KLHDC7B as a novel diagnostic biomarker in urine exosomal mRNA promotes bladder urothelial carcinoma cell proliferation and migration, inhibits apoptosis.

Bladder cancer is a common kind of urinary system cancer, in which bladder urothelial carcinoma (BLCA) comprises approximately 90% of all bladder cancer types. In our previous study, we discovered KLHDC7B in urine exosomal messenger RNA (mRNA) as a prospective molecular marker for bladder cancer detection. To systematically study the role and mechanism of KLHDC7B in BLCA, we focused on the most common type of BLCA in this study. First, we used RNA sequencing to discover that KLHDC7B was considerably increased in BLCA patients' urine exosomes compared to healthy controls. Then, we validated this result in an independent cohort and identified it as an effective tool for diagnosing and distinguishing high-grade and low-grade BLCA. Finally, we studied the role and mechanism of KLHDC7B in BLCA at the cellular level, providing a functional basis for its expression as a novel laboratory diagnostic biomarker for BLCA exosomal mRNA, which has important theoretical and clinical significance.

Lymphocyte migration regulation related proteins in urine exosomes may serve as a potential biomarker for lung cancer diagnosis.

BACKGROUND: The migration of lymphocytes shares many similarities in mode and mechanism with the metastasis of lung cancer tumor cells. But changes in the expression of lymphocyte migration regulation related proteins in urine exosomes remain unclear. This study is to investigate the expression changes of lymphocyte migration regulation related proteins in urine exosomes of lung cancer patients, and further verify their correlation with the development and progression of lung cancer. METHODS: Urine exosomes were collected from lung cancer patients and healthy people aged 15-79 years. Mass spectrometry was used to screen and explore the expression changes of lymphocyte migration regulation related proteins in healthy people of different ages. Enzyme-linked immunosorbent assay and western blotting were used to detect the expression changes of lymphocyte migration regulation related proteins in lung cancer patients. RESULTS: Analyzing the data of urine exosome proteomics, a total of 12 lymphocyte related proteins were identified, 5 of which were lymphocyte migration regulation related proteins. Among these proteins, WASL and STK10 proteins showed a gradual decrease in expression with age, and WNK1 protein showed a gradual increase. Lung cancer patients had reduced expression of WASL and increased expression of STK10 and WNK1 proteins in urine exosomes compared to normal people. Urine exosome WASL, STK10, and WNK1 were diagnosed with lung cancer, with a combined AUC of 0.760. CONCLUSIONS: Lymphocyte migration regulation related proteins were differentially expressed in the urine exosome of lung cancer patients, and WASL, STK10 and WNK1 may serve as potential biomarkers for lung cancer diagnosis.

Contribution of thick ascending limb and distal convoluted tubule to glucose-induced hypercalciuria in healthy controls.

Carbohydrates increase kidney stone risk and increase urine calcium and magnesium. We hypothesize that the effects of glucose as an allosteric modulator of calcium-sensing receptors may mediate this effect. Six healthy subjects were on a low-sodium diet before consuming 100 g of glucose beverage. Timed fasting (3) and postglucose (6) urine and blood samples were collected every 30 min. Urine composition and serum markers were measured and microvesicular abundance of tubular transport proteins (NHE3, NKCC2, NCC, and TRPV5) were quantified. Postglucose, serum glucose, and insulin rose rapidly with a parallel increase in calcium and magnesium excretion and no change in fractional excretion of sodium. Both serum parathyroid hormone (PTH) and urine TRPV5 fell in the postglucose periods. The rise in the calcium and magnesium excretion likely occurred primarily in the thick ascending limb where they are both under control of the calcium-sensing receptor. The fall in PTH and TRPV5 support the role of glucose as an allosteric modulator of calcium-sensing receptor.NEW & NOTEWORTHY Sugar increases urine calcium and magnesium as well as kidney stone and bone disease risk. Our study provided new insights into the underlying mechanism as we gave healthy subjects an oral glucose load and used newer tools such as fractional excretion of lithium, serum parathyroid hormone, and microvesicular abundance of tubular transport proteins to characterize the mechanism and identify the thick ascending limb with possible calcium-sensing receptor mediation as a likely contributor to this mechanism.

Identification of a Non-Invasive Urinary Exosomal Biomarker for Diabetic Nephropathy Using Data-Independent Acquisition Proteomics.

