Urinary Extracellular Vesicles Research Articles

Clinical, biochemical, and miRNA profile of subjects with positive screening of primary aldosteronism and nonclassic apparent mineralocorticoid excess.

To identify and characterize clinical and biochemical parameters of subjects with both PA and NCAME conditions (NCAME&PA) and study the miRNA cargo in their urinary extracellular vesicles as potential biomarkers for this novel condition. We performed a cross-sectional study of 206 Chilean adult subjects from a primary care cohort. We measured blood pressure (BP), cortisol (F), cortisone (E), aldosterone, plasma renin activity (PRA), microalbuminuria (MAC), plasma NGAL, MMP9, fractional-potassium-excretion (FEK). Subjects were classified as NCAME&PA, PA, NCAME, essential hypertensives (EH), or healthy controls (CTL). EV-miRNAs were quantified by Taqman-qPCR. We found that 30.6% subjects had an abnormal endocrine phenotype: NCAME&PA (6.8%), PA (11.2%) or NCAME (12.6%), and the prevalence of AHT was 92.9%, 82.6%, and 65%, respectively. NCAME&PA subjects had both lower cortisone (p < 0.05) and lower PRA (p < 0.0001), higher FEK (p = 0.02) and higher MAC (p = 0.01) than EH or CTL. NCAME&PA subjects had also higher NGAL levels than CTL and PA (p < 0.05). Exosome miR-192, miR-133a and miR-21 expression decreased with phenotype severity and correlated with BP and PRA (p < 0.05). We identified adult subjects with a combined condition of NCAME and PA associated with higher BP, increased renal and endothelial damage markers than control and EH. Additionally, we observed a differential expression of a specific miRNAs, suggesting a potential role of these miRNAs associated to this novel combined phenotype.

PetSavers research

Jack Lawson, a lecturer in small animal internal medicine at the Royal Veterinary College, discusses his PetSavers funded research: Urinary extracellular vesicles as a source of biomarkers in feline chronic kidney disease and hypertension.

REDUCED NACL COTRANSPORTER ABUNDANCE AND PHOSPHORYLATION IN URINARY EXTRACELLULAR VESICLES IN RESPONSE TO KCL SUPPLEMENTS IN HEALTHY ADULTS MAINTAINED ON A HIGH-NA+ LOW-K+ DIET: A

Objective: A renal-K switch mechanism has been established in rodent models and demonstrates that a low K+ intake acts as a trigger that links K+ and Na+-dependent blood pressure. This mechanism involves activation (phosphorylation) of the NaCl-cotransporter NCC in the distal convoluted tubule in response to low-K+ intake and suppression (dephosphorylation) after a high-K+ intake. As a first step to establishing a similar mechanism in humans, this study aimed to examine if the abundance of NCC and pNCC in urinary extracellular vesicles (uEVs) of healthy adults on a high-Na+ diet changes in response to alterations in K+ intake. Design and method: The study design was a double-blind, randomised and placebo-controlled trial. Healthy adults maintained on a high-Na+ (∼200mmol/day), low-K+ (∼60mmol/day) diet underwent a run-in period of 1-week followed by a cross-over study, with 5 days supplementary KCl (active phase, Span K, 24mmol thrice daily) or 5 days placebo administered in random order and separated by 2 days washout. NCC and pNCC were measured in uEVs after each phase. Participants who met dietary compliance (no less than 80%) and increased urinary K+ in the active phase were included in analyses. Results: Among the 18 (out of 28) participants who met inclusion criteria, supplementary KCl administration (versus placebo) in the active phase was associated with higher levels of plasma K+ (p &lt; 0.001) and 24-Hour urine levels of K+ (p &lt; 0.001), Cl- (p &lt; 0.001) and aldosterone (p &lt; 0.001), and lower levels of 24-Hour urinary Na+/K+ (p &lt; 0.001), NCC (p &lt; 0.01) and pNCC (p &lt; 0.05); whilst the ratio of pNCC/NCC remained unchanged. Correlation analyses revealed an inverse correlation of plasma K+ with NCC (R2 = 0.11, p = 0.05) and δurinary K+ with δpNCC (R2 = 0.23, p = 0.04). Ambulatory systolic blood pressure positively correlated with timed urinary Na+/K+ (R2 = 0.08, p = 0.04) during interventions.δ Conclusions: KCl supplementation in healthy adult subjects maintained on a high-Na+ low-K+ diet was associated with a fall in NCC and pNCC, consistent with the renal-K switch mechanism, and offers a useful target for dietary intervention.

