Urinary mRNA Research Articles

Urinary sediment mRNA as a potent biomarker of IgA nephropathy

BackgroundThe quantification of mRNA expression in urinary sediments is a reliable biomarker for various diseases. However, few studies have investigated the clinical relevance of urinary mRNA levels in IgA nephropathy (IgAN). Thus, we investigated the expression of urinary mRNAs and their clinical significance in IgAN.MethodsOverall, 200 patients with biopsy-proven IgAN, 48 disease controls, and 76 healthy controls were enrolled. We identified the differential expression of mRNAs in renal tissue between patients with IgAN and normal subjects using the Gene Expression Omnibus dataset and selected candidate mRNAs. mRNA expression in the urinary sediment was measured using quantitative real-time polymerase chain reaction. Associations between urinary mRNA levels and clinicopathological parameters were analyzed and the predictive value of mRNAs for disease progression was evaluated.ResultsThe urinary expression of CCL2, CD14, DNMT1, FKBP5, Nephrin, and IL-6 was significantly upregulated in patients with IgAN compared with healthy controls. C3, FLOT1, and Podocin levels were significantly correlated with renal function, where C3, FLOT1, and TfR levels were significantly correlated with urinary protein excretion. During follow-up, 26 (13.0%) patients with IgAN experienced disease progression, defined as a greater than 50% reduction in the estimated glomerular filtration rate or progression to end-stage renal disease. Urinary mRNA levels of FLOT1 (HR 3.706, 95% CI 1.373–10.005, P = 0.010) were independently associated with an increased risk of disease progression.ConclusionsOur results suggest that urinary sediment mRNAs are a useful biomarker in IgAN patients. Further studies with larger sample sizes and longer follow-up durations are required.

Unveiling potential: urinary exosomal mRNAs as non-invasive biomarkers for early prostate cancer diagnosis

BackgroundThis study investigated the use of urinary exosomal mRNA as a potential biomarker for the early detection of prostate cancer (PCa).MethodsNext-generation sequencing was utilized to analyze exosomal RNA from 10 individuals with confirmed PCa and 10 individuals without cancer. Subsequent validation through qRT-PCR in a larger sample of 43 PCa patients and 92 healthy controls revealed distinct mRNA signatures associated with PCa.ResultsNotably, mRNAs for RAB5B, WWP1, HIST2H2BF, ZFY, MARK2, PASK, RBM10, and NRSN2 showed promise as diagnostic markers, with AUC values between 0.799 and 0.906 and significance p values. Combining RAB5B and WWP1 in an exoRNA diagnostic model outperformed traditional PSA tests, achieving an AUC of 0.923, 81.4% sensitivity, and 89.1% specificity.ConclusionsThese findings highlight the potential of urinary exosomal mRNA profiling, particularly focusing on RAB5B and WWP1, as a valuable strategy for improving the early detection of PCa.

Over-expression of KRT17 and MDK genes at mRNA levels in urine-exfoliated cells is associated with early non-invasive diagnosis of non-muscle-invasive bladder cancer

IntroductionCurrent diagnostic approaches for bladder cancer (BLCA) are often invasive or lack the required sensitivity and specificity. This underscores the need for an early non-invasive diagnostic test for BLCA. This work aimed to explore the potential of molecular markers in urine-exfoliated cells for the diagnosis of non-muscle-invasive bladder cancer (NMIBC). Materials and methodsUrine specimens (n = 140) were collected from NMIBC patients (n = 68) and control subjects (31 healthy volunteers and 41 non-cancer patients with common urological diseases [CUD]. Total RNA was extracted from the cells isolated from urine specimens. mRNA expression of selected genes: CDC20, KRT15, FOXM1, CXCR2, UPK1B, MDK, KRT20, and KRT17 was determined using RT-qPCR. The receiver operating characteristic (ROC) curve was then plotted to obtain the area under the curve (AUC), specificity, and sensitivity of the urinary mRNA markers. ResultsThe expression of CDC20, MDK, UPK1B, FOXM1, KRT17, and KRT20 was up-regulated in samples obtained from low- and high-grade pathological grades of NMIBC compared to that measured in healthy subjects. Notably, MDK and KRT17 mRNA levels in the low- and high-grade cases were substantially higher than in normal and CUD groups. The AUC of the KRT17 and MDK combination in diagnosing NMIBC was 0.92, surpassing that of single genes. The sensitivity and specificity of the KRT17 and MDK combination were 74% and 94%, respectively. In diagnosing low-grade from healthy and CUD groups, analysis of the KRT17 and MDK combination yielded AUCs of 0.94 and 0.95, respectively, with sensitivities of 85% and 97%, and specificities of 93% and 85%. ConclusionThe findings of this study revealed that KRT17 and MDK together are potential urine-based biomarkers for early diagnosis of NMIBC.

