of graft kidney. Although an early protocol biopsy is indispensable for depicting the severity of pathologic lesions in subclinical acute rejection (subAR), it is not acceptable in some cases and cannot be performed because of its invasive nature. Therefore, we examined the detection of noninvasive biomarkers that are closely related to the pathology of subAR in protocol biopsies three months after kidney transplantation. In this study, the urinary level of microtubule-associated protein 1 light chain 3 (LC3), monocyte chemotactic protein-1 (MCP-1), liver-type fatty acid-binding protein (L-FABP), neutrophil gelatinase-associated lipocalin (NGAL), and human epididymis secretory protein 4 (HE4) were measured three months after kidney transplantation. Urine samples of 80 patients undergoing kidney transplantation between August 2014 to September 2016, were prospectively collected after three months. SubAR was observed in 11 patients (13.8%) in protocol biopsy. The urinary levels of LC3, MCP-1, NGAL, and HE4 were significantly higher in patients with subAR than in those without, while those of L-FABP did not differ between the two groups. Multivariate regression models, receiver-operating characteristics (ROC), and areas under ROC curves (AUC) were used to identify predicted values of subAR. Urinary HE4 levels were able to better identify subAR (AUC = 0.808) than the other four urinary biomarkers. In conclusion, urinary HE4 is increased in kidney transplant recipients of subAR three months after kidney transplantation, suggesting that HE4 has the potential to be used as a novel clinical biomarker for predicting subAR.

Taurine is a sulfur containing nutrient that has been shown to protect against oxidative stress, which has been implicated in the pathophysiology leading to late graft failure after renal transplantation. We prospectively investigated whether high urinary taurine excretion, reflecting high taurine intake, is associated with low risk for development of late graft failure in renal transplant recipients (RTR). Urinary taurine excretion was measured in a longitudinal cohort of 678 stable RTR. Prospective associations were assessed using Cox regression analyses. Graft failure was defined as the start of dialysis or re-transplantation. In RTR (58% male, 53 ± 13 years old, estimated glomerular filtration rate (eGFR) 45 ± 19 mL/min/1.73 m 2 ), urinary taurine excretion (533 (210-946) µmol/24 h) was significantly associated with serum free sulfhydryl groups (β = 0.126; P = 0.001). During median follow-up for 5.3 (4.5-6.0) years, 83 (12%) patients developed graft failure. In Cox regression analyses, urinary taurine excretion was inversely associated with graft failure (hazard ratio: 0.74 (0.67-0.82); P < 0.001). This association remained significant independent of potential confounders. High urinary taurine excretion is associated with low risk of late graft failure in RTR. Therefore, increasing taurine intake may potentially support graft survival in RTR. Further studies are warranted to determine the underlying mechanisms and the potential of taurine supplementation.

Enzyme activity may be more pathophysiologically relevant than enzyme quantity and is regulated by changes in conformational status that are undetectable by traditional proteomic approaches. Further, enzyme activity may provide insights into rapid physiological responses to inflammation/injury that are not dependent on de novo protein transcription. Activity-based protein profiling (ABPP) is a chemical proteomic approach designed to characterize and identify active enzymes within complex biological samples. Activity probes have been developed to interrogate multiple enzyme families with broad applicability, including but not limited to serine hydrolases, cysteine proteases, matrix metalloproteases, nitrilases, caspases, and histone deacetylases. The goal of this overview is to describe the overall rationale, approach, methods, challenges, and potential applications of ABPP to transplantation research. To do so, we present a case example of urine serine hydrolase ABPP in kidney transplant rejection to illustrate the utility and workflow of this analytical approach. Ultimately, developing novel transplant therapeutics is critically dependent on understanding the pathophysiological processes that result in loss of transplant function. ABPP offers a new dimension for characterizing dynamic changes in clinical samples. The capacity to identify and measure relevant enzyme activities provides fresh opportunities for understanding these processes and may help identify markers of disease activity for the development of novel diagnostics and real-time monitoring of patients. Finally, these insights into enzyme activity may also help to identify new transplant therapeutics, such as enzyme-specific inhibitors.

