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Maynard DM, Gochuico BR, Pri Chen H, Bleck CKE, Zerfas PM, Introne WJ, Gahl WA, Malicdan MCV. Insights into the renal pathophysiology in Hermansky-Pudlak syndrome-1 from urinary extracellular vesicle proteomics and a new mouse model. FEBS Lett 2024. [PMID: 39739361 DOI: 10.1002/1873-3468.15088] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2024] [Revised: 11/07/2024] [Accepted: 12/01/2024] [Indexed: 01/02/2025]
Abstract
Hermansky-Pudlak syndrome type 1 (HPS-1) is a rare, autosomal recessive disorder caused by defects in the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Impaired kidney function is among its clinical manifestations. To investigate HPS-1 renal involvement, we employed 1D-gel-LC-MS/MS and compared the protein composition of urinary extracellular vesicles (uEVs) from HPS-1 patients to normal control individuals. We identified 1029 proteins, 149 of which were altered in HPS-1 uEVs. Ingenuity Pathway Analysis revealed disruptions in mitochondrial function and the LXR/RXR pathway that regulates lipid metabolism, which is supported by our novel Hps1 knockout mouse. Serum concentration of the LXR/RXR pathway protein ApoA1 in our patient cohort was positively correlated with kidney function (with the estimated glomerular filtration rate or eGFR). uEVs can be used to study epithelial cell protein trafficking in HPS-1 and may provide outcome measures for HPS-1 therapeutic interventions.
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Affiliation(s)
- Dawn M Maynard
- Section on Human Biochemical Genetics, Medical Genetics Branch, NHGRI, National Institutes of Health, Bethesda, MD, USA
| | - Bernadette R Gochuico
- Section on Human Biochemical Genetics, Medical Genetics Branch, NHGRI, National Institutes of Health, Bethesda, MD, USA
| | - Hadass Pri Chen
- Section on Human Biochemical Genetics, Medical Genetics Branch, NHGRI, National Institutes of Health, Bethesda, MD, USA
| | - Christopher K E Bleck
- National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA
| | - Patricia M Zerfas
- Office of Research Services, Office of the Director, National Institutes of Health, Bethesda, MD, USA
| | - Wendy J Introne
- Section on Human Biochemical Genetics, Medical Genetics Branch, NHGRI, National Institutes of Health, Bethesda, MD, USA
| | - William A Gahl
- Section on Human Biochemical Genetics, Medical Genetics Branch, NHGRI, National Institutes of Health, Bethesda, MD, USA
| | - May C V Malicdan
- Section on Human Biochemical Genetics, Medical Genetics Branch, NHGRI, National Institutes of Health, Bethesda, MD, USA
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2
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Hayama Y, Kuribayashi-Okuma E, Fujii N, Ochiai-Homma F, Yamazaki O, Tsurutani Y, Nishikawa T, Shibata S. ENaCγ in Urinary Extracellular Vesicles as an Indicator of MR Signaling in Primary Aldosteronism. Hypertension 2024; 81:2457-2467. [PMID: 39319458 DOI: 10.1161/hypertensionaha.124.23379] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2024] [Accepted: 09/12/2024] [Indexed: 09/26/2024]
Abstract
BACKGROUND Aldosterone and the MR (mineralocorticoid receptor) are important therapeutic targets for hypertension and cardiovascular diseases. However, biomarkers of tissue MR signaling are not fully established. Extracellular vesicles released from eukaryotic cells can provide information on tissue signaling. Using samples from patients with primary aldosteronism (PA), we explored the potential of urinary extracellular vesicles (uEVs) as a noninvasive indicator of MR signaling to guide treatment. METHODS We analyzed proteins contained in PA uEVs by liquid chromatography tandem mass spectrometry. We narrowed down candidate biomarkers by referring to an existing database of urinary exosomes. The results were validated through Western blot analysis involving 63 patients with PA and 11 healthy volunteers. RESULTS We identified a total of 1940 proteins in PA uEVs. Comparative analysis with the existing database narrowed down the pathways enriched in PA uEVs, which were related to diabetic complications, Rac1 signaling, and aldosterone-regulated sodium reabsorption. A closer look at the identified proteins revealed ENaCγ (epithelial Na+ channel γ) peptides near the proteolytic cleavage sites, and Western blot analysis confirmed the predominant presence of cleaved ENaCγ, a marker of aldosterone signaling in renal tubules. In PA uEVs, cleaved ENaCγ showed a 4.8-fold increase compared with healthy volunteers and was significantly correlated with the aldosterone-to-renin ratio, aldosterone levels, and fractional excretion of K+. Targeted treatment in PA reduced the abundance of cleaved ENaCγ, suggesting a causal role for MR in its induction. CONCLUSIONS This study provides a list of proteins contained in PA uEVs and suggests that ENaCγ in uEVs is a promising biomarker for renal MR signaling.