A universal urinary cell gene signature of acute rejection in kidney allografts

IntroductionAcute 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. MethodsWe 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. ResultsThe 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). ConclusionThe urinary cell three-gene signature score discriminates AR due to TCMR, ABMR or MR from NR biopsies in human kidney allograft recipients.

Urinary exosomal mRNAs as biomarkers for predicting the therapeutic effect of renin-angiotensin system inhibitors in IgA nephropathy patients

BackgroundRenin-angiotensin system inhibitors (RASi) treatment is the basic therapy for IgA nephropathy (IgAN) patients. However, there is few of biomarker that can predict the efficacy of RASi. This study aimed to find urinary exosomal mRNAs related to the therapeutic effect of RASi in the treatment of proteinuria in IgAN patients. Methods: We divided IgAN patients in screening cohort into A1 (proteinuria increase at 3 months), B1 (proteinuria decrease less than 50 % at 3 months), C1 (proteinuria decrease more than 50 % at 3 months) groups according to changes of proteinuria after treatment. The urinary exosomes were collected before biopsy, RNAs were extracted and analyzed with the microarray assay. The candidate genes were screened by differentially expressed genes (DEGs) analysis and then validated by quantitative real-time polymerase chain reaction (qPCR) in a validation cohort. A receiver operating characteristic (ROC) curve was used to evaluate gene performance in predicting therapeutic effect on RASi reducing proteinuria in IgAN patients. Results: ECE1 and PDE1A mRNAs were significantly different among the three groups, and were gradually decreased among A1, B1 and C1 groups. In the validation cohort, the level of urinary exosomal ECE1 and PDE1A mRNAs were also significantly lower in A2 group compared with C2 group(ECE1, P < 0.001;PDE1A, P < 0.01). Besides, the level of ECE1 mRNA was also lower in B2 group compared with C2 group (P < 0.01). The ROC curve verified that urinary exosomal ECE1 and PDE1A gene level predicted RASi efficacy in IgAN patients with area under curve (AUC) 0.68 and 0.63 respectively. Conclusion: Urinary exosomal ECE1 and PDE1A mRNAs expression can serve as potential biomarkers for predicting the RASi efficacy to reduce proteinuria in IgAN patients.

#2493 Podocyturia: a novel biomarker of ageing

Abstract Background and Aims Urinary podocyte shedding (podocyturia) is a biomarker of glomerular injury in young and old individuals. Podocyturia is associated with diabetes and hypertension, and it is an early indicator of systemic cardiovascular disease. Importantly, podocyturia has also been noted in healthy adults and may indicate physiologic podocyte turnover. Whether age is a predictor of podocyte loss, independently of diabetes and hypertension, is still unclear. The aim of this study was to examine the association between age and urinary podocin levels. Method This was a secondary analysis of data from 5 studies, including 868 individuals, 18-90 years of age. Data on urinary podocin mRNA levels was present on all subjects, as measured by CFX96 Touch™ RT-PCR detection system (Biorad, USA). Descriptive statistics, Chi-square test, ANOVA, and post hoc analysis were performed. Linear regression was conducted with gender, diabetes status, and hypertension status as covariates. Significance was set at p &amp;lt; 0.05. Results The mean age of the total sample was 55.10 ± 14.84 years; 46% were female, 26.4% had diabetes, and 37.9% had hypertension. There were significant differences in mean podocin levels by age group (F = 25.27, p ≤ 0.001) as follows. Podocin levels were significantly lower in Group 1 (age 18-35 years; 0.79 ± 0.67 million copies), compared to Group 2 (36-55 years; 2.21 ± 2.15 million copies) and Group 3 (over 56 years; 2.86 ± 3.18 million copies). Moreover, mean podocin levels differed significantly between Group 2 and Group 3 (p = 0.003). Age was a predictor of podocyturia, independently of diabetes and hypertension (p &amp;lt; 0.001). Conclusion Podocyturia was present in younger adults. There was a significant rise in podocyturia in middle-aged adults (36-55 years), denoting a transitional phase. There were higher podocyturia levels in older adults compared to the youngest and middle-aged adults. These differences in podocin by age were observed while accounting for diabetes and hypertension.