Diabetic nephropathy (DN), as the one of most common complications of diabetes, is generally diagnosed based on a longstanding duration, albuminuria, and decreased kidney function. Some patients with the comorbidities of diabetes and other primary renal diseases have similar clinical features to DN, which is defined as non-diabetic renal disease (NDRD). It is necessary to distinguish between DN and NDRD, considering they differ in their pathological characteristics, treatment regimes, and prognosis. Renal biopsy provides a gold standard; however, it is difficult for this to be conducted in all patients. Therefore, it is necessary to discover non-invasive biomarkers that can distinguish between DN and NDRD. In this research, the urinary exosomes were isolated from the midstream morning urine based on ultracentrifugation combined with 0.22 µm membrane filtration. Data-independent acquisition-based quantitative proteomics were used to define the proteome profile of urinary exosomes from DN (n = 12) and NDRD (n = 15) patients diagnosed with renal biopsy and Type 2 diabetes mellitus (T2DM) patients without renal damage (n = 9), as well as healthy people (n = 12). In each sample, 3372 ± 722.1 proteins were identified on average. We isolated 371 urinary exosome proteins that were significantly and differentially expressed between DN and NDRD patients, and bioinformatic analysis revealed them to be mainly enriched in the immune and metabolic pathways. The use of least absolute shrinkage and selection operator (LASSO) logistic regression further identified phytanoyl-CoA dioxygenase domain containing 1 (PHYHD1) as the differential diagnostic biomarker, the efficacy of which was verified with another cohort including eight DN patients, five NDRD patients, seven T2DM patients, and nine healthy people. Additionally, a concentration above 1.203 µg/L was established for DN based on the ELISA method. Furthermore, of the 19 significantly different expressed urinary exosome proteins selected by using the protein-protein interaction network and LASSO logistic regression, 13 of them were significantly related to clinical indicators that could reflect the level of renal function and hyperglycemic management.

Differential expression of coagulation pathway-related proteins in diabetic urine exosomes.

BACKGROUND: Coagulation function monitoring is important for the occurrence and development of diabetes. A total of 16 related proteins are involved in coagulation, but how these proteins change in diabetic urine exosomes is unclear. To explore the expression changes of coagulation-related proteins in urine exosomes and their possible roles in the pathogenesis of diabetes, we performed proteomic analysis and finally applied them to the noninvasive monitoring of diabetes. METHODS: Subject urine samples were collected. LC-MS/MS was used to collect the information on coagulation-related proteins in urine exosomes. ELISA, mass spectrometry and western blotting were used to further verify the differential protein expression in urine exosomes. Correlations with clinical indicators were explored, and receiver operating characteristic (ROC) curves were drawn to evaluate the value of differential proteins in diabetes monitoring. RESULTS: Analyzing urine exosome proteomics data, eight coagulation-related proteins were found in this study. Among them, F2 was elevated in urine exosomes of diabetic patients compared with healthy controls. The results of ELISA, mass spectrometry and western blotting further verified the changes in F2. Correlation analysis showed that the expression of urine exosome F2 was correlated with clinical lipid metabolism indexes, and the concentration of F2 was strongly positively correlated with blood TG levels (P < 0.05). ROC curve analysis showed that F2 protein in urine exosomes had a good monitoring value for diabetes. CONCLUSION: Coagulation-related proteins were expressed in urine exosomes. Among them, F2 was increased in diabetic urine exosomes and may be a potential biomarker for monitoring diabetic changes.

Urinary exosomes derived circRNAs as biomarkers for chronic renal fibrosis.

