The genetic source tracking of human urinary exosomes

The genetic origins of nanoscale extracellular vesicles in our body fluids remains unclear. Here, we perform a tracking analysis of urinary exosomes via RNA sequencing, revealing that urine exosomes mostly express tissue-specific genes for the bladder and have close cell-genetic relationships to the endothelial cell, basal cell, monocyte, and dendritic cell. Tracking the differentially expressed genes of cancers and corresponding enrichment analysis show urine exosomes are intensively involved in immune activities, indicating that they may be harnessed as reliable biomarkers of noninvasive liquid biopsy in cancer genomic diagnostics and precision medicine.

Urinary Exosomes Diagnosis of Urological Tumors: A Systematic Review and Meta-Analysis

PurposeExosomes could be released directly into the urine by the urological tumoral cells, so testing urinary exosomes has great potential for non-invasive diagnosis and monitor of urological tumors. The objective of this study is to systematically review and meta-analysis of urinary exosome for urological tumors diagnosis.Materials and MethodsA systematic review of the recent English-language literature was conducted according to the PRISMA statement recommendations (CRD42021250613) using PubMed, Embase, Cochrane Library, Web of Science, and Scopus databases up to April 30, 2021. Risk-of-bias assessment was performed according to the QUADAS 2 tool. The true diagnostic value of urinary exosomes by calculating the number of true positive, false positive, true negative, and false negative, diagnoses by extracting specificity and sensitivity data from the selected literature.ResultsSixteen eligible studies enrolling 3224 patients were identified. The pooled sensitivity and specificity of urinary exosomes as a diagnostic tool in urological tumors were 83% and 88%, respectively. The area under the summary receiver operating characteristic curve was 0.92 (95% CI: 0.89–0.94). Further subgroup analyses showed that our results were stable irrespective of the urinary exosome content type and tumor type.ConclusionUrinary exosomes may serve as novel non-invasive biomarkers for urological cancer detection. Future clinical trial designs must validate and explore their utility in treatment decision-making.Systematic Review Registration[ https://www.crd.york.ac.uk/prospero/], identifier [CRD42021250613].

Abstract MP36: Human Urinary Exosomes Contain Functional ACE2

The Ang II convertase and SARS-COV-2 co-receptor ACE2 is highly expressed on proximal tubules within the kidney. ACE2 is also present in urine and reportedly correlates with various renal pathologies that may reflect enhanced shedding of the peptidase through activation of ADAMs. Indeed, 95 kDa ACE2 is typically detected in urine consistent with a shorter, soluble form of the peptidase; however, the full-length, membrane-bound form of ACE2 (120 kDa) is also evident in urine which is difficult to reconcile with ACE2 shedding. To account for these isoforms, we evaluated ACE2 expression in exosomes isolated from human urine. Morning collections from males [50 to 64 years of age, non-smokers] were immediately processed for exosome isolation by cibacron blue binding of albumin followed by 0.2 μmicron filtration to remove microvesicles and apoptotic bodies, Amicon 100 kDa concentration, and ultracentrifugation (UC) to pellet exosomes. Analysis of the UC pellet fraction revealed the exosomal markers ALIX, CD63 and HSP70, as well as the proximal tubule peptidases neprilysin (NEP) and ACE2. Exosomal ACE2 content was 45 ± 11 ng/mL (mean ± SEM; N=5) by ELISA and exosomal activity hydrolyzed Ang II to Ang-(1-7) that was abolished by the ACE2 inhibitor MLN4760. Fluorescent nanotracking analysis (f-NTA) with Alexa Fluor antibodies and CellMask Deep Red membrane stain (CMDR) demonstrate a similar density of ACE2+ and NEP+ exosomes that were ~50% of total urinary exosomes (*P<0.05 vs. CD63+, N=3) while particle sizes were comparable and in the expected range of exosomes (100-150 nm). We conclude that human urinary exosomes express functional ACE2 which may originate from proximal tubule release.