Efficacy of Integrated Risk Score Using Omics-Based Biomarkers for the Prediction of Acute Rejection in Kidney Transplantation: A Randomized Prospective Pilot Study.

Acute rejection (AR) is critical for long-term graft survival in kidney transplant recipients (KTRs). This study aimed to evaluate the efficacy of the integrated risk score of omics-based biomarkers in predicting AR in KTRs. This prospective, randomized, controlled, multicenter, pilot study enrolled 40 patients who recently underwent high-immunologic-risk kidney transplantation (KT). Five omics biomarkers were measured, namely, blood mRNA (three-gene signature), urinary exosomal miRNA (three-gene signature), urinary mRNA (six-gene signature), and two urinary exosomal proteins (hemopexin and tetraspanin-1) at 2 weeks and every 4 weeks after KT for 1 year. An integrated risk score was generated by summing each biomarker up. The biomarker group was informed about the integrated risk scores and used to adjust immunosuppression, but not the control group. The outcomes were graft function and frequency of graft biopsy. Sixteen patients in the biomarker group and nineteen in the control group completed the study. The mean estimated glomerular filtration rate after KT did not differ between the groups. Graft biopsy was performed in two patients (12.5%) and nine (47.4%) in the biomarker and control groups, respectively, with the proportion being significantly lower in the biomarker group (p = 0.027). One patient (6.3%) in the biomarker group and two (10.5%) in the control group were diagnosed with AR, and the AR incidence did not differ between the groups. The tacrolimus trough level was significantly lower in the biomarker group than in the control group at 1 year after KT (p = 0.006). Integrated omics biomarker monitoring may help prevent unnecessary or high-complication-risk biopsy and enables tailored immunosuppression by predicting the risk of AR in KTRs.

474 An Investigation of Novel Urinary Cell mRNA Profiles for Noninvasive Diagnosis of Acute Rejection in Kidney Transplant Recipients

OBJECTIVES/GOALS: RNA-seq of urine and kidney allograft biopsies (bx) found that urinary cell immune landscape reflects intragraft molecular events and we discovered a shared set of 127 mRNAs in urine matched to T cell mediated and antibody mediated rejection bx. We prioritized ITM2A, SLAMF6 and IKZF3 mRNAs and herein investigate if these accurately predict rejection. METHODS/STUDY POPULATION: We collected urine samples from adult kidney allograft (KA) recipients at the time of KA bx. KA bx were classified by pathologists by Banff criteria. Total RNA was isolated from KA bx-matched urine samples. Absolute copy numbers of ITM2A, SLAMF6, and IKZF3 mRNAs and 18S rRNA were measured using our customized RT-qPCR assays. Logistic regression used to derive an equation for a combined signature score of 18S-normalized urinary cell mRNA levels of ITM2A, IKZF3, and SLAMF6 that best predicts Acute Rejection (AR= both T cell mediated rejection and antibody mediated rejection). Area under the ROC curve (AUC) was calculated to discriminate between AR and No Rejection (NR) biopsies for 18S-normalized urinary cell levels of ITM2A, IKZF3 and SLAMF6 and the composite signature score. AUCs were compared by DeLong Method. RESULTS/ANTICIPATED RESULTS: Urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNAs in urine discriminated KA recipients with AR biopsies (n=95) from those with NR biopsies (n=160) (All P values &lt;0.05, Mann-Whitney test) and the AUC was 0.69 (95%CI, 0.62 to 0.76) for ITM2A, 0.61 (95%CI, 0.53 to 0.68) for IKZF3, and 0.60 (95%CI, 0.53 to 0.68) for SLAMF6. The derived combination signature score of urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNA discriminated KA recipients with AR from those with NR (P&lt;0.0001, Mann Whitney test) and the combined signature score AUC was 0.72 (95%CI, 0.65 to 0.79). The combination signature score discriminated AR vs NR better than IKZF3 and SLAMF6 alone, but was not significantly different than ITM2A alone (DeLong method). (Additional results/figures to be included in poster) DISCUSSION/SIGNIFICANCE: Our RNA-seq offered a unique opportunity to diagnose AR by measuring the mRNAs in urine. We now found that urinary cell mRNA levels of ITM2A, IKZF3, SLAMF6 and the combined signature are diagnostic of AR, a major and serious post-transplant complication. This allows for much-needed KA molecular surveillance and personalization of immunosuppression.