BACKGROUND: Chronic renal disease (CKD) is a common and irreversible loss of renal function. Renal fibrosis reflected the degree of renal dysfunction. However, the current biomarkers only characterize the renal function instead of indicating the fibrosis degree. The potential diagnostic value of urinary exosomes derived circRNAs for renal fibrosis needs to be further studied. METHODS: Urine exosomes from 3 chronic kidney disease (CKD) patients without renal fibrosis and 3 renal fibrotic patients were collected and human circRNAs microarray analysis were performed to detect the circRNAs expression profile. 110 biopsy-proven CKD patients and 54 healthy controls were enrolled and urine exosomes derived RNA was isolated. The expression of hsa_circ_0036649 was measured and the correlation with renal function parameter and pathological indicators was performed. The receiver operating characteristic (ROC) curve for the diagnosis of renal fibrosis was calculated. RESULTS: Human circRNAs microarray showed 365 circRNAs up expressed and 195 circRNAs down expressed in renal fibrotic patients compared to none fibrosis CKD patients. The expression of hsa_circ_0036649 was decreased in renal fibrotic patients according to RT-PCR and correlated with serum creatinine, blood urea nitrogen (BUN), estimated glomerular filtration rate and cystatin c. Further, the expression of hsa_circ_0036649 was correlated with the score of tubulointerstitial fibrosis (TIF) and the score of glomerular sclerosis. The ROC curve showed that hsa_circ_0036649 may predict renal fibrosis at a cut-off value of 0.597 with a sensitivity of 45.5% and specificity of 87.9%. CONCLUSION: Expression of urinary exosomes derived hsa_circ_0036649 associated with the degree of renal fibrosis. Its potential role as a biomarker in CKD remained to be supported by further follow-up studies.Key MessagescircRNAs profile in urine exosomes in renal fibrosis patients was revealed.The expression of urine exosomes derived hsa_circ_0036649 was correlated to renal function and fibrosis degree.circRNAs derived from urinary exosomes may become a new research direction for biomarkers of renal fibrosis.

Urine Exosomal AMACR Is a Novel Biomarker for Prostate Cancer Detection at Initial Biopsy.

Objectives: The aim of this study is to identify and validate urine exosomal AMACR (UE-A) as a novel biomarker to improve the detection of prostate cancer (PCa) and clinically significant PCa (Gleason score ≥ 7) at initial prostate biopsy. Methods: A total of 289 first-catch urine samples after the digital rectal exam (DRE) were collected from patients who underwent prostatic biopsy, and 17 patients were excluded due to incomplete clinical information. Urine exosomes were purified, and urinary exosomal AMACR (UE-A) was measured by enzyme-linked immunosorbent assay (ELISA). The diagnostic performance of UE-A was evaluated by receiver operating characteristic (ROC) analysis, decision curve analysis (DCA), and waterfall plots. Results: The expression of AMACR in PCa and csPCa was significantly higher than that in BPH and non-aggressive (p < 0.001). The UE-A presented good performance in distinguishing PCa from BPH or BPH plus non-significant PCa (nsPCa) from csPCa with an area under the ROC curve (AUC) of 0.832 and 0.78, respectively. The performance of UE-A was further validated in a multi-center cohort of patients with an AUC of 0.800 for detecting PCa and 0.749 for detecting csPCa. The clinical utility assessed by DCA showed that the benefit of patients using UE-A was superior to PSA, f/t PSA, and PSAD in both the training cohort and the validation cohort in terms of all threshold probabilities. Setting 95% sensitivity as the cutoff value, UE-A could avoid 27.57% of unnecessary biopsies, with only 4 (1.47%) csPCa patients missed. Conclusions: We demonstrated the great performance of UE-A for the early diagnosis of PCa and csPCa. UE-A could be a novel non-invasive diagnostic biomarker to improve the detection of PCa and csPCa.

Differential Expression of Urinary Exosomal miRNA in Idiopathic Membranous Nephropathy and Evaluation of its Diagnostic Value.

Urinary exosomal miRNA is an ideal non-invasive biomarker of renal disease, but little is known about its ability to diagnose idiopathic membranous nephropathy (IMN). The purpose of this study was to explore the clinical value of urinary exosomal miRNAs in IMN. Urine samples were collected from 36 IMN patients and 36 healthy subjects. Some samples were used to analyze the miRNA profiles of urinary exosomes by high-throughput sequencing. The remaining cases were verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, the serum of the patients and healthy people was collected, and the clinical parameters were detected. Through high-throughput sequencing of samples, it was found that 20 miRNAs were markedly down-regulated. MiR-9-5p and miR-30b-5p were selected for verification, and the results were consistent with those of high-throughput sequencing. MiR-9-5p was correlated with the level of triglyceride and estimated glomerular filtration rate. MiR-30b-5p was related to the levels of anti-phospholipase A2 receptor antibody, serum albumin, ß 2-microglobulin and the ratio of global sclerosis/observed glomeruli number. The analysis of Receiver Operating Characteristic curves revealed that miR-30b-5p and miR-9-5p showed a potential diagnostic value for IMN. This study showed that there were significant differences in urinary exosome miRNA profiles between IMN patients and healthy persons. MiR-30b-5p and miR-9-5p may become new non-invasive biomarkers of IMN.

iTRAQ-Based Proteomic Analysis of APP Transgenic Mouse Urine Exosomes.

