Reference gene panel for urinary exosome-based molecular diagnostics in patients with kidney disease.

BACKGROUND: Kidney disease is a severe complication of diabetes that often leads to end-stage renal disease. Early diagnosis is crucial for prevention or delay. However, the current diagnostic methods, with their limitations in detecting the disease in its early stages, underscore the urgency and importance of finding new solutions. miRNAs encapsulated inside urinary exosomes (UEs) have potential as early biomarkers for kidney diseases. The need for reference miRNAs for accurate interpretation currently limits their translational potential. AIM: To identify consistently expressing reference miRNAs from UEs of controls and patients with type 2 diabetesmellitus (T2DM) and biopsy-confirmed kidney diseases. METHODS: miRNA profiling was performed on UEs from 31 human urine samples using a rigorous and unbiased method. The UEs were isolated from urine samples collected from healthy individuals (n = 6), patients with T2DM (n = 13), and T2DM patients who also had kidney diseases (including diabetic nephropathy, n = 5; membranous nephropathy, n = 5; and IgA nephropathy, n = 2) through differential ultracentrifugation. After characterizing the UEs, miRNA expression profiling using microarray technology was conducted. RESULTS: Microarray data analysis identified 14 miRNAs that were consistently expressed in UEs from 31 human samples, representing various kidney conditions: diabetic controls, diabetic nephropathy, membrane nephropathy, IgA nephropathy, and healthy controls. Through in silico analysis, we determined that 10 of these miRNAs had significant potential to serve as reference genes in UEs. CONCLUSION: We identified uniformly expressing UE miRNAs that could serve as reference genes kidney disease biomarkers.

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

Background: 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. Method: 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). Result: 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. Conclusion: 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.

Evaluating the Urinary Exosome microRNA Profile of von Hippel Lindau Syndrome Patients with Clear Cell Renal Cell Carcinoma.

INTRODUCTION: Renal cell carcinoma is one of the ten more common malignant tumors worldwide, with a high incidence and mortality rate. Kidney cancer frequently presents at an advanced stage, and it is almost invariably fatal. Much progress has been made in identifying molecular targets for therapy in the hope of improving survival rates, but still, we have no good markers for early detection or progression of the disease. Von Hippel Lindau syndrome (VHL) is an autosomal dominant cancer hereditary syndrome in which affected individuals are at risk of developing bilateral and multifocal renal cell carcinomas (RCC) as well as other tumors. These patients provide an ideal platform to investigate the potential of urinary exosomal miRNA biomarkers in the early development of ccRCC, as these patients are regularly imaged and tumors are actively monitored until the tumor reaches 3 cm before surgical excision. This allows for pre- and post-surgical urine collection and comparison to excised tumor tissues. Studying different biomarkers in urine can provide comprehensive molecular profiling available to patients and physicians and can be a great source of additional tumor genetic information. METHODS: Pre- and postoperative urine samples were obtained from a cohort of VHL patients undergoing surveillance and surgical excision of ccRCCs, and exosomes were extracted. MicroRNA-Seq analysis was performed on miRNA extracted from both urine-derived exosomes and FFPE material from excised ccRCCs. RESULTS: MicroRNA-Seq analysis highlighted a significant difference in the urinary exosome-derived miRNA expression profiles between VHL patients and normal control individuals. This included decreased expression of the miR-320 family, such as miR-320a, known to be decreased in sporadic ccRCC and suppressed by the HIF1α transcription factor activated by the loss of the VHL gene. MiR-542-5p represented a potential marker of VHL-associated ccRCC that was lowly expressed in normal control urinary exosomes, significantly increased in the preoperative urinary exosomes of tumor-bearing VHL patients, and subsequently reduced to normal levels of expression after tumor excision. In concordance with this, the expression of miR-542-5p was increased in the VHL-associated ccRCC in comparison to the normal kidney. CONCLUSIONS: This study shows the potential for miRNA profiling of exosomes from readily available biofluids to both distinguish VHL patient urine from normal control urine microRNAs and to provide biomarkers for the presence of VHL syndrome-associated ccRCC. Further validation studies are necessary to demonstrate the utility of urinary exosome-derived miRNAs as biomarkers in kidney cancer.