Background: Individuals who have kidney disease or kidney transplants need routine assessment of their kidney damage and function, which are largely measured based on histological examination of kidney biopsies, blood test, and urinalysis. These methods are practically difficult or inconvenient, and expensive. The objective of this study was to develop a model to estimate the kidney damage and function by surface-enhanced Raman spectroscopy (SERS)., Methods: Urine samples were collected from two previous studies: renal allograft recipient Lewis rats receiving anti-TGF-β antibody or control antibody treatment and obese diabetic ZSF1 rats with kidney disease fed with whole grape powder-containing chow or control chow. Silver nanoparticle-based SERS spectra of urine were measured. SERS spectra were analyzed using principal component analysis (PCA) combined with linear discriminant analysis (LDA) and partial least squires (PLS) analysis., Results: PCA/LDA separated anti-TGF-β antibody-treated group from control group with 90% sensitivity and 70% specificity in kidney transplants, and grape-fed group from controls with 72.7% sensitivity and 60% specificity in diabetic kidneys. The receiver operating characteristic curves showed that the integration area under the curve was 0.850 ± 0.095 (p = 0.008) in kidney transplant groups and 0.800 ± 0.097 (p = 0.02) in diabetic kidney groups. PLS predicted the biochemical parameters of kidney function using the SERS spectra, resulting in R 2  = 0.8246 (p < 0.001,urine protein), R 2  = 0.8438 (p < 0.001, urine creatinine), R 2  = 0.9265 (p < 0.001, urea), R 2  = 0.8719 (p < 0.001, serum creatinine), and R 2  = 0.6014 (p < 0.001, urine protein to creatinine ratio)., Conclusion: Urine SERS spectral analysis suggesting that it may become a convenient method for rapid assessment of renal impairment.

To improve early renal allograft function, it is important to develop a noninvasive diagnostic method for acute T cell-mediated rejection (TCMR). This study aims to explore potential noninvasive urinary biomarkers to screen for acute TCMR in kidney transplant recipients (KTRs) using untargeted metabolomic profiling. Urinary metabolites, collected from KTRs with stable graft function (STA) or acute TCMR episodes, were analyzed using liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analyses were performed to discriminate differences in urinary metabolites between the two groups. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic performance of potential urinary biomarkers. Statistical analysis revealed the differences in urinary metabolites between the two groups and indicated several statistically significant metabolic features suitable for potential biomarkers. By comparing the retention times and mass fragmentation patterns of the chemicals in metabolite databases, samples, and standards, six of these features were clearly identified. ROC curve analysis showed the best performance of the training set (area under the curve value, 0.926; sensitivity, 90.0%; specificity, 84.6%) using a panel of five potential biomarkers: guanidoacetic acid, methylimidazoleacetic acid, dopamine, 4-guanidinobutyric acid, and L-tryptophan. The diagnostic accuracy of this model was 62.5% for an independent test dataset. LC-MS-based untargeted metabolomic profiling is a promising method to discriminate between acute TCMR and STA groups. Our model, based on a panel of five potential biomarkers, needs to be further validated in larger scale studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Subclinical inflammation is an important predictor of death-censored graft loss, and its treatment has been shown to improve graft outcomes. Urine CXCL10 outperforms standard post-transplant surveillance in observational studies, by detecting subclinical rejection and early clinical rejection before graft functional decline in kidney transplant recipients., Methods and Analysis: This is a phase ii/iii multicentre, international randomised controlled parallel group trial to determine if the early treatment of rejection, as detected by urine CXCL10, will improve kidney allograft outcomes. Incident adult kidney transplant patients (n~420) will be enrolled to undergo routine urine CXCL10 monitoring postkidney transplant. Patients at high risk of rejection, defined as confirmed elevated urine CXCL10 level, will be randomised 1:1 stratified by centre (n=250). The intervention arm (n=125) will undergo a study biopsy to check for subclinical rejection