Alzheimer's disease (AD) is a common dementia disease in the elderly. To get a better understanding of the pathophysiology, we performed a proteomic analysis of the urine exosomes (U-exo) in AD model mice (J20). The polymer precipitation method was used to isolate U-exo from the urine of 3-month-old J20 and wild-type (WT) mice. Neuron-derived exosome (N-exo) was isolated from U-exo by immunoprecipitation. iTRAQ-based MALDI TOF MS/MS was used for proteomic analysis. The results showed that compared to WT, the levels of 61 and 92 proteins were increased in the J20 U-exo and N-exo, respectively. Gene ontology enrichment analysis demonstrated that the sphingolipid catabolic process, ceramide catabolic process, membrane lipid catabolic process, Aß clearance, and Aß metabolic process were highly enriched in U-exo and N-exo. Among these, Asah1 was shown to be the key protein in lipid metabolism, and clusterin, ApoE, neprilysin, and ACE were related to Aß metabolism and clearance. Furthermore, protein-protein interaction analysis identified four protein complexes where clusterin and ApoE participated as partner proteins. Thus, J20 U-exo and N-exo contain proteins related to lipid- and Aß-metabolism in the early stages of AD, providing a new insight into the underlying pathological mechanism of early AD.

Combination of Urine Exosomal mRNAs and lncRNAs as Novel Diagnostic Biomarkers for Bladder Cancer.

BACKGROUND: The recent discovery of miRNAs and lncRNAs in urine exosomes has emerged as promising diagnostic biomarkers for bladder cancer (BCa). However, mRNAs as the direct products of transcription has not been well evaluated in exosomes as biomarkers for BCa diagnosis. The purpose of this study was to identify tumor progression-related mRNAs and lncRNAs in urine exosomes that could be used for detection of BCa. METHODS: RNA-sequencing was performed to identify tumor progression-related biomarkers in three matched superficial tumor and deep infiltrating tumor regions of muscle-invasive bladder cancer (MIBC) specimens, differently expressed mRNAs and lncRNAs were validated in TCGA dataset (n = 391) in the discovery stage. Then candidate RNAs were chosen for evaluation in urine exosomes of a training cohort (10 BCa and 10 healthy controls) and a validation cohort (80 BCa and 80 healthy controls) using RT-qPCR. The diagnostic potential of the candidates were evaluated by receiver operating characteristic (ROC) curves. RESULTS: RNA sequencing revealed 8 mRNAs and 32 lncRNAs that were significantly upregulated in deep infiltrating tumor region. After validation in TCGA database, 10 markedly dysregulated RNAs were selected for further investigation in urine exosomes, of which five (mRNAs: KLHDC7B, CASP14, and PRSS1; lncRNAs: MIR205HG and GAS5) were verified to be significantly dysregulated. The combination of the five RNAs had the highest AUC to disguising the BCa (0.924, 95% CI, 0.875-0.974) or early stage BCa patients (0.910, 95% CI, 0.850 to 0.971) from HCs. The expression levels of these five RNAs were correlated with tumor stage, grade, and hematuria degrees. CONCLUSIONS: These findings highlight the potential of urine exosomal mRNAs and lncRNAs profiling in the early diagnosis and provide new insights into the molecular mechanisms involved in BCa.

MicroRNAs as disease progression biomarkers and therapeutic targets in experimental autoimmune encephalomyelitis model of multiple sclerosis.