Detection of Urinary Exosomal HSD11B2 mRNA Expression: A Useful Novel Tool for the Diagnostic Approach of Dysfunctional 11β-HSD2-Related Hypertension

ObjectiveApparent mineralocorticoid excess (AME) is an autosomal recessive disorder caused by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme deficiency, traditionally assessed by measuring either the urinary cortisol metabolites ratio (tetrahydrocortisol+allotetrahydrocortisol/tetrahydrocortisone, THF+5αTHF/THE) or the urinary cortisol/cortisone (F/E) ratio. Exosomal mRNA is an emerging diagnostic tool due to its stability in body fluids and its biological regulatory function. It is unknown whether urinary exosomal HSD11B2 mRNA is related to steroid ratio or the HSD11B2 662 C&gt;G genotype (corresponding to a 221 A&gt;G substitution) in patients with AME and essential hypertension (EH).Aim of the StudyTo detect and quantify HSD11B2 mRNA from urinary exosomes in samples from family members affected by AME and EH, and to evaluate the relationship between exosomal HSD11B2 mRNA, steroid ratio, 662C&gt;G genotype, and hypertension.MethodsIn this observational case–control study, urinary steroid ratios and biochemical parameters were measured. Urinary exosomes were extracted from urine and exosomal HSD11B2 mRNA was quantified by Droplet Digital PCR (ddPCR). B2M (β-2 microglobulin) gene was selected as the reference housekeeping gene.ResultsAmong family members affected by AME, exosomal urinary HSD11B2 mRNA expression was strictly related to genotypes. The two homozygous mutant probands showed the highest HSD11B2 mRNA levels (median 169, range 118–220 copies/µl) that progressively decreased in 221 AG heterozygous with hypertension (108, range 92–124 copies/µl), 221 AG heterozygous normotensives (23.35, range 8–38.7 copies/µl), and wild-type 221 AA subjects (5.5, range 4.5–14 copies/µl). Heterozygous hypertensive subjects had more HSD11B2 mRNA than heterozygous normotensive subjects. The F/E urinary ratio correlated with HSD11B2 mRNA copy number (p &lt; 0.05); HSD11B2 mRNA strongly decreased while THF+5αTHF/THE increased in the two probands after therapy. In the AME family, HSD11B2 copy number correlated with both F/E and THF+5αTHF/THE ratios, whereas in EH patients, a high F/E ratio reflected a reduced HSD11B2 mRNA expression.ConclusionsHSD11B2 mRNA is detectable and quantifiable in urinary exosomes; its expression varies according to the 662 C&gt;G genotype with the highest levels in homozygous mutant subjects. The HSD11B2 mRNA overexpression in AME could be due to a compensatory mechanism of the enzyme impairment. Exosomal mRNA is a useful tool to investigate HSD11B2 dysregulation in hypertension.

Proteomic Profiling of Tissue Exosomes Indicates Continuous Release of Malignant Exosomes in Urinary Bladder Cancer Patients, Even with Pathologically Undetectable Tumour

Invasive urothelial bladder cancer (UBC) has high recurrence rates even after radical cystectomy (RC). Exosomes are membrane-bound nanovesicles, which have been shown to contribute to carcinogenesis and metastasis. We previously showed that urinary exosomes display a malignant profile in UBC patients despite the absence of detectable tumour. Here, we investigated exosomes from sampling sites close to or distant from the former tumour, aiming to understand the effect of the tumour on the local milieu. Ten patients scheduled for cystectomy after transurethral bladder resection (TUR-B), without remaining detectable tumour, were included. Exosomes were isolated from tissue explants of both the previous tumour site and distant bladder tissue. Proteins were quantified by mass spectrometry in seven patients. Exosomes from the previous tumour site were enriched in inflammatory but not cancer-related pathways compared to distant tissue. However, the 69 most abundant proteins in tissue-derived exosomes regardless of site, 20 of which were also found in urinary exosomes from our previous study, were enriched for cancer-related metabolic pathways and associated with poor prognosis in an external mRNA dataset. The enrichment of cancer-related pathways in the most abundant proteins, regardless of sampling site, confirms our hypothesis that despite the absence of detectable tumour, the entire bladder releases exosomes that contribute to metastasis and highlights the need for early RC.