CircSCAF8 promotes growth and metastasis of prostate cancer through the circSCAF8-miR-140-3p/miR-335-LIF pathway

Circular RNAs (circRNAs) have been increasingly linked to cancer progression. However, the detailed biological functions of circRNAs in prostate cancer (PCa) remain unclear. Using high-throughput circRNA sequencing, we previously identified 18 urine extracellular vesicle circRNAs that were increased in patients with PCa compared with those with benign prostatic hyperplasia. Spearman correlation analysis of the expression levels of the 18 circRNAs between the tumor tissue and matched urine extracellular vesicles in 30 PCa patients showed that circSCAF8 had the highest R2 (R2 = 0.635, P < 0.001). The Cox proportional hazards regression model was used to estimate the effect of circSCAF8 on progression-free survival. The in vitro and in vivo functional experiments were implemented to investigate the effects of circSCAF8 on the phenotype of PCa. We found that the knockdown of circSCAF8 in PCa cells suppressed the proliferation, migration, and invasion ability, while overexpression of circSCAF8 had the opposite effects. Similar results were observed in vivo. In a cohort of 85 patients who had undergone radical prostatectomy, circSCAF8 expression in PCa tissues was a powerful predictor of progression-free survival (HR = 2.14, P = 0.022). Mechanistically, circSCAF8 can function by binding to both miR-140-3p and miR-335 to regulate LIF expression and activate the LIF-STAT3 pathway that leads to the growth and metastasis of PCa. Collectively, our findings demonstrate that circSCAF8 contributes to PCa progression through the circSCAF8-miR-140-3p/miR-335-LIF pathway.

A Simple and Easy Evaluation Method for Urinary Extracellular Vesicles Quality

A Simple and Easy Evaluation Method for Urinary Extracellular Vesicles Quality

Extracellular vesicles in kidney disease.

Extracellular vesicles are released by the majority of cell types and circulate in body fluids. They function as a long-distance cell-to-cell communication mechanism that modulates the gene expression profile and fate of target cells. Increasing evidence has established a central role of extracellular vesicles in kidney physiology and pathology. Urinary extracellular vesicles mediate crosstalk between glomerular and tubular cells and between different segments of the tubule, whereas circulating extracellular vesicles mediate organ crosstalk and are involved in the amplification of kidney damage and inflammation. The molecular profile of extracellular vesicles reflects the type and pathophysiological status of the originating cell so could potentially be exploited for diagnostic and prognostic purposes. In addition, robust preclinical data suggest that administration of exogenous extracellular vesicles could promote kidney regeneration and reduce inflammation and fibrosis in acute and chronic kidney diseases. Stem cells are thought to be the most promising source of extracellular vesicles with regenerative activity. Extracellular vesicles are also attractive candidates for drug delivery and various engineering strategies are being investigated to alter their cargo and increase their efficacy. However, rigorous standardization and scalable production strategies will be necessary to enable the clinical application of extracellular vesicles as potential therapeutics.

The Preliminary Exploration of What Role miRNAs Derived From Urinary Exosomes Play in Kidney Stone Formation

To identify dysregulated microRNAs (miRNAs) derived from urinary small Extracellular Vesicles (sEVs) and to explore the potential roles sEVs miRNAs play in nephrolithiasis. First morning voids were collected from participants. Urinary exosomes were isolated via sequential differential centrifugation and ultracentrifugation. Next-generation sequencing was conducted to analyze the transcriptome of urine sEVs from three Calcium oxalate (CaOx) stone patients and three age-/sex-matched healthy controls. Bioinformatics analysis was performed to explore how dysregulated miRNAs were associated with stone formation. The miRNA sequencing results of sEVs derived from epithelial cells (GSE110509) were compared with the dysregulated miRNAs of our study, and finally we found three common miRNAs. 54 dysregulated miRNAs were found between control and stone groups, among which 14 miRNAs were down-regulated and 40 miRNAs were up-regulated in stone groups compared to control groups. Gene ontology (GO) analysis indicated that dysregulated miRNAs were enriched in oxidative stress (Biological Process), focal adhesion (Cellular Component), and cell adhesion molecule binding (Molecular Function). Kyoto Encyclopedia of Genes and Genomes pathways analysis demonstrated that dysregulated miRNAs were common enriched in MAPK signaling pathway, focal adhesion and AGE-RAGE signaling pathway. The expression of miR-223-3p, miR-127-3p, and miR-214-5p were significantly different in either the sEVs derived from cells (GSE110509) or the sEVs derived from urine. QPCR confirmed that the expression of miR-223-3p was higher in urinary sEVs derived from CaOx stone patients. The expression of miR-223-3p is significantly higher in urinary sEVs derived from CaOx stone patients and may affect stone formation via regulating oxidative stress, focal adhesion and inflammation process.