Urinary Exosomal miR-17-5p Accelerates Bladder Cancer Invasion by Repressing its Target Gene ARID4B and Regulating the Immune Microenvironment.

BACKGROUND: Urothelial bladder cancer (BCa) is a common malignant tumor of the urinary system. It has been identified that exosomal miRNAs contribute to the development of BCa. However, its significance and mechanism in the malignant biological behavior of BCa remain unclear. In this study, the influence of exosomal miRNAs on BCa progression was investigated. METHODS: High-throughput sequencing was conducted to analyze the microRNA-expression profile in urinary exosomes to screen out the key miRNA of muscle-invasive bladder cancer (MIBC). Then, candidate miRNA expression was verified and validated in urinary exosomes and tissue samples. To address the potential role of the candidate miRNA, we overexpressed and knocked down the candidate miRNA and explored its activity in BCa cell lines. Furthermore, the target gene of the selected miRNA was predicted and validated. RESULTS: The expression profile of miRNAs revealed increased expression of miR-17-5p in MIBC urinary exosomes, and this was later confirmed in urinary exosomes and tissue samples. Cell function studies revealed that exosomal miR-17-5p significantly promoted the growth and invasion of BCa cells. Bioinformatics and luciferase experiments demonstrated that the ARID4B mRNA 3' UTR might be the binding site for miR-17-5p. Low ARID4B levels were linked to high-grade BCa patients and were associated with a better prognosis. CONCLUSION: Elevated miR-17-5p contributes to BCa progression by targeting ARID4B and influencing the immune system. Based on these findings, miR-17-5p has the potential to be a new therapeutic target for the treatment of BCa.

Urinary exosomes from patients with diabetic kidney disease induced podocyte apoptosis via microRNA-145-5p/Srgap2 and the RhoA/ROCK pathway.

Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease without early diagnostic and specific therapeutic approaches. Podocyte apoptosis and loss play important roles in the pathological process of DKD. This study aimed to explore whether urinary exosomes from type 2 diabetes patients with DKD could induce podocyte apoptosis and the underlying pathological mechanisms. The exosomes were isolated from the urine samples of patients with DKD (DKD-Exo). Later, they were taken up and internalized by MPC5 cells. MPC5 cells were co-cultured with DKD-Exo (45 µg/ml) for 24 h in the presence or absence of microRNA-145-5p (miR-145-5p) inhibitor, fasudil and pcDNA-Srgap2 transfection. MiR-145-5p and Srgap2 expression was evaluated using real-time quantitative PCR. The protein levels of Srgap2, Bcl-2, Bax, and cleaved caspase-3, as well as ROCK activity were determined using Western blotting. Cell apoptosis was measured using flow cytometry and the TUNEL assay. miR-145-5p expression in MPC5 cells exposed to DKD-Exo was markedly upregulated. miR-145-5p negatively regulated Srgap2 levels. Exposure of MPC5 cells to DKD-Exo reduced Srgap2 expression and activated ROCK, which was partly reversed by the presence of the miR-145-5p inhibitor or Srgap2 overexpression. The apoptosis of MPC5 cells exposed to DKD-Exo increased significantly, which was counteracted by the addition of the miR-145-5p inhibitor and fasudil. The results showed that urinary exosomal miR-145-5p from patients with DKD induced podocyte apoptosis by inhibiting Srgap2 and activating the RhoA/ROCK pathway, suggesting that urinary exosomal miR-145-5p is involved in the pathological process of DKD and could become a noninvasive diagnostic biomarker for DKD.

Urinary exosome proteins PAK6 and EGFR as noninvasive diagnostic biomarkers of diabetic nephropathy.