Cross-sectional and longitudinal analyses of urinary extracellular vesicle mRNA markers in urothelial bladder cancer patients

We designed this multi-center prospective study with the following objectives: (1) the cross-sectional validation of extracellular vesicles (EV) mRNA markers to detect urothelial bladder cancer (UBC) before transurethral resection of bladder cancer (TURBT), and (2) the longitudinal validation of EV mRNA markers to monitor non-muscle invasive bladder cancer (NMIBC) recurrence after TURBT. EV mRNA markers evaluated in this study were KRT17, GPRC5A, and SLC2A1 in addition to two additional markers from literatures, MDK and CXCR2, and measured by quantitative RT-PCR with normalization by a reference gene (ALDOB). Diagnostic performances of EV mRNA markers were compared to conventional markers. Regarding the first objective, we confirmed that EV mRNA biomarkers in urine were higher in UBC patients, particularly those with higher stage/grade tumors, than in those without UBC (n = 278 in total) and the diagnostic performance of EV mRNA MDK and KRT17 outperformed conventional biomarkers with AUC 0.760 and 0.730, respectively. Concerning the second objective, we prospectively analyzed the time courses of EV mRNA markers while NMIBC patients (n = 189) (median follow-up 19 months). The expression of EV mRNA KRT17 was significantly high in patients with recurrence, while it gradually decreased over time in those without recurrence (p < 0.01).

Urinary exosomal mRNA as a biomarker for the diagnosis of bladder cancer.

To study the diagnostic value of mRNA expression in urinary exocrine body in bladder cancer. From February 2022 to December 2022, 60 patients diagnosed with bladder cancer by pathology in the Department of Urology, Affiliated Hospital of Chengde Medical University were selected as the case group. In total, 40 healthy subjects receiving physical examinations were selected as the control group. 100 mL of morning urine samples were collected from the subjects in both groups based on the same standard. Three subjects were randomly selected from each group. Urinary exosomes were extracted by differential ultracentrifugation. High-throughput sequencing (RNA-seq) was used to detect mRNA expression profiles in urinary exosomes and identify differentially expressed genes. Bioinformatic analysis was performed to predict major biological functions of differentially expressed genes and related signaling pathways. RT-PCR validated expression levels of differentially expressed genes in urinary exosomes between the two groups. ROC curves evaluated the diagnostic value of differential genes for bladder cancer. Spearman's correlation analysis determined correlations between differentially expressed genes and the occurrence of bladder cancer. ROC curves speculated the diagnostic value of using combined differentially expressed genes. Compared with normal subjects, there were 189 significantly differentially expressed genes in urinary exosomes of bladder cancer patients, including 33 up-regulated and 156 down-regulated. According to go and kyoto encyclopedia of genes and genomes (KEGG) analysis, the above differentially expressed genes may participate in the occurrence and development of bladder cancer through the MAPK pathway, PPAP signaling pathway, PI3K Akt signaling pathway and Hippo signaling pathway, affect protein and lipid metabolism, RNase activity, polysaccharide synthesis, signal transduction and other biological processes, and participate in cell proliferation, death, movement and adhesion, as well as cell differentiation and signal transduction. RT-PCR verified that the expression of tmeff1, SDPR, ACBD7, SCG2 and COL6A2 in the two groups of samples was statistically significant ( P < 0.05). The ROC curve showed that the area under curve area under the curve of the five differential genes were 0.6934, 0.7746, 0.7239, 0.6396 and 0.6610, respectively. The sensitivity was 42.11%, 64.86%, 47.37%, 73.53% and 76.47%, and the specificity was 90%, 81.36%, 96.36%, 61.02% and 58.18%, respectively. Spearman correlation analysis showed that tmeff1, SDPR and acbd7 were associated with the occurrence of bladder cancer. The ROC curve of the combined diagnosis of the three and the two combined diagnoses suggested that the area under the curve of the combined diagnosis of SDPR and acbd7 was 0.7945, the sensitivity was 89.09%, and the specificity was 60.53%. The gene expression profile in urinary exosomes of bladder cancer patients has changed significantly, and the differential genes may play an important biological role in the occurrence and development of bladder cancer. The combined detection of urinary exosome SDPR and ACBD7 has a certain diagnostic value for bladder cancer.