and biopsy-proven rejection will be treated per protocol. The control arm (n=125) will undergo routine post-transplant monitoring. The primary outcome at 12 months is a composite of death-censored graft loss, clinical biopsy-proven acute rejection, de novo donor-specific antibody, inflammation in areas of interstitial fibrosis and tubular atrophy (Banff i-IFTA, chronic active T-cell mediated rejection) and subclinical tubulitis on 12-month surveillance biopsy. The secondary outcomes include decline of graft function, microvascular inflammation at 12 months, development of IFTA at 12 months, days from transplantation to clinical biopsy-proven rejection, albuminuria, EuroQol five-dimension five-level instrument, cost-effectiveness analysis of the urine CXCL10 monitoring strategy and the urine CXCL10 kinetics in response to rejection therapy., Ethics and Dissemination: The study has been approved by the University of Manitoba Health Research Ethics Board (HS20861, B2017:076) and the local research ethics boards of participating centres. Recruitment commenced in March 2018 and results are expected to be published in 2023. De-identified data may be shared with other researchers according to international guidelines (International Committee of Medical Journal Editors [ICJME])., Trial Registration Number: NCT03206801; Pre-results., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Long-term survival in orthotopic liver transplant (OLT) recipients remains impaired because of many contributing factors, including a low pretransplant muscle mass (or sarcopenia). However, influence of posttransplant muscle mass on survival is currently unknown. We hypothesized that posttransplant urinary creatinine excretion rate (CER), an established noninvasive marker of total body muscle mass, is associated with long-term survival after OLT. In a single-center cohort study of 382 adult OLT recipients, mean ± standard deviation CER at 1 year posttransplantation was 13.3 ± 3.7 mmol/24 h in men and 9.4 ± 2.6 mmol/24 h in women. During median follow-up for 9.8 y (interquartile range 6.4-15.0 y), 104 (27.2%) OLT recipients died and 44 (11.5%) developed graft failure. In Cox regression analyses, as continuous variable, low CER was associated with increased risk for mortality (HR = 0.43, 95% CI: 0.26-0.71, P = .001) and graft failure (HR = 0.42, 95% CI: 0.20-0.90, P = .03), independent of age, sex, and body surface area. Similarly, OLT recipients in the lowest tertile had an increased risk for mortality (HR = 2.69; 95% CI: 1.47-4.91, P = .001) and graft failure (HR = 2.77, 95% CI: 1.04-7.39, P = .04), compared to OLT recipients in the highest tertile. We conclude that 1 year posttransplant low total body muscle mass is associated with long-term risk of mortality and graft failure in OLT recipients., (© 2018 The Authors. American Journal of Transplantation published by Wiley Periodicals, Inc. on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Transplant glomerulopathy (TG) represents a major cause of long-term allograft failure and is the leading cause of overall post-transplant proteinuria. The extent to which histopathologic features predicts prognostication is uncertain., Methods: A single-center retrospective cohort with biopsy-proven TG was investigated. Renal biopsies were scored according to Banff 2017. The primary outcome was death-censored graft failure defined as return to dialysis or estimated glomerular filtration rate (eGFR) decreased to <15 mL/min/1.73 m2. The prognostic significance of clinical and histopathologic parameters was determined using Cox proportional hazards models., Results: Data from 180 cases were available for analysis with a median follow-up of 5.0 (2.6-8.2) years. In multivariable models, ci + ct score (HR 3.1; 95% CI 2.0-4.9), cg score (HR 1.7; 95% CI 1.1-2.8), eGFR (HR 2.1; 95% CI 1.4-3.2) and proteinuria (HR 2.4; 95% CI 1.6-3.7) were independent predictors of the primary outcome. Mesangial Immunoglobulin A deposition did not significantly affect allograft survival. The only significant pathologic factors for the severity of proteinuria were cg and g + ptc (adjusted R2 = 0.46) as determined by multivariable stepwise linear regression analysis., Conclusions: Severe ci + ct and cg at biopsy were predictors of unfavorable allograft prognosis in TG patients even after taking into consideration clinical characteristics. Histologic severity of cg and g + ptc was significantly associated with clinical proteinuria., (© 2019 S. Karger AG, Basel.)