Multiple sclerosis is an autoimmune neurodegenerative disease of the central nervous system characterized by pronounced inflammatory infiltrates entering the brain, spinal cord and optic nerve leading to demyelination. Focal demyelination is associated with relapsing-remitting multiple sclerosis, while progressive forms of the disease show axonal degeneration and neuronal loss. The tests currently used in the clinical diagnosis and management of multiple sclerosis have limitations due to specificity and sensitivity. MicroRNAs (miRNAs) are dysregulated in many diseases and disorders including demyelinating and neuroinflammatory diseases. A review of recent studies with the experimental autoimmune encephalomyelitis animal model (mostly female mice 6-12 weeks of age) has confirmed miRNAs as biomarkers of experimental autoimmune encephalomyelitis disease and importantly at the pre-onset (asymptomatic) stage when assessed in blood plasma and urine exosomes, and spinal cord tissue. The expression of certain miRNAs was also dysregulated at the onset and peak of disease in blood plasma and urine exosomes, brain and spinal cord tissue, and at the post-peak (chronic) stage of experimental autoimmune encephalomyelitis disease in spinal cord tissue. Therapies using miRNA mimics or inhibitors were found to delay the induction and alleviate the severity of experimental autoimmune encephalomyelitis disease. Interestingly, experimental autoimmune encephalomyelitis disease severity was reduced by overexpression of miR-146a, miR-23b, miR-497, miR-26a, and miR-20b, or by suppression of miR-182, miR-181c, miR-223, miR-155, and miR-873. Further studies are warranted on determining more fully miRNA profiles in blood plasma and urine exosomes of experimental autoimmune encephalomyelitis animals since they could serve as biomarkers of asymptomatic multiple sclerosis and disease course. Additionally, studies should be performed with male mice of a similar age, and with aged male and female mice.

Augmentation of Urinary Lactoferrin Enhances Host Innate Immune Clearance of Uropathogenic Escherichia coli.

Urinary tract infection (UTI) is a prominent global health care burden. Although UTI is readily treated with antibiotics in healthy adults, complicated cases in immune-compromised individuals and the emerging antibiotic resistance of several uropathogens have accelerated the need for new treatment strategies. Here, we surveyed the composition of urinary exosomes in a mouse model of uropathgenic Escherichia coli (UPEC) UTI to identify specific urinary tract defense constituents for therapeutic development. We found an enrichment of the iron-binding glycoprotein lactoferrin in the urinary exosomes of infected mice. In subsequent in vitro studies, we identified human bladder epithelial cells as a source of lactoferrin during UPEC infection. We further established that exogenous treatment with human lactoferrin (hLf) reduces UPEC epithelial adherence and enhances neutrophil antimicrobial functions including bacterial killing and extracellular trap production. Notably, a single intravesicular dose of hLf drastically reduced bladder bacterial burden and neutrophil infiltration in our murine UTI model. We propose that lactoferrin is an important modulator of innate immune responses in the urinary tract and has potential application in novel therapeutic design for UTI.

Expression signatures of exosomal long non-coding RNAs in urine serve as novel non-invasive biomarkers for diagnosis and recurrence prediction of bladder cancer.

Recently, expression signatures of exosomal long non-coding RNAs (lncRNAs) have been proposed as potential non-invasive biomarkers for cancer detection. In this study, we aimed to develop a urinary exosome (UE)-derived lncRNA panel for diagnosis and recurrence prediction of bladder cancer (BC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to screen and evaluate the expressions of eight candidate lncRNAs in a training set (208 urine samples) and a validation set (160 urine samples). A panel consisting of three differently expressed lncRNAs (MALAT1, PCAT-1 and SPRY4-IT1) was established for BC diagnosis in the training set, showing an area under the receiver-operating characteristic (ROC) curve (AUC) of 0.854. Subsequently, the performance of the panel was further verified with an AUC of 0.813 in the validation set, which was significantly higher than that of urine cytology (0.619). In addition, Kaplan-Meier analysis suggested that the up-regulation of PCAT-1 and MALAT1 was associated with poor recurrence-free survival (RFS) of non-muscle-invasive BC (NMIBC) (p < 0.001 and p = 0.002, respectively), and multivariate Cox proportional hazards regression analysis revealed that exosomal PCAT-1 overexpression was an independent prognostic factor for the RFS of NMIBC (p = 0.018). Collectively, our findings indicated that UE-derived lncRNAs possessed considerable clinical value in the diagnosis and prognosis of BC.

Urinary exosomal expression of activator of G protein signaling 3 in polycystic kidney disease.