Exosomal UMOD gene expression and urinary uromodulin level as early noninvasive diagnostic biomarkers for diabetic nephropathyin type 2 diabetic patients.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Exosomes are promising biomarkers for disease diagnosis and uromodulin is a kidney-specific protein. So, this study was designed to investigate the change in the gene expression of urinary exosomal uromodulin mRNA and urinary uromodulin level and determine the diagnostic potential of these noninvasive biomarkers in the early stage of diabetic nephropathy in type 2 diabetic patients. This study included 100 participants; urinary exosomes were isolated using polyethylene glycol (PEG). Gene expression of exosomal uromodulin mRNA was determined by quantitative real-time polymerase chain reaction (q-RT-PCR). The urinary uromodulin levels were determined by an enzyme-linked immunosorbent assay (ELISA). In this study, the gene expression of exosomal uromodulin (UMOD) mRNA and the level of urinary uromodulin showed a significant increase in all diabetic groups with and without nephropathy compared to the control group. The exosomal UMOD mRNA showed a significant positive correlation with urinary uromodulin in all groups. Multiple logistic regression showed that urinary uromodulin was an independent determinant for DN. A diagnostic model of two indicators, exosomal UMOD mRNA and urinary uromodulin, can significantly predict DN. The area under the curve is 0.095, with a 95% confidence interval of 0.98-1, and 0.81, with a 95% confidence interval of 0.69-0.92, for the exosomal UMOD mRNA and urinary uromodulin, respectively. Urinary exosomal mRNA of UMOD and urinary uromodulin levels are progressively elevated in an early stage of DN, even before the microalbuminuria stage, so they could be used as early predictors for DN.

Urinary podocyte stress marker as a prognostic indicator for diabetic kidney disease

BackgroundDiabetic kidney diseases (DKD) is a the most common cause of end-stage kidney disease (ESKD) around the world. Previous studies suggest that urinary podocyte stress biomarker, e.g. podocin:nephrin mRNA ratio, is a surrogate marker of podocyte injury in non-diabetic kidney diseases.MethodWe studied 118 patients with biopsy-proved DKD and 13 non-diabetic controls. Their urinary mRNA levels of nephrin, podocin, and aquaporin-2 (AQP2) were quantified. Renal events, defined as death, dialysis, or 40% reduction in glomerular filtration rate, were determined at 12 months.ResultsUrinary podocin:nephrin mRNA ratio of DKD was significantly higher than the control group (p = 0.0019), while urinary nephrin:AQP2 or podocin:AQP2 ratios were not different between groups. In DKD, urinary podocin:nephrin mRNA ratio correlated with the severity of tubulointerstitial fibrosis (r = 0.254, p = 0.006). and was associated with the renal event-free survival in 12 months (unadjusted hazard ratio [HR], 1.523; 95% confidence interval [CI] 1.157–2.006; p = 0.003). After adjusting for clinical and pathological factors, urinary podocin:nephrin mRNA ratio have a trend to predict renal event-free survival (adjusted HR, 1.327; 95%CI 0.980–1.797; p = 0.067), but the result did not reach statistical significance.ConclusionUrinary podocin:nephrin mRNA ratio has a marginal prognostic value in biopsy-proven DKD. Further validation is required for DKD patients without kidney biopsy.