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Affiliation(s)
- Yuto Hayama
- Division of Nephrology, Department of Internal Medicine, Teikyo University School of Medicine, Japan (Y.H., E.K.-O., F.O.-H., O.Y., S.S.)
| | - Emiko Kuribayashi-Okuma
- Division of Nephrology, Department of Internal Medicine, Teikyo University School of Medicine, Japan (Y.H., E.K.-O., F.O.-H., O.Y., S.S.)
| | - Norihiko Fujii
- Radioisotope Research Center (N.F.), Teikyo University, Japan
| | - Fumika Ochiai-Homma
- Division of Nephrology, Department of Internal Medicine, Teikyo University School of Medicine, Japan (Y.H., E.K.-O., F.O.-H., O.Y., S.S.)
| | - Osamu Yamazaki
- Division of Nephrology, Department of Internal Medicine, Teikyo University School of Medicine, Japan (Y.H., E.K.-O., F.O.-H., O.Y., S.S.)
| | - Yuya Tsurutani
- Endocrionology and Diabetes Center, Yokohama Rosai Hospital, Japan (Y.T., T.N.)
| | - Tetsuo Nishikawa
- Endocrionology and Diabetes Center, Yokohama Rosai Hospital, Japan (Y.T., T.N.)
| | - Shigeru Shibata
- Division of Nephrology, Department of Internal Medicine, Teikyo University School of Medicine, Japan (Y.H., E.K.-O., F.O.-H., O.Y., S.S.)
- Health Science Research Sector, Advanced Comprehensive Research Organization (ACRO) (S.S.), Teikyo University, Japan
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3
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Hallal SM, Sida LA, Tűzesi Á, Shivalingam B, Sim H, Buckland ME, Satgunaseelan L, Alexander KL. Size matters: Biomolecular compositions of small and large extracellular vesicles in the urine of glioblastoma patients. JOURNAL OF EXTRACELLULAR BIOLOGY 2024; 3:e70021. [PMID: 39554867 PMCID: PMC11565258 DOI: 10.1002/jex2.70021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/17/2024] [Revised: 10/10/2024] [Accepted: 10/24/2024] [Indexed: 11/19/2024]
Abstract
The promise of urinary extracellular vesicles (uEVs) in biomarker discovery is emerging. However, the characteristics and compositions of different uEV subpopulations across normal physiological and pathological states require rigorous explication. We recently reported proteomic signatures of small (s)-uEVs (<200 nm membranous nanoparticles) and described putative biomarkers corresponding to the diagnosis, tumour burden and recurrence of the lethal adult primary brain tumour, glioblastoma. Here, we comprehensively characterise uEV populations with significantly different mean and mode particle sizes obtained by differential centrifugation at 100,000 × g (100K-uEVs; smaller) and 17,000 × g (17K-uEVs; larger) using Fourier-transform infrared spectroscopy and quantitative data-independent acquisition mass spectrometry. We show distinct differences in protein and lipid content, prominent protein secondary structures, and proteome distributions between uEV populations that can distinguish glioblastoma patients from healthy controls and correspond to clinically relevant tumour changes (i.e., recurrence and treatment resistance). Among the key findings is a putative seven-protein biomarker panel associated with 17K-uEVs that could distinguish all glioblastoma patients from healthy controls and accurately classify 98.2% of glioblastoma samples. These novel, significant findings demonstrate that both uEV populations offer individual and combined biomarker potential. Further research is warranted to elucidate the complete diagnostic, prognostic, and predictive capabilities of often-neglected 17K-uEV populations.
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Affiliation(s)
- Susannah M. Hallal
- Neurosurgery DepartmentChris O'Brien LifehouseCamperdownNSWAustralia
- Department of NeuropathologyRoyal Prince Alfred HospitalCamperdownNSWAustralia
- School of Medical SciencesThe University of SydneyCamperdownNSWAustralia
| | - Liam A. Sida
- School of Medical SciencesThe University of SydneyCamperdownNSWAustralia
| | - Ágota Tűzesi
- Department of NeuropathologyRoyal Prince Alfred HospitalCamperdownNSWAustralia
- School of Medical SciencesThe University of SydneyCamperdownNSWAustralia
| | - Brindha Shivalingam
- Neurosurgery DepartmentChris O'Brien LifehouseCamperdownNSWAustralia
- Neurosurgery DepartmentRoyal Prince Alfred HospitalCamperdownNSWAustralia
- Sydney Medical School, Faculty of Medicine and Health SciencesThe University of SydneyCamperdownNSWAustralia
| | - Hao‐Wen Sim
- Department of Medical OncologyChris O'Brien LifehouseCamperdownNSWAustralia
- NHMRC Clinical Trials CentreThe University of SydneyCamperdownNSWAustralia
- Faculty of Medicine and HealthUniversity of New South WalesKensingtonNSWAustralia
| | - Michael E. Buckland
- Department of NeuropathologyRoyal Prince Alfred HospitalCamperdownNSWAustralia
- School of Medical SciencesThe University