OBJECTIVE: PKD is a genetic disease that is characterized by abnormally proliferative epithelial cells in the kidney and liver. Urinary exosomes have been previously examined as a source of unique proteins that may be used to diagnose and monitor the progression of PKD. Previous studies by our group have shown that AGS3, which is a receptor-independent regulator G-proteins, was markedly upregulated in RTECs during kidney injury including PKD. In this study, our goal was to determine whether AGS3 could be measured in exosomes using animals and humans with PKD. RESULTS: In our study, urinary exosomes were isolated from PCK rats and the control Sprague-Dawley (SD) rats. AGS3 expression was significantly increased (P < 0.05) in PKD versus SD rats at 16 weeks of age. This increase was detectable in a time-dependent manner from 8 weeks of age and peaked at ~ 16-20 weeks (length of study). Similarly, in exosomes from human urine samples with PKD, AGS3 expression was significantly increased (P < 0.05) compared to healthy human controls where AGS3 was largely undetectable. In conclusion, the detection of AGS3 in urinary exosomes may be a novel biomarker for PKD, and provide new insight into the biology of tubular epithelial cell function during cystic disease progression.

Integrated Kidney Exosome Analysis for the Detection of Kidney Transplant Rejection.

Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are noninvasive alternatives but are late markers with low specificity. We report a urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as a surrogate marker for inflammation. We optimized iKEA to detect T-cell-derived EVs and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients and achieved high detection accuracy (91.1%). Fast, noninvasive, and cost-effective, iKEA could offer new opportunities in managing transplant recipients, perhaps even in a home setting.

Proteomic analyses of Urine Exosomes reveal New Biomarkers of Diabetes in Pregnancy.

OBJECTIVE: To evaluate 24 hour urine exosome protein content changes among pregnant US subjects with diabetes and obesity during early pregnancy. METHODS: The exosome proteome content from 24 hour urine samples of pregnant subjects with gestational diabetes mellitus (GDM, N=8) and pre-gestational Type 2 diabetes (PGD, N = 10) were compared with control samples (CTRL, N = 10) obtained at week 20 of pregnancy. Differences in exosome protein load between groups was identified by liquid chromatography/mass spectrometry, analyzed by linear regression in negative binomial distribution, visualized in MetaboAnalyst (version 3.0), and validated by western immunoblotting. RESULTS: At the 20th week of pregnancy, we identified 646, 734 and 856 proteins in exosomes from 24 hour urine samples of patients from the CTRL, GDM and PGD groups, respectively. S100 calcium binding protein A9, damage associated molecular pattern (DAMP) signal, was found to be significantly increased in both GDM and PGD subjects. In GDM subjects the peptide counts for S100A9 protein independently correlated with maternal obesity and macrosomia of the newborn infants. Early to late pregnancy developmental changes in the GDM group were shown to utilize pathways and protein expression levels differently from those in PGD or CTRL groups. CONCLUSIONS: Urinary exosome proteomic analysis non-invasively provides insights into maternal changes during diabetic pregnancy. Exosome biomarkers early in pregnancy can be potentially used to better understand pathophysiologic mechanisms of diabetes at a cellular level, and to distinguish between gestational and pre-gestational diabetes at the pathway level. This information can aid intervention efforts to improve pregnancy outcomes in women with diabetes.

Perturbations in the urinary exosome in transplant rejection.

BACKGROUND: Urine exosomes are small vesicles exocytosed into the urine by all renal epithelial cell types under normal physiologic and disease states. Urine exosomal proteins may mirror disease specific proteome perturbations in kidney injury. Analysis methodologies for the exosomal fraction of the urinary proteome were developed for comparing the urinary exosomal fraction versus unfractionated proteome for biomarker discovery. METHODS: Urine exosomes were isolated by centrifugal filtration of urine samples collected from kidney transplant patients with and without acute rejection (AR), which were biopsy matched. The proteomes of unfractionated whole urine (Uw) and urine exosomes (Ue) underwent mass spectroscopy-based quantitative proteomics analysis. The proteome data were analyzed for significant differential protein abundances in AR. RESULTS: A total of 1018 proteins were identified in Uw and 349 proteins in Ue. Two hundred seventy-nine overlapped between the two urinary compartments and 70 proteins were unique to the Ue compartment. Of 349 exosomal proteins identified from transplant patients, 220 had not been previously identified in the normal Ue fraction. Eleven Ue proteins, functionally involved in an inflammatory and stress response, were more abundant in urine samples from patients with AR, three of which are exclusive to the Ue fraction. Ue AR-specific biomarkers (1) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw. CONCLUSION: A rapid urinary exosome isolation method and quantitative measurement of enriched Ue proteins was applied. Perturbed proteins in the exosomal compartment of urine collected from kidney transplant patients were specific to inflammatory responses, and were not observed in the Ue fraction from normal healthy subjects. Ue-specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring AR.