Generation and analysis of pseudohypoaldosteronism type II knock-in mice caused by a nonsense KLHL3 mutation in the Kelch domain.

Mutations in the Kelch-like 3 (KLHL3) gene are the most common cause of inherited pseudohypoaldosteronism type II (PHAII) featuring thiazide-sensitive hypertension and hyperkalemic metabolic acidosis. Although Klhl3R528H/+ knock-in (KI) mice carrying a missense mutation in the Kelch repeat domain have been reported, nonsense KLHL3mutations in the same domain that cause PHAII have not been fully investigated in vivo. We generated and analyzed Klhl3KI mice harboring a nonsense W523X mutation (corresponding to the human KLHL3W470X mutation). Both heterozygous and homozygous Klhl3W523X/+ KI mice exhibited typical PHAII with low-renin hypertension, hyperkalemia with reduced renal potassium excretion, and hyperchloremic metabolic acidosis. Their kidney tissues showed the presence of Klhl3mRNA and increased Klhl3 protein levels along with enhanced downstream Wnk1/4-Spak/Osr1-N(k)cc phosphorylation. Increased protein expression of total Spak, phosphor(p-)Spak, total Ncc, and p-Ncc from urinary extracellular vesicles (uEVs) also confirmed the activation of the Wnk-mediated Ncc pathway. In vitro studies showed that the human KLHL3W470X mutation resulted in increased KLHL3 protein stability and disrupted its binding affinity for WNK1/4, leading to the attenuated degradation and increased abundance of total WNKs. In conclusion, nonsense Klhl3W523X/+ mice recapitulating PHAII phenotypes exhibit Klhl3 protein stability, abrogating its binding to Wnks, with enhanced Ncc expression in the kidney tissue and even in uEVs. Activation of the WNK-mediated Na+ -Cl- co-transporter reiterated the in vivo pathogenic role of nonsense KLHL3mutations in PHAII.

Acute Intravenous NaCl and Volume Expansion Reduces Sodium-Chloride Cotransporter Abundance and Phosphorylation in Urinary Extracellular Vesicles.

Sodium chloride (NaCl) loading and volume expansion suppress the renin-angiotensin-aldosterone system to reduce renal tubular reabsorption of NaCl and water, but effects on the sodium-chloride cotransporter (NCC) and relevant renal transmembrane proteins that are responsible for this modulation in humans are less well investigated. We used urinary extracellular vesicles (uEVs) as an indirect readout to assess renal transmembrane proteins involved in NaCl and water homeostasis in 44 patients with hypertension who had repeatedly raised aldosterone/renin ratios undergoing infusion of 2 L of 0.9% saline over 4 hours. When measured by mass spectrometry in 13 patients, significant decreases were observed in NCC (median fold change [FC]=0.70); pendrin (FC=0.84); AQP2 (FC=0.62); and uEV markers, including ALIX (FC=0.65) and TSG101 (FC=0.66). Immunoblotting reproduced the reduction in NCC (FC=0.54), AQP2 (FC=0.42), ALIX (FC=0.52), and TSG101 (FC=0.55) in the remaining 31 patients, and demonstrated a significant decrease in phosphorylated NCC (pNCC; FC=0.49). However, after correction for ALIX, the reductions in NCC (FC=0.90) and pNCC (FC=1.00) were no longer apparent, whereas the significant decrease in AQP2 persisted (FC=0.62). We conclude that (1) decreases in NCC and pNCC, induced by acute NaCl loading and volume expansion, may be due to diluted post-test urines; (2) the lack of change of NCC and pNCC when corrected for ALIX, despite a fall in plasma aldosterone, may be due to the lack of change in plasma K+; and (3) the decrease in AQP2 may be due to a decrease in vasopressin in response to volume expansion.