OBJECTIVE: The actin cytoskeleton plays an essential role in maintaining podocyte functions. However, whether the urinary exosome proteins related to the regulation of the actin cytoskeleton are changed in diabetic nephropathy (DN) is still unknown. This study was to investigate the possibility that related proteins can be applied as diagnostic biomarkers for DN. METHODS: Urinary exosomes were obtained from 144 participants (Discovery phase: n = 72; Validation phase: n = 72) by size exclusion chromatography methods. Proteomic analysis of urinary exosome by LC-MS/MS. Western blot and ELISA were applied to validate the selected urinary exosome proteins. The clinical value of selected urinary exosome proteins was evaluated using correlation and receiver operating characteristic curve analyses. RESULTS: Fifteen urinary proteins related to the regulation of the actin cytoskeleton were identified in urinary exosomes. Three upregulated proteins were selected, including Serine/threonine-protein kinase PAK6 (PAK6), Epidermal growth factor receptor (EGFR), and SHC-transforming protein 1(SHC1). The expression level of PAK6 and EGFR was negatively correlated with estimated glomerular filtration rate and positively correlated with serum creatinine levels. For diagnosing DN in the discovery phase: the area under curve (AUC) of PAK6 was 0.903, EGFR was 0.842, and the combination of two proteins was 0.912. These better performances were also observed in the validation phase (For PAK6: AUC = 0.829; For EGFR: AUC = 0.797; For PAK6 + EGFR: AUC = 0.897). CONCLUSIONS: Urinary exosome proteins PAK6 and EGFR may be promising and noninvasive biomarkers for diagnosing DN.

Urinary exosomes: a promising biomarker of drug-induced nephrotoxicity.

Drug-induced nephrotoxicity (DIN) is a big concern for clinical medication, but the clinical use of certain nephrotoxic drugs is still inevitable. Current testing methods make it hard to detect early renal injury accurately. In addition to understanding the pathogenesis and risk factors of drug-induced nephrotoxicity, it is crucial to identify specific renal injury biomarkers for early detection of DIN. Urine is an ideal sample source for biomarkers related to kidney disease, and urinary exosomes have great potential as biomarkers for predicting DIN, which has attracted the attention of many scholars. In the present paper, we will first introduce the mechanism of DIN and the biogenesis of urinary exosomes. Finally, we will discuss the changes in urinary exosomes in DIN and compare them with other predictive indicators to enrich and boost the development of biomarkers of DIN.

Sex differences in FASN protein concentrations in urinary exosomes related to serum triglycerides levels in healthy adults.

BACKGROUND: Dysregulation of lipid metabolism is the most prominent metabolic alteration observed in obesity, cancer, and cardiovascular diseases. The present study aimed to explore the sex differences associated with lipid metabolism in urinary exosome proteins, and evaluate the correlation of urinary exosome proteins with serum lipid biomarkers. METHODS: The key enzymes regulating lipid metabolism in healthy adults were screened using urinary exosome data. Urinary exosomes were isolated from 120 healthy subjects and the expression of urinary proteins was assessed by Western blotting and ELISA. The correlation between urinary protein concentrations and the levels of serum lipid biomarkers was analyzed using correlation analysis. RESULTS: Three urinary exosome proteins, namely fatty acid synthase (FASN), phosphoenolpyruvate carboxykinase (PCK1), and ATP-citrate synthase (ACLY) were identified, and only FASN showed sex differences. Sex differences were also observed in the serum triglyceride (TG) levels. Healthy males had higher FASN levels than females, and a moderate positive correlation was found between FASN concentrations and serum TG levels in healthy males (r = 0.479, P < 0.05). FASN concentrations in different age groups were positively correlated with the level of serum TG (18 ~ 30 years, r = 0.502; 31 ~ 44 years, r = 0.587; 45 ~ 59 years, r = 0.654; all P < 0.05). In addition, FASN concentrations was positively related to the increase in serum TG levels (range:1.0 ~ 1.7 mmol/L; r = 0.574, P < 0.05). CONCLUSIONS: Sex differences were observed in urinary exosome FASN protein levels in healthy adults. FASN protein levels positively correlated with increased serum TG levels. FASN may serve as a novel biomarker to evaluate fatty acid synthesis in the human body.