Background: We performed a study to identify differences in the urinary microbiome associated with chronic allograft dysfunction (CAD) and compared the urinary microbiome of male and female transplant recipients with CAD., Methods: This case-control study enrolled 67 patients within the Deterioration of Kidney Allograft Function (DeKAF) Genomics cohort at two transplant centers. CAD was defined as a greater than 25% rise in serum creatinine relative to a 3 month post-transplant baseline. Urine samples from patients with and without CAD were analyzed using 16S V4 bacterial ribosomal DNA sequences., Results: Corynebacterium was more prevalent in female and male patients with CAD compared to non-CAD female patients (P = 0.0005). A total 21 distinct Operational Taxonomic Unit (OTUs) were identified as significantly different when comparing CAD and non-CAD patients using Kruskal-Wallis (P < 0.01). A subset analysis of female patients with CAD compared to non-CAD females identified similar differentially abundant OTUs, including the genera Corynebacterium and Staphylococcus (Kruskal-Wallis; P = 0.01; P = 0.004, respectively). Male CAD vs female CAD analysis showed greater abundance of phylum Proteobacteria in males., Conclusion: There were differences in the urinary microbiome when comparing female and male CAD patients with their female non-CAD counterparts and these differences persisted in the subset analysis limited to female patients only., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Despite modern immunosuppressive therapy, allograft rejection remains a major cause of solid organ transplant dysfunction. For clinical care, organ transplant function is routinely monitored by measuring biomarkers that, depending on the organ transplanted, include serum creatinine, N-terminal pro-hormone of brain natriuretic peptide (NT-proBNP), and aspartate aminotransferase. All can be measured easily in clinical chemistry laboratories. The main problem with these biomarkers is that they have a low sensitivity for the detection of allograft damage and are nonspecific for the detection of allograft rejection. To diagnose rejection, histologic examination of grafted tissue is necessary, which requires an invasive biopsy procedure. There is thus an unmet need in transplantation medicine for biomarkers that are specific for rejection, identify graft injury at an early stage, and may eventually overcome the need for a transplant biopsy. Recently, tremendous progress in the field of biomarkers has been made. In this narrative review, the potential of donor-derived cell-free DNA (ddcfDNA), cell-free nucleosomes, and extracellular vesicles to act as next-generation biomarkers for solid organ transplant is discussed. Based on the fact that cell content is released during rejection, these markers could serve as very specific biomarkers for allograft injury and rejection. These markers have the potential to improve rejection monitoring, evaluate the response to antirejection therapy, and may decrease the need for invasive procedures.

Background: Acute rejection is hazardous to graft survival in kidney transplant recipients (KTRs). We aimed to identify novel biomarkers for early diagnosis of acute T cell-mediated rejection (TCMR) in urinary exosomes of KTRs., Methods: Among 458 graft biopsies enrolled in a cross-sectional multicenter study, 22 patients with stable graft function (STA) who had not shown pathologic abnormality and 25 patients who diagnosed biopsy-proven TCMR were analyzed. We performed proteomic analysis using nano-ultra performance liquid chromatography-tandem mass spectrometry (nano-UPLC-MS/MS) to identify candidate biomarkers for early TCMR diagnosis on urinary exosomes. We confirmed the protein levels of each candidate biomarker by western blot analysis., Results: A total of 169 urinary exosome proteins were identified by nano-UPLC-MS/MS. Forty-six proteins showed increased expression in STA patients, while 17 proteins were increased in TCMR patients. Among them, we selected five proteins as candidate biomarkers for early diagnosis of TCMR according to significance, degree of quantity variance, and information from the ExoCarta database. We confirmed the proteomic expression levels of five candidate biomarkers by western blot analysis in each patient. Of all candidate biomarkers, tetraspanin-1 and hemopexin were significantly higher in TCMR patients (STA:TCMR ratio = 1:1.8, P = 0.009, and 1:3.5, P = 0.046, respectively)., Conclusions: Tetraspanin-1 and hemopexin were detected in KTR urine and could act as potential diagnostic proteins for TCMR., Competing Interests: The authors have declared that no competing interests exist.