An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure

BackgroundUrinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine.Methods3 EV isolation methods were compared: ultracentrifugation, magnetic bead-based, and size-exclusion chromatography from 0.5 mL or 1 mL of rat and human urine. EV yield and mass spectrometry signals (Q-ToF and Triple Quad) were evaluated from each method. Metabolomic profiling was performed on EVs isolated from the urine of rats exposed to ionizing radiation 1-, 14-, 30- or 90-days post-exposure, and human urine from patients receiving thoracic radiotherapy for the treatment of lung cancer pre- and post-treatment.ResultsSize-exclusion chromatography is the preferred method for EV isolation from 0.5 mL of urine. Mass spectrometry-based metabolomic analyses of EV cargo identified biochemical changes induced by radiation, including altered nucleotide, folate, and lipid metabolism. We have provided standard operating procedures for implementation of these methods in other laboratories.ConclusionsWe demonstrate that EVs can be isolated from small volumes of urine and analytically investigated for their biochemical contents to detect radiation induced metabolomic changes. These findings lay a groundwork for future development of methods to monitor response to radiotherapy and can be extended to an array of molecular phenotyping studies aimed at characterizing EV cargo.

Elevated Urinary Rab10 Phosphorylation in Idiopathic Parkinson Disease.

BackgroundPathogenic leucine‐rich repeat kinase 2 LRRK2 mutations may increase LRRK2 kinase activity and Rab substrate phosphorylation. Genetic association studies link variation in LRRK2 to idiopathic Parkinson disease (iPD) risk.ObjectivesThrough measurements of the LRRK2 kinase substrate pT73‐Rab10 in urinary extracellular vesicles, this study seeks to understand how LRRK2 kinase activity might change with iPD progression.MethodsUsing an immunoblotting approach validated in LRRK2 transgenic mice, the ratio of pT73‐Rab10 to total Rab10 protein was measured in extracellular vesicles from a cross‐section of G2019S LRRK2 mutation carriers (N = 45 participants) as well as 485 urine samples from a novel longitudinal cohort of iPD and controls (N = 85 participants). Generalized estimating equations were used to conduct analyses with commonly used clinical scales.ResultsAlthough the G2019S LRRK2 mutation did not increase pT73‐Rab10 levels, the ratio of pT73‐Rab10 to total Rab10 nominally increased over baseline in iPD urine vesicle samples with time, but did not increase in age‐matched controls (1.34‐fold vs. 1.05‐fold, 95% confidence interval [CI], 0.004–0.56; P = 0.046; Welch's t test). Effect estimates adjusting for sex, age, disease duration, diagnosis, and baseline clinical scores identified increasing total Movement Disorder Society‐Sponsored Revision of the Unified (MDS‐UPDRS) scores (β = 0.77; CI, 0.52–1.01; P = 0.0001) with each fold increase of pT73‐Rab10 to total Rab10. Lower Montreal Cognitive Assessment (MoCA) score in iPD is also associated with increased pT73‐Rab10.ConclusionsThese results provide initial insights into peripheral LRRK2‐dependent Rab phosphorylation, measured in biobanked urine, where higher levels of pT73‐Rab10 are associated with worse disease progression. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

MO619: Landscape RNA Profiling of Urinary Extracellular Vesicles in Patients with Diabetic Nephropathy