Potential role for vitamin D vs. intermittent fasting in controlling aquaporin-1 and aquaporin-3 expression in HFD-induced urinary bladder alterations in rats.

Background: High-fat diet-induced obesity is linked to suppression of aquaporins (AQPs) expression in different tissues. Both vitamin D and intermittent fasting were identified to enhance AQPs expression. In the urinary bladder, AQP-1 and AQP-3 mRNA transcripts were identified. Vitamin D has an impact on a variety of genes that encode proteins that control cell proliferation, differentiation, and death. Aim: To assess potential benefits of vitamin D and intermittent fasting (IF) and to explore alterations to the urinary bladder triggered by high-fat diet (HFD) in a rat model of obesity. Methods: Each of the 4 groups contained six adult male albino rats; control: a standard rodent chew for 12 weeks, HFD: HFD and fructose were administered orally via gastric gavage for 12 weeks, and vitamin D: HFD and fructose were administered orally for 8 weeks, then 4 weeks of intraperitoneal injection of vitamin D (5 microns/Kg/2 days) and IF group: Received intraperitoneal injections of vitamin D (5 microns/Kg/2 days) for 4 weeks after consumption of HFD and fructose orally for 8 weeks. The serum lipid profile was conducted at end of the experiment. In the bladder homogenates, the levels of oxidative stress indicators were assessed. Quantitative real-time PCR was performed on recently collected bladder samples. AQP-1 and AQP-3 immunohistochemistry was done. Results: When compared to the HFD group, the vitamin D and IF groups both demonstrated a substantial improvement in histopathological, immunohistochemical, biochemical, and molecular markers. Conclusion: In all examined parameters, IF exceeded vitamin D as a preventive factor for the urinary bladder deterioration.

Urinary Sediment Podocin mRNA Excretion in Healthy Adults

Urinary Sediment Podocin mRNA Excretion in Healthy Adults

A Systematic Investigation on the Impact of Invasive Kidney Allograft Biopsy on Urinary Cell mRNA Profiles

A Systematic Investigation on the Impact of Invasive Kidney Allograft Biopsy on Urinary Cell mRNA Profiles

Urinary mRNA Profile in Recipients of Allogeneic Hematopoietic Cell Transplant with AKI

Urinary mRNA Profile in Recipients of Allogeneic Hematopoietic Cell Transplant with AKI

Evaluation of Immunocompetence and Biomarkers of Tolerance in Chimeric and Immunosuppression-free Kidney Allograft Recipients.

Thirty-seven patients have received a living-donor kidney transplant in a phase 2 study designed to induce tolerance with facilitated allogeneic hematopoietic stem cell transplant. The study protocol is based on tolerogenic CD8 + /T-cell receptor - facilitating cells (FCR001; also including hematopoietic stem cells and αβ-T-cell receptor + T cells) and low-dose, nonmyeloablative conditioning. Persistent chimerism allowing full immunosuppression (IS) withdrawal was achieved in 26 patients (time off IS 36-123 mo). We evaluated biomarkers of tolerance through urinary cell mRNA profiling and immunocompetence to respond to vaccination in these patients. We also assessed kidney function and metabolic parameters compared with standard-of-care patients on IS. Persistently chimeric patients retained chimerism after removal of IS and remained rejection free without donor HLA-specific antibody development. The presence of donor chimerism at >50% correlated with a signature of tolerance in urinary cell mRNA profiles, with a uniquely elevated increase in the ratio of cytotoxic T lymphocyte-associated protein 4 to granzyme B mRNA. Tolerance was associated with protection from recurrence of immune-mediated causes of kidney disease. Tolerant participants were safely vaccinated, developed protective immune responses, and did not lose chimerism after vaccination. When compared with kidney transplant recipients treated with standard IS, tolerant participants showed stable kidney function and reduced medication use for hypertension and hyperlipidemia. These results suggest that elimination of IS has distinct advantages in living-donor kidney allograft recipients.