Metabolic profiling of urinary exosomes for systemic lupus erythematosus discrimination based on HPL-SEC/MALDI-TOF MS.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease which leads to the formation of immune complex deposits in multiple organs and has heterogeneous clinical manifestations. Currently, exosomes for liquid biopsy have been applied in diagnosis and monitoring of diseases, whereas SLE discrimination based on exosomes at the metabolic level is rarely reported. Herein, we constructed a protocol for metabolomic study of urinary exosomes from SLE patients and healthy controls (HCs) with high efficiency and throughput. Exosomes were first obtained by high-performance liquid size-exclusion chromatography (HPL-SEC), and then metabolic fingerprints of urinary exosomes were extracted by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with high throughput and high efficency. With the statistical analysis by orthogonal partial least-squares discriminant analysis (OPLS-DA) model, SLE patients were efficiently distinguished from HCs, the area under the curve (AUC) of the receiver characteristic curve (ROC) was 1.00, and the accuracy of the unsupervised clustering heatmap was 90.32%. In addition, potential biomarkers and related metabolic pathways were analyzed. This method, with the characteristics of high throughput, high efficiency, and high accuracy, will provide the broad prospect of exosome-driven precision medicine and large-scale screening in clinical applications.

Changes in urinary exosomal protein CALM1 may serve as an early noninvasive biomarker for diagnosing diabetic kidney disease.

BACKGROUND: The risk of the development and progression of diabetic kidney disease (DKD) was increased by abnormal calcium release. However, it is still unknown whether calcium signal pathway-related proteins are changed in urinary exosomes. This study aims to explore the changes in urinary exosomal proteins, which may provide novel biomarkers for diagnosing DKD. METHODS: Urinary exosomes were isolated from 132 participants by size exclusion chromatography method and 72 participants were tested by LC-MS/MS (Discovery phase). Correlation and multivariate logistics analysis were applied to evaluate selected urinary proteins. Western blot and ELISA were used to validate the selected protein (Validation phase: n = 60). The diagnostic performance of the selected biomarker was evaluated by receiver operating characteristic curve analyses between the discovery and validation phases. RESULTS: Sixteen calcium signal pathway-related proteins were identified, however, only Calmodulin-1(CALM1) was continuously increased. Different expression of CALM1 was found in patients with different level of estimated glomerular filtration rate (eGFR) in two cohorts. The level of CALM1 was correlated with eGFR and serum creatinine levels in two cohorts. Multivariate analysis revealed that serum albumin (ALB) levels and CALM1 were independent risk factors for DKD. A diagnostic model based on CALM1 and serum ALB levels that could significantly distinguish DKD was established and validated. CONCLUSIONS: Significant changes in calcium signal pathway-related urinary exosomal proteins were observed. The CALM1 may serve as an early noninvasive biomarker for diagnosing DKD.

Liquid Biopsy at the Frontier of Kidney Diseases: Application of Exosomes in Diagnostics and Therapeutics.

In the era of precision medicine, liquid biopsy techniques, especially the use of urine analysis, represent a paradigm shift in the identification of biomarkers, with considerable implications for clinical practice in the field of nephrology. In kidney diseases, the use of this non-invasive tool to identify specific and sensitive biomarkers other than plasma creatinine and the glomerular filtration rate is becoming crucial for the diagnosis and assessment of a patient's condition. In recent years, studies have drawn attention to the importance of exosomes for diagnostic and therapeutic purposes in kidney diseases. Exosomes are nano-sized extracellular vesicles with a lipid bilayer structure, composed of a variety of biologically active substances. In the context of kidney diseases, studies have demonstrated that exosomes are valuable carriers of information and are delivery vectors, rendering them appealing candidates as biomarkers and drug delivery vehicles with beneficial therapeutic outcomes for kidney diseases. This review summarizes the applications of exosomes in kidney diseases, emphasizing the current biomarkers of renal diseases identified from urinary exosomes and the therapeutic applications of exosomes with reference to drug delivery and immunomodulation. Finally, we discuss the challenges encountered when using exosomes for therapeutic purposes and how these may affect its clinical applications.

Urinary exosomal metabolites: Overlooked clue for predicting cardiovascular risk.

Over the last decade, increasing research has focused on urinary exosomes (UEs) in biological fluids and their relationship with physiological and pathological processes. UEs are membranous vesicles with a size of 40-100 nm, containing a number of bioactive molecules such as proteins, lipids, mRNAs, and miRNAs. These vesicles are an inexpensive non-invasive source that can be used in clinical settings to differentiate healthy patients from diseased patients, thereby serving as potential biomarkers for the early identification of disease. Recent studies have reported the isolation of small molecules called exosomal metabolites from individuals' urine with different diseases. These metabolites could utilize for a variety of purposes, such as the discovery of biomarkers, investigation of mechanisms related to disease development, and importantly prediction of cardiovascular diseases (CVDs) risk factors, including thrombosis, inflammation, oxidative stress, hyperlipidemia as well as homocysteine. It has been indicated that alteration in urinary metabolites of N1-methylnicotinamide, 4-aminohippuric acid, and citric acid can be valuable in predicting cardiovascular risk factors, providing a novel approach to evaluating the pathological status of CVDs. Since the UEs metabolome has been clearly and precisely so far unexplored in CVDs, the present study has specifically addressed the role of the mentioned metabolites in the prediction of CVDs risk factors.