Activation of the local renin-angiotensin system (RAS) is an independent risk factor for the development of proteinuria and left ventricular hypertrophy (LVH) more commonly seen in masked hypertensives. It has been reported that urinary angiotensinogen (UAGT) level provides a specific index of the intrarenal RAS status. The aim of this study was to evaluate the association between UAGT and left ventricular mass index (LVMI) and urinary albumin-creatinine ratio (UACR) in renal transplant recipients (RTRs) with masked hypertension (HT). A total of 116 non-diabetic-treated hypertensive RTRs were included in this study. The patients were divided into two groups: masked hypertensives and controlled hypertensives. Forty-two (36.2%) of RTRs had masked HT. Mean UACR and LVMI levels were higher in RTRs with masked HT than in RTRs with controlled HT (P < 0.001). UAGT level was also higher in masked hypertensives compared to controlled hypertensives (P < 0.001). Multivariable regression analysis showed that UAGT was positively correlated with UACR (β = 0.024, P = 0.001) and LVMI (β = 0.082, P = 0.001) in masked hypertensives. Consequently, masked HT was considerably frequent (36.2%) in treated hypertensive RTRs and high UAGT levels accompanied by high albuminuria and LVMI levels were seen in these patients. Overproduction of the UAGT may play a pivotal role in the development of LVH and proteinuria in masked hypertensives., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Allograft rejection is still an important complication after kidney transplantation. Currently, monitoring of these patients mostly relies on the measurement of serum creatinine and clinical evaluation. The gold standard for diagnosing allograft rejection, i.e. performing a renal biopsy is invasive and expensive. So far no adequate biomarkers are available for routine use., Objectives: We aimed to develop a urine metabolite constellation that is characteristic for acute renal allograft rejection., Methods: NMR-Spectroscopy was applied to a training cohort of transplant recipients with and without acute rejection., Results: We obtained a metabolite constellation of four metabolites that shows promising performance to detect renal allograft rejection in the cohorts used (AUC of 0.72 and 0.74, respectively)., Conclusion: A metabolite constellation was defined with the potential for further development of an in-vitro diagnostic test that can support physicians in their clinical assessment of a kidney transplant patient.

Pediatric kidney transplantation is lifesaving, but long-term allograft survival is still limited by injury processes mediated by alloimmune inflammation that may otherwise be clinically silent. Chemokines associated with alloimmune inflammation may offer prognostic value early post-transplant by identifying patients at increased risk of poor graft outcomes. We conducted a single-center prospective cohort study of consecutive pediatric kidney transplant recipients (<19 years). Urinary CCL2 and CXCL10 measured at 6 months post-transplant were evaluated for association with long-term eGFR decline, allograft survival, and concomitant acute cellular rejection histology. Thirty-eight patients with a mean age of 12.4 ± 4.6 years were evaluated. Urinary CCL2 was associated with eGFR decline until 6 months (ρ -0.43; P < .01), but not at later time points. Urinary CXCL10 was associated with eGFR decline at 36 months (ρ -0.49; P < .01), risk of 50% eGFR decline (HR = 1.04; P = .02), risk of allograft loss (HR = 1.05; P = .01), borderline rejection or rejection episodes 6-12 months post-transplant (r .41; P = .02), and Banff i + t score (r .47, P < .01). CCL2 and CXCL10 were also correlated with one another (ρ 0.54; P < .01). CCL2 and CXCL10 provide differing, but complementary, information that may be useful for early non-invasive prognostic testing in pediatric kidney transplant recipients., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Noninvasive monitoring methods of immune status are preferred by transplant recipients. The present study investigated whether urinary C‑X‑C motif chemokine 13 (CXCL13) had the potential to reflect ongoing immune processes within renal allografts. Using an ELISA assay, the level of urinary CXCL13 was quantified in a total of 146 renal allograft recipients and 40 healthy controls at