Abstract BACKGROUND AND AIMS Recent studies have revealed that urinary extracellular vesicles (uEV) are secreted with rich biological information useful for understanding the pathophysiological conditions and identifying potential biomarkers for kidney disease. Diabetic nephropathy (DN) has become the major cause of CKD worldwide, while the features of uEV in DN were poorly understood. The present study aims to provide the landscape and in-depth analysis of the transcriptome characteristics of uEV in DN. METHOD Urine was collected from a total of 40 subjects, including healthy controls (HC, n = 14), type 2 DM (T2DM, n = 16), and renal biopsy-confirmed DN (n = 10). uEV was isolated by differential centrifugation and total RNA was extracted for long RNA sequencing. A model based on CIBERSORT was calculated to trace the genetic source of uEV toward resident cells of urinary system. Differentially expressed genes (DEG) between DN and HC and DN and T2DM were applied to gene ontology (GO), Reactome pathway enrichment, as well as gene set enrichment analysis (GSEA). Correlation of the transcriptome profiling between uEV and kidney tissue in DN was also explored. RESULTS About 67%∼68% of transcripts in RNA-seq were mRNA, 11%∼12% were LncRNA and 7%∼10% were CircRNA, and the abundance of mRNA was greater than other RNA types. By CIBERSORT model applying marker genes of podocytes, epithelial cells of tubule of different segments, loop of Henle, and collecting duct, and bladder transitional epithelial cells. The uEV was shown to originate mainly from the collecting duct epithelial cells of the kidney and bladder transitional epithelial cells (Fig. 1a). Confocal laser microscopy showed the co-localization of CD63 and AQP2 (Fig. 1b) in kidney tissues. GO and Reactome analysis were further performed in upregulated mRNA between DN and T2DM and DN and HC to explore the mRNA profiling in terms of the biological significance. Impressively, ‘extracellular exosome’ as well as other terms associated with constitution of EV, ‘plasma membrane’ and ‘extracellular space’ were enriched by GO cellular component (CC) analysis in DN. Besides, Reactome analysis identified pathways of ‘immune system’, ‘extracellular matrix organization’ and ‘degradation of extracellular matrix’ reflecting the pathogenesis mechanism of DN (Fig. 1c). Additionally, microarray data of DN glomeruli and tubule from the GEO database were analyzed in comparison to uEV RNA-seq data. GSEA showed that great similarities were found in the top 10 terms of Reactome pathways (Fig. 2). CONCLUSION In conclusion, this study provides a landscape transcriptome profile, the genetic trace and the biological function terms of uEV in DN. uEV holds potentials in reflecting the transcriptome characteristics of renal tissue, which is of significance for understanding the pathogenesis of DN and biomarker discovery.

Urinary extracellular vesicles contain mature transcriptome enriched in circular and long noncoding RNAs with functional significance in prostate cancer.

Long noncoding (lnc)RNAs modulate gene expression alongside presenting unexpected source of neoantigens. Despite their immense interest, their ability to be transferred and control adjacent cells is unknown. Extracellular Vesicles (EVs) offer a protective environment for nucleic acids, with pro and antitumourigenic functions by controlling the immune response. In contrast to extracellular nonvesicular RNA, few studies have addressed the full RNA content within human fluids’ EVs and have compared them with their tissue of origin. Here, we performed Total RNA‐Sequencing on six Formalin‐Fixed‐Paraffin‐Embedded (FFPE) prostate cancer (PCa) tumour tissues and their paired urinary (u)EVs to provide the first whole transcriptome comparison from the same patients. UEVs contain simplified transcriptome with intron‐free cytoplasmic transcripts and enriched lnc/circular (circ)RNAs, strikingly common to an independent 20 patients’ urinary cohort. Our full cellular and EVs transcriptome comparison within three PCa cell lines identified a set of overlapping 14 uEV‐circRNAs characterized as essential for prostate cell proliferation in vitro and 28 uEV‐lncRNAs belonging to the cancer‐related lncRNA census (CLC2). In addition, we found 15 uEV‐lncRNAs, predicted to encode 768 high‐affinity neoantigens, and for which three of the encoded‐ORF produced detectable unmodified peptides by mass spectrometry. Our dual analysis of EVs‐lnc/circRNAs both in urines’ and in vitro’s EVs provides a fundamental resource for future uEV‐lnc/circRNAs phenotypic characterization involved in PCa.