Therapeutic potential of urine exosomes derived from rats with diabetic kidney disease.

Kidney disease is prevalent in diabetes. Urinary exosomes (uE) from animal models and patients with Diabetic nephropathy (DN) showed increased levels of miRs with reno-protective potential. We examined whether urinary loss of such miRs is associated with their reduced renal levels in DN patients. We also tested whether injecting uE can leverage kidney disease in rats. In this study (study-1) we performed microarray profiling of miRNA in uE and renal tissues in DN patients and subjects with diabetes without DN (controls). In study-2, diabetes was induced in Wistar rats by Streptozotocin (i.p. 50 mg/kg of body weight). Urinary exosomes were collected at 6th, 7th and 8th weeks, and injected back into the rats (100ug/biweekly, uE-treated n=7) via tail vein on weeks 9 and 10. Equal volume of vehicle was injected in controls (vehicle, n=7). uE from the human and rat showed the presence of exosome-specific proteins by immunoblotting. Microarray profiling revealed a set of 15 miRs having high levels in the uE, while lower in renal biopsies, from DN, compared to controls (n=5-9/group). Bioinformatic analysis also confirmed the Renoprotective potential of these miRs. Taqman qPCR confirmed the opposite regulation of miR-200c-3p and miR-24-3p in paired uE and renal biopsy samples from DN patients (n=15), relative to non-DN controls. A rise in 28 miRs levels, including miR-200c-3p, miR-24-3p, miR-30a-3p and miR-23a-3p were observed in the uE of DN rats, collected between 6th-8th weeks, relative to baseline (before diabetes induction). uE- treated DN rats had significantly reduced urine albumin-to-creatinine ratio, attenuated renal pathology, and lower miR-24-3p target fibrotic/inflammatory genes (TGF-beta, and Collagen IV), relative to vehicle treated DN rats. In uE treated rats, the renal expression of miR-24-3p, miR-30a-3p, let-7a-5p and miR-23a-3p was increased, relative to vehicle control. Patients with diabetic nephropathy had reduced renal levels, while higher uE abundance of miRs with reno-protective potential. Reverting the urinary loss of miRs by injecting uE attenuated renal pathology in diabetic rats.

TRAIL and EGFR Pathways Targeting microRNAs are Predominantly Regulated in Human Diabetic Nephropathy.

BACKGROUND: Unbiased microRNA profiling of renal tissue and urinary extracellular vesicles (uEVs) from diabetic nephropathy (DN) subjects may unravel novel targets with diagnostic and therapeutic potential. Here we used the miRNA profile of uEVs and renal biopsies from DN subjects available on the GEO database. METHODS: The miR expression profiles of kidney tissue (GSE51674) and urinary exosomes (GSE48318) from DN and control subjects were obtained by GEO2R tools from Gene Expression Omnibus (GEO) databases. Differentially expressed miRNAs in DN samples, relative to controls, were identified using a bioinformatic pipeline. Targets of miRs commonly regulated in both sample types were predicted by miRWalk, followed by functional gene enrichment analysis. Gene targets were identified by MiRTarBase, TargetScan and MiRDB. RESULTS: Eight miRs, including let-7c, miR-10a, miR-10b and miR-181c, were significantly regulated in kidney tissue and uEVs in DN subjects versus controls. The top 10 significant pathways targeted by these miRs included TRAIL, EGFR, Proteoglycan syndecan, VEGF and Integrin Pathway. Gene target analysis by miRwalk upon validation using ShinyGO 70 targets with significant miRNA-mRNA interaction. CONCLUSION: In silico analysis showed that miRs targeting TRAIL and EGFR signaling are predominately regulated in uEVs and renal tissue of DN subjects. After wet-lab validation, the identified miRstarget pairs may be explored for their diagnostic and/or therapeutic potential in diabetic nephropathy.