scheduled intervals and at the time of the indicated or protocol biopsy. The results of the present study revealed that urinary CXCL13/creatinine (Cr) was lower in normal transplants compared with in those with acute tubular necrosis (ATN; P=0.001), chronic allograft nephropathy (CAN; P=0.01), and acute rejection (AR; P<0.0001), which was associated with a good diagnostic performance for AR [area under the curve (AUC)=0.818, P<0.0001). In addition, urinary CXCL13/Cr levels in patients with AR were also higher than that of patients with graft dysfunction but no rejection, including ATN and CAN (P=0.034). Notably, urinary CXCL13 distinguished between acute antibody‑mediated rejection (ABMR) and acute cellular rejection, with an AUC of 0.856. Furthermore, patients with steroid‑resistant AR exhibited significantly increased urinary CXCL13/Cr levels than patients with reversible AR (P=0.001). Additionally, elevated levels of urinary CXCL13/Cr within the first month of transplant were predictive of graft function at 3 and 6 months (P=0.044 and P=0.04, respectively). Collectively, the findings of the present study indicated that the noninvasive investigation of urinary CXCL13/Cr may be valuable for the detection of AR, particularly ABMR. In addition, high urinary CXCL13/Cr levels predicted a poor response to steroid treatment and compromised graft function.

Urinary CXCL10 and metabolites are biomarkers independently associated with TCMR. We sought to test whether these biomarkers fluctuate in association with histological severity of TCMR over short time frames. Forty-nine pairs of renal biopsies obtained 1-3 months apart from 40 pediatric renal transplant recipients were each scored for TCMR acuity score (i + t; Banff criteria). Urinary CXCL10:Cr and TCMR MDS were obtained at each biopsy and were tested for association with changes between biopsies in acuity, estimated GFR (ΔeGFR), and 12-month ΔeGFR. Sequential biopsies were obtained 1.8 ± 0.8 months apart. Biopsy 1 was usually obtained under protocol (75%), and 62% percent had evidence of TCMR. Using each biopsy pair for comparison, ΔeGFR did not predict change in acuity. By contrast, change in acuity was significantly correlated with change in urinary CXCL10:Cr (ρ 0.45, P = .003) and MDS (ρ 0.29, P = .04) between biopsies. The 12-month ΔeGFR was not predicted by TCMR acuity or CXCL10:Cr at Biopsy 2; however, an inverse correlation was seen with urinary MDS (ρ -0.35; P = .02). Changes in eGFR correlate poorly with evolving TCMR acuity on histology. Urinary biomarkers may be superior for non-invasive monitoring of rejection, including histological response to therapy, and may be prognostic for medium-term function., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Acute renal allograft rejection is a common complication after renal transplantation that often leads to chronic rejection and ultimate graft loss. While renal allograft biopsy remains the gold standard for diagnosis of acute rejection, the possibility of biopsy-associated complications cannot be overlooked. The development of noninvasive methods for accurate detection of acute renal allograft rejection is thus of significant clinical importance., Methods: Gas chromatography-mass spectrometry (GC/MS) was employed for analysis of urine metabolites in 15 renal allograft recipients with acute rejection and 15 stable renal transplant recipients. Partial least squares (PLS) regression and leave-one-out analyses were performed to ascertain whether the metabolites identified could be exploited to distinguish acute rejection from stable groups as well as their sensitivity and specificity., Results: Overall, 14 metabolites were significantly altered in the acute rejection group (11 and 3 metabolites displayed higher and lower levels, respectively) relative to the stable transplant group. Data from PLS and leave-one-out analyses revealed that the differential metabolites identified not only distinguished acute rejection from stable transplant recipients but also showed high sensitivity and specificity for diagnosis of renal allograft recipients with acute rejection., Conclusion: Urine metabolites identified with GC/MS can effectively distinguish acute rejection from stable transplant recipients, supporting the potential utility of metabolome analysis in non-invasive diagnosis of acute rejection.