Renal gluconeogenesis induction by glucagon and insulin receptor deletion in humans

Gluconeogenesis is one of the key processes through which the kidney contributes to glucose homeostasis. Recently, we provided evidence to suggest that early insulin resistance may impair fast‐fed regulation of Phosphoenolpyruvate Carboxykinase (PEPCK), rate‐limiting enzyme for gluconeogenesis in humans. To confirm this and to elucidate a link between insulin resistance and fast‐fed regulation of PEPCK in the kidneys, we studied primary proximal tubule cells (hPT) and urinary extracellular vesicles (uEVs) from humans. We demonstrate that fasted hPTs had higher PEPCK and insulin receptor substrate‐2 (IRS2) mRNA levels than fed cells. The fast‐fed regulation was, however, attenuated in insulin receptor knockdown hPTs (IRKO). The insulin receptor knockdown was confirmed by a blunted insulin‐induced suppression in PEPCK and PGC1α mRNA and p‐IR and p‐AKT protein expression in IRKO cells. Exosomes secreted by the wild‐type or IRKO hPT showed similar regulation as observed in the respective hPT. Similarly, in human uE, the relative abundance of IRS‐2 mRNA (to IRS1) was higher in the fasted state relative to the fed condition. However, the fast‐fed difference was absent in subjects with early insulin resistance. These subjects had higher circulating glucagon levels relative to subjects with optimal insulin sensitivity. Furthermore, in hPT cells, glucagon significantly induced PEPCK mRNA, IRS2 mRNA, and gluconeogenesis. Insulin receptor deletion further increased their levels. Together the data suggest that reduced insulin sensitivity and high glucagon in early insulin resistance may impair renal gluconeogenesis via IRS2 regulation. Funding from ICMR‐CARE and SGPGI intramural to ST and CSIR to RS.

Sphingomyelin‐enriched extracellular vesicles from hypertensive diabetic mice regulate calcium mobilization and amiloride‐sensitive transepithelial current in mouse cortical collecting duct cells

Uncontrolled hypertension (HTN) and diabetes mellitus (DM) are two major public health burdens contributing to increased hospitalization, readmission costs, and adverse clinical outcomes. Moreover, DM is a leading cause of death and long‐term morbidity. Overexpression and activity of epithelial sodium channels (ENaC) in the kidney results in hypertension, but the mechanism for its regulation in the diabetic kidney is not entirely understood. Cholesterol is known to increase ENaC activity in cultured kidney cells in a calcium and PI(4,5)P2‐dependent mechanism. Presumably ENaC is regulated by other lipids in pathophysiology. Extracellular vesicles (EVs) are nano‐sized vesicles that play an important role in intercellular communication and intracellular signaling. Here, we investigated whether EVs isolated from the urine of hypertensive diabetic db/db mice or diabetic db/db mice augment ENaC activity by inhibiting calcium mobilization in mpkCCD cells. Adult diabetic db/db mice were salt loaded (4% NaCl) for 7 days to induce hypertension. Blood pressure was measured by tail‐cuff, and urine from db/db mice with systolic blood pressure of greater than 140mmHg or from normotensive db/db mice were used to isolate EVs. Nanoparticle tracking analysis was performed to determine EV concentration. EVs from hypertensive diabetic db/db mice (N=5) compared to diabetic db/db mice (N=5) suppressed ionomycin‐induced calcium mobilization in mpkCCD cells cultured on glass bottom 35 mm dishes (N=3 experiments, p&lt;0.05) which was measured by confocal microscopy and augmented amiloride‐sensitive transepithelial current in mpKCCD cells cultured in 12mm diameter permeable supports (N=6 permeable supports per group) measured by an epithelial volt ohm meter (EVOM). Sphingomyelin were found to be enriched in urinary EVs from hypertensive diabetic db/db mice compared to diabetic only db/db mice. Exogenous application of sphingomyelin to mpkCCD cells mimicked the suppression of calcium mobilization and increase in amiloride‐sensitive transepithelial current in these cells. Taken together, these data suggest differences in the packaged cargo of urinary EVs between HTN and DM mice that presumably plays a role in the regulation of ENaC activity.