Development and nationwide validation of kidney graft injury markers using urinary exosomes and microvesicles (complete English translation of the Japanese version)

BackgroundNon-invasive, prompt, and proper detection tools for kidney graft injuries (KGIs) are awaited to ensure graft longevity. We screened diagnostic biomarkers for KGIs following kidney transplantation using extracellular vesicles (EVs; exosomes and microvesicles) from the urine samples of patients.MethodsOne hundred and twenty-seven kidney recipients at 11 Japanese institutions were enrolled in this study; urine samples were obtained prior to protocol/episode biopsies. EVs were isolated from urine samples, and EV RNA markers were assayed using quantitative reverse transcription polymerase chain reaction. Diagnostic performance of EV RNA markers and diagnostic formulas comprising these markers were evaluated by comparison with the corresponding pathological diagnoses.ResultsEV CXCL9, CXCL10, and UMOD were elevated in T-cell-mediated rejection samples compared with other KGI samples, while SPNS2 was elevated in chronic antibody-mediated rejection (cABMR) samples. A diagnostic formula developed through Sparse Logistic Regression analysis using EV RNA markers allowed us to accurately (with an area under the receiver operator characteristic curve [AUC] of 0.875) distinguish cABMR from other KGI samples. EV B4GALT1 and SPNS2 were also elevated in cABMR, and a diagnostic formula using these markers was able to distinguish between cABMR and chronic calcineurin toxicity accurately (AUC 0.886). In interstitial fibrosis and tubular atrophy (IFTA) urine samples and those with high Banff chronicity score sums (BChS), POTEM levels may reflect disease severity, and diagnostic formulas using POTEM detected IFTA (AUC 0.830) and high BChS (AUC 0.850).ConclusionsKGIs could be diagnosed with urinary EV mRNA analysis with relatively high accuracy.

319 Optimizing Urinary cell mRNA profiling of kidney allograft recipients: Development of a home processing protocol for noninvasive diagnosis of T cell mediated rejection and BK virus nephropathy

OBJECTIVES/GOALS: Development of a user friendly home kit that enables kidney transplant recipients to process urine at home and post the lysate containing RNA to a Core Laboratory would simplify urinary cell mRNA profiling and facilitate longitudinal monitoring. We report our home processing protocol and investigation of its diagnostic performance characteristics. METHODS/STUDY POPULATION: We developed a home processing protocol (HPP) consisting of urine filtration and lysis of urinary cells, both performed at home by the kidney transplant recipients (KTR) themselves, followed by isolation of total RNA from the lysate and mRNA enrichment using a silica-membrane-based cartridge, both performed at the Core Laboratory. Using the HPP, total RNA was isolated from kidney allograft biopsy-matched urines and absolute copy numbers of CD3εmRNA, CXCL10 mRNA, and 18S rRNA, components of the Clinical Trials in Organ Transplantation 04 (CTOT-04) three-gene TCMR diagnostic signature, and urinary cell BKV VP1 mRNA copy number, were measured using customized RT-qPCR assays. RESULTS/ANTICIPATED RESULTS: CTOT-04 three-gene TCMR diagnostic signature scores in urine processed using HPP discriminated KTR with TCMR (12 TCMR biopsies from 11 KTR) from KTR with no TCMR/BKVN (29 No TCMR/No BKVN biopsies from 29 KTR) (P=0.0005, Mann-Whitney test), and AUROC was 0.84 (95% CI, 0.69 to 0.98). TCMR was diagnosed with 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 urine processed with HPP discriminated KTR with BKVN (n=7) from KTR with no TCMR/BKVN (n=29) and AUROC was 1.0 (95% CI, 1.00 to 1.00). BKVN was diagnosed with a sensitivity of 86% (95% CI, 42 to 99) at specificity of 100% (95% CI, 85 to 100) with the previously validated cutpoint of 6.5x10^8 BKV VP1 mRNA copies/μg of RNA (P DISCUSSION/SIGNIFICANCE: Urine processed using the HPP predicted TCMR and BKVN in KTR. The HPP represents not only a significant advance towards portability of urinary cell mRNA profiling but also should improve patient management by minimizing visits for urine collection.