Urinary exosome tsRNAs as novel markers for diagnosis and prediction of lupus nephritis.

Objective: Lupus nephritis (LN) is one of the most severe organ manifestations of systemic lupus erythematosus (SLE). Early identification of renal disease in SLE is important. Renal biopsy is currently recognized as the gold standard for diagnosing LN, however, it is invasive and inconvenient for dynamic monitoring. Urine has been considered more promising and valuable than blood in identifying inflamed kidney tissue. Here, we determine whether the signatures of tRNA-derived small noncoding RNA (tsRNA) in urinary exosomes can serve as novel biomarkers for the diagnosis of LN. Methods: tsRNA sequencing was performed in exosome extracted from pooled urine of 20 LN patients and 20 SLE without LN, and the top 10 upregulated tsRNAs were screened as candidate markers of LN. The candidate urinary exosomal tsRNAs were primarily elected by TaqMan probe-based quantitative reverse transcription-PCR (RT-PCR) in 40 samples (20 LN and 20 SLE without LN) in the training phase. In the validation phase, selected tsRNAs from the training phase were further confirmed in a larger cohort (54 LN patients and 39 SLE without LN). Receiver operating characteristic curve (ROC) analysis was conducted to evaluate the diagnostic efficacy. Results: Upregulated levels of tRF3-Ile-AAT-1 and tiRNA5-Lys-CTT-1 in the urinary exosomes were observed in LN compared with SLE without LN (P < 0.0001 and P < 0.001) and healthy controls (P < 0.01 and P < 0.01), with the area under the curve (AUC) of 0.777 (95% CI: 0.681-0.874, sensitivity 79.63%, specificity 66.69%) and 0.715 (95% CI: 0.610-0.820, sensitivity 66.96%, specificity 76.92%) for discriminating LN from SLE without LN patients. SLE patients with mild activity and moderate to severe activity had higher levels of urinary exosome derived tRF3-Ile AAT-1 (P = 0.035 and P < 0.001) and tiRNA5-Lys-CTT-1 (P = 0.021 and P < 0.001) compared with patients with no activity. Moreover, bioinformatics analysis revealed that both of the tsRNAs regulate the immune process by modulating metabolism and signal pathway. Conclusion: In this study, we demonstrated that urinary exosome tsRNAs can be served as noninvasive biomarkers for the efficient diagnosis and prediction of nephritis in SLE.

Urinary exosomes: Diagnostic impact with a bioinformatic approach.

Exosomes are tiny membrane-enveloped vesicles of endosomal origin, typically 40-120nm in diameter, produced by most cells in both normal and pathological situations. These exosomes can be isolated from all biofluids, including urine. In this context, many researchers have focused on the analysis of urinary exosomes because urine can be collected in large quantities, regularly, and with minimal effort. Exosomes contain phospholipids, cholesterol, proteins, glycoconjugates, nucleic acids, and metabolites. Because all organs and tissues produce exosomes, their molecular cargo can provide first-hand information about the physiological and biological state of the site of origin. Many potential disease biomarker candidates have already been identified in urinary exosomes. In this chapter, we performed a bibliometric analysis of the keywords "exosome(s)" and "urine" to identify related terms, diseases and molecular/biological processes, and other related terms. This yielded interesting results suggesting that exosomes in urine may play a role in the pathogenesis of various diseases. Moreover, this chapter discusses exosomes isolation and characterization methodologies and highlight the importance of urinary exosomes and their role in the diagnosis, prognosis and therapy of various diseases. We offer a bibliometric approach and an in-depth analysis on several exosomes' isolation techniques, diagnostic potential for urogenital and specific non-urogenital diseases, as well as an overview of miRNAs significance on urinary exosomes, conferring a more complete status to this review, something that was still lacking in the current literature.

Urinary extracellular vesicles carry multiple activators and regulators of coagulation.