Urinary interleukin-9 in youth with type 1 diabetes mellitus

AimsInterleukin-9 (IL-9) attenuates podocyte injury in experimental kidney disease, but its role in diabetic nephropathy is unknown. We sought to relate urinary IL-9 levels to the release of podocyte-derived extracellular vesicles (EVs) in youth with type 1 diabetes. We related urinary IL-9 levels to clinical variables and studied interactions between urinary IL-9, vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) on urinary albumin/creatinine ratio (ACR) a functional measure of podocyte injury.MethodsWe performed an analysis of urine samples and clinical data from a cohort of youth with type 1 diabetes (n = 53). Cytokines were measured using a Luminex platform (Eve Technologies), and nanoscale flow cytometry was employed to quantify urinary podocyte-derived EVs. All urinary measures were normalized to urinary creatinine.ResultsMean age was 14.7 ± 1.6 years, and the mean time from diagnosis was 6.7 ± 2.9 years. Mean HbA1c was 70.3 ± 13.9 mmol/mol, mean ACR was 1.3 ± 1.9 mg/mmol, and mean eGFR was 140.3 ± 32.6 ml/min/1.73 m2. IL-9 was inversely related to podocyte EVs (r = − 0.56, p = 0.003). IL-9 was also inversely related to blood glucose, HbA1C and eGFR (r = − 0.44, p = 0.002; r = − 0.41, p = 0.003; r = − 0.49, p < 0.001, respectively) and positively correlated with systolic BP (r = 0.30, p = 0.04). There was a significant interaction between IL-9, EVs and ACR (p = 0.0143), and the relationship between IL-9 and ACR depended on VEGF (p = 0.0083), TNFα (p = 0.0231) and IL-6 levels (p = 0.0178).ConclusionsIL-9 is associated with podocyte injury in early type 1 diabetes, and there are complex interactions between urinary IL-9, inflammatory cytokines and ACR.

Microbiomes of Urine and the Prostate Are Linked to Human Prostate Cancer Risk Groups

BackgroundBacteria play a suspected role in the development of several cancer types, and associations between the presence of particular bacteria and prostate cancer have been reported. ObjectiveTo provide improved characterisation of the prostate and urine microbiome and to investigate the prognostic potential of the bacteria present. Design, setting, and participantsMicrobiome profiles were interrogated in sample collections of patient urine (sediment microscopy: n = 318, 16S ribosomal amplicon sequencing: n = 46; and extracellular vesicle RNA-seq: n = 40) and cancer tissue (n = 204). Outcome measurements and statistical analysisMicrobiomes were assessed using anaerobic culture, population-level 16S analysis, RNA-seq, and whole genome DNA sequencing. Results and limitationsWe demonstrate an association between the presence of bacteria in urine sediments and higher D’Amico risk prostate cancer (discovery, n = 215 patients, p < 0.001; validation, n = 103, p < 0.001, χ2 test for trend). Characterisation of the bacterial community led to the (1) identification of four novel bacteria (Porphyromonas sp. nov., Varibaculum sp. nov., Peptoniphilus sp. nov., and Fenollaria sp. nov.) that were frequently found in patient urine, and (2) definition of a patient subgroup associated with metastasis development (p = 0.015, log-rank test). The presence of five specific anaerobic genera, which includes three of the novel isolates, was associated with cancer risk group, in urine sediment (p = 0.045, log-rank test), urine extracellular vesicles (p = 0.039), and cancer tissue (p = 0.035), with a meta-analysis hazard ratio for disease progression of 2.60 (95% confidence interval: 1.39–4.85; p = 0.003; Cox regression). A limitation is that functional links to cancer development are not yet established. ConclusionsThis study characterises prostate and urine microbiomes, and indicates that specific anaerobic bacteria genera have prognostic potential. Patient summaryIn this study, we investigated the presence of bacteria in patient urine and the prostate. We identified four novel bacteria and suggest a potential prognostic utility for the microbiome in prostate cancer.

A Model to Detect Significant Prostate Cancer Integrating Urinary Peptide and Extracellular Vesicle RNA Data.