MP63-02 PROSPECTIVE MULTI-INSTITUTIONAL VALIDATION OF a 3-GENE mRNA BIOMARKER PANEL FOR DETECTION AND SURVEILLANCE OF NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP63-02 PROSPECTIVE MULTI-INSTITUTIONAL VALIDATION OF a 3-GENE mRNA BIOMARKER PANEL FOR DETECTION AND SURVEILLANCE OF NON-MUSCLE INVASIVE BLADDER CANCER Zachary Kornberg, Eugene Shkolyar, Kathleen E. Mach, Timothy J. Lee, Vinh LA, Ihna Yoo, Gabriela Rodriguez, Amy D. Dobberfuhl, Eila C. Skinner, John T. Leppert, and Joseph C. Liao Zachary KornbergZachary Kornberg More articles by this author , Eugene ShkolyarEugene Shkolyar More articles by this author , Kathleen E. MachKathleen E. Mach More articles by this author , Timothy J. LeeTimothy J. Lee More articles by this author , Vinh LAVinh LA More articles by this author , Ihna YooIhna Yoo More articles by this author , Gabriela RodriguezGabriela Rodriguez More articles by this author , Amy D. DobberfuhlAmy D. Dobberfuhl More articles by this author , Eila C. SkinnerEila C. Skinner More articles by this author , John T. LeppertJohn T. Leppert More articles by this author , and Joseph C. LiaoJoseph C. Liao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003321.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Risk stratification for patients with non-muscle invasive bladder cancer (NMIBC) is essential for treatment planning. Urinary biomarkers based on mRNA are a promising non-invasive modality for potential augmentation of current detection and surveillance methods. Previously we reported a 3-gene mRNA panel strongly associated with bladder cancer (BCa) showing promise as a screening and surveillance assay. Here we validate the performance of this assay prospectively in a multi-institution cohort to further demonstrate its potential utility in the management of NMIBC. METHODS: Urine samples were collected from patients undergoing BCa screening and surveillance between 2016 and 2022 at Stanford University and the Veterans Affairs Palo Alto Health Care System. Patients with lesions concerning for bladder cancer on cystoscopy underwent transurethral resection, with stage and grade determined by histopathologic diagnosis. BCa incidence were categorized by AUA risk stratification. Voided urine samples were collected, centrifuged, and RNA was isolated from the pellet. qPCR was performed to evaluate expression of ROBO1, CRH, and IGF2. Assay positivity was evaluated via previously published formula, and sensitivity and specificity for the detection of any BCa, as well as increased-risk disease (AUA intermediate and high-risk NMIBC), were determined. RESULTS: 216 patients provided urine samples at time of diagnostic cystoscopy, and 233 patients provided 587 samples at surveillance cystoscopies. In the detection cohort, 88 new cancers were diagnosed. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the biomarker panel for any BCa was 1, 0.43, 0.53, and 1 respectively. In the surveillance cohort 189 recurrences occurred. For detection of any recurrence, the sensitivity, specificity, NPV, and PPV of the biomarker panel were 0.97, 0.24, 0.41, and 0.96, and for increased-risk disease, were 0.98, 0.25, 0.36, and 0.96 respectively. Using this assay, 59 cystoscopies (25%) could have been avoided in the detection group without missing a single incidence of BCa, and 96 cystoscopies (17%) could have been avoided in the surveillance group while missing only 4 increased-risk BCas. CONCLUSIONS: The 3-gene urinary mRNA biomarker panel shows high sensitivity for detection of NMIBC in an extended patient cohort. With further validation in prospective trials, this panel may facilitate biomarker-informed BCa screening and surveillance. Source of Funding: Department of Veterans Affairs BLR&D Merit Review I01 BX004962 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e871 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Zachary Kornberg More articles by this author Eugene Shkolyar More articles by this author Kathleen E. Mach More articles by this author Timothy J. Lee More articles by this author Vinh LA More articles by this author Ihna Yoo More articles by this author Gabriela Rodriguez More articles by this author Amy D. Dobberfuhl More articles by this author Eila C. Skinner More articles by this author John T. Leppert More articles by this author Joseph C. Liao More articles by this author Expand All Advertisement PDF downloadLoading ...