Cells shape their extracellular milieu by secreting intracellular products into the environment including extracellular vesicles which are lipid-bilayer limited membrane particles. These vesicles carry out a range of functions, including regulation of coagulation, via multiple contributor mechanisms. Urinary extracellular vesicles are secreted by various cells, lining the urinary space, including the nephron and bladder. They are known to have procoagulant properties, however, the details of this function, beyond tissue factor are not well known. The aim of the study was to access the role of urinary extracellular vesicles in impacting coagulation upon supplementation to plasma. This could indicate their physiological function upon kidney injury or pathology. Supplementation to standard human plasma and plasmas deficient in various coagulation factors was used for this purpose, and calibrated automated thrombogram (CAT®) was the major technique applied. We found that these vesicles contain multiple coagulation-related factors, and their lipid composition affects coagulation activities of plasma upon supplementation. Remarkably, these vesicles can restore thrombin generation in FVII, FVIII, FIX and FXI -deficient plasmas. This study explores the multiple roles of urinary extracellular vesicles in coagulation in in vitro blood coagulation and implies their importance in its regulation by several mechanisms.

Urinary exosomal long non-coding RNAs as noninvasive biomarkers for diagnosis of bladder cancer by RNA sequencing.

Introduction: Cystoscopy is the standard methodology for diagnosis of bladder cancer (BC), but it is invasive and relatively expensive. Previous studies have found that urinary exosomal long non-coding RNAs (lncRNAs) may act as potential noninvasive biomarkers for diagnosis. Here we identified urinary exosomal lncRNAs that are differentially expressed between BC and controls, and established a panel for diagnosis of BC. Methods: We performed RNA sequencing in urinary exosomes of 7 controls and 7 patients, subsequently the differentially expressed lncRNAs were detected in training cohort (50 controls and 50 patients) and validation cohort (43 controls and 43 patients). The diagnostic power of lncRNAs for BC was calculated by the area under curve (AUC). The panel for diagnosis of BC was calculated by logistic regression. Results: The results of RNA sequencing in urinary exosomes showed that 240 upregulated lncRNAs and 275 downregulated lncRNAs were differentially expressed. The levels of MKLN1-AS, TALAM1, TTN-AS1 and UCA1 in BC patients were higher than that in controls in the training and validation cohort by real-time PCR. Using logistic regression, with the combination of these four lncRNAs and NMP22, we identified a panel of five parameters capable of classifying BC patients versus controls on the basis of the training cohort (AUC=0.850). Moreover, the performance of the panel exhibited better performance than either single parameter in the validation cohort. Conclusion: Collectively, this study confirmed the diagnostic value of lncRNAs for BC by high-throughout urinary exosomal RNA sequencing.

Naturally Equipped Urinary Exosomes Coated Poly (2-ethyl-2-oxazoline)-Poly (D, L-lactide) Nanocarriers for the Pre-Clinical Translation of Breast Cancer.

Recently, biomimetic nanoparticles for tumor-targeted therapy have attracted intensifying interest. Although exosomes are an excellent biomimetic material, numerous challenges are still hindering its clinical applications, such as low yield, insufficient targeting efficiency, and high cost. In this work, urinary exosomes (UEs) with high expression of CD9 and CD47 were extracted from breast cancer patients by a non-invasive method. Here, a nanotechnology approach is reported for tumor homologous targeting via CD9 and phagocytosis escape via CD47 through UE-coated poly (2-ethyl-2-oxazoline)-poly (D, L-lactide) (PEOz-PLA) nanoparticles (UEPP NPs). The cytotoxic agent doxorubicin (DOX)-loaded UEPP (UEPP-D) NPs with an initial particle size of 61.5 nm showed a burst release under acidic condition mimicking the tumor microenvironment. In vitro experiments revealed that UEPP-D NPs exhibited significantly improved cellular uptake, cytotoxicity, and apoptosis in MCF-7 cell lines as compared to DOX-loaded PEOz-PLA nanoparticles (PP-D NPs) and free DOX. More importantly, anti-phagocytosis and pharmacokinetic studies confirmed that UEPP-D NPs had superior immune escape ability and significantly prolonged the drug's bloodstream circulation in vivo. Finally, UEPP-D NPs showed a markedly higher antitumor efficacy and lower side-toxicity in MCF-7 tumor bearing nude mice model. Thus, this versatile nano-system with immune escape, homologous targeting, and rapid response release characteristics could be a promising tool for breast cancer treatment.