Simple SummaryProstate cancer is one of the leading causes of cancer-related death in men in the world, but a large proportion of men that are diagnosed with prostate cancer do not have a form of the disease that will cause them long term harm. Therefore, there is a need to accurately predict the aggressiveness of the disease without taking an invasive biopsy. In this study, we develop a test that can predict whether a patient has prostate cancer and how aggressive that cancer is. This test combines clinical measurements, levels of four genes collected from a fraction of the urine, and levels of six peptides found in urine. We found that this test, deemed ‘ExoSpec’, has the potential to improve the pathway for men with a clinical suspicion of prostate cancer and could reduce the requirement for biopsies by 30%.There is a clinical need to improve assessment of biopsy-naïve patients for the presence of clinically significant prostate cancer (PCa). In this study, we investigated whether the robust integration of expression data from urinary extracellular vesicle RNA (EV-RNA) with urine proteomic metabolites can accurately predict PCa biopsy outcome. Urine samples collected within the Movember GAP1 Urine Biomarker study (n = 192) were analysed by both mass spectrometry-based urine-proteomics and NanoString gene-expression analysis (167 gene-probes). Cross-validated LASSO penalised regression and Random Forests identified a combination of clinical and urinary biomarkers for predictive modelling of significant disease (Gleason Score (Gs) ≥ 3 + 4). Four predictive models were developed: ‘MassSpec’ (CE-MS proteomics), ‘EV-RNA’, and ‘SoC’ (standard of care) clinical data models, alongside a fully integrated omics-model, deemed ‘ExoSpec’. ExoSpec (incorporating four gene transcripts, six peptides, and two clinical variables) is the best model for predicting Gs ≥ 3 + 4 at initial biopsy (AUC = 0.83, 95% CI: 0.77–0.88) and is superior to a standard of care (SoC) model utilising clinical data alone (AUC = 0.71, p < 0.001, 1000 resamples). As the ExoSpec Risk Score increases, the likelihood of higher-grade PCa on biopsy is significantly greater (OR = 2.8, 95% CI: 2.1–3.7). The decision curve analyses reveals that ExoSpec provides a net benefit over SoC and could reduce unnecessary biopsies by 30%.

Analysis of microRNAs in Small Urinary Extracellular Vesicles and Their Potential Roles in Pathogenesis of Renal ANCA-Associated Vasculitis.

Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) represents an autoimmunity disease characterized by high mortality. For successful treatment, the detailed knowledge of its complex pathogenesis and the set of biomarkers for differential diagnostics are desired. Analysis of molecular content of small urinary extracellular vesicles (uEV) offers the possibility to find markers in the form of microRNAs (miRNAs) and study the pathways involved in pathogenesis. We used next-generation sequencing in the first preliminary study to detect the miRNAs with altered expression in uEVs of patients with AAV in comparison with age-matched controls. We confirmed the results using single-target quantitative polymerase chain reaction tests on different sets of samples and found five miRNAs (miR-30a-5p, miR-31-3p, miR-99a-5p, miR-106b-5p, miR-182-5p) with highly elevated levels in uEVs of patients. We performed the comparison of their targets with the differentially expressed proteins in uEVs of patients included in the first phase. We realized that upregulated miRNAs and proteins in uEVs in AAV patients target different biological pathways. The only overlap was detected in pathways regulating the actin cytoskeleton assembly and thus potentially affecting the glomerular functions. The associations of upregulated miRNAs with pathways that were neglected as components of complex AAV pathogenesis, e.g., the epidermal growth factor receptor signaling pathway, were found.

The Role of Urinary Extracellular Vesicles Sodium Chloride Cotransporter in Subtyping Primary Aldosteronism.

BackgroundAdrenal venous sampling (AVS) is recognized as the gold standard for subtyping primary aldosteronism (PA), but its invasive nature and technical challenges limit its availability. A recent study reported that sodium chloride cotransporter (NCC) in urinary extracellular vesicles (uEVs) is a promising marker for assessing the biological activity of aldosterone and can be treated as a potential biomarker of PA. The current study was conducted to verify the hypothesis that the expression of NCC and its phosphorylated form (pNCC) in uEVs are different in various subtypes and genotypes of PA and can be used to select AVS candidates.MethodsA total of 50 patients with PA were enrolled in the study. Urinary extracellular vesicles (uEVs) were isolated from spot urine samples using ultracentrifugation. NCC and pNCC expressions were tested in patients diagnosed with PA who underwent AVS. Sanger sequencing of KCNJ5 was performed on DNA extracted from adrenal adenoma.ResultspNCC (1.89 folds, P<.0001) and NCC (1.82 folds, P=0.0002) was more abundant in the uEVs in the high lateralization index (h-LI, ≥ 4) group than in the low LI (l-LI, < 4) group. Carriers of the somatic KCNJ5 mutations, compared with non-carriers, had more abundant pNCC expression (2.16 folds, P=0.0039). Positive correlation between pNCC abundance and plasma aldosterone level was found in this study (R = 0.1220, P = 0.0129).ConclusionsThe expression of pNCC in uEVs in patients with PA with various subtypes and genotypes was different. It can be used as biomarker of AVS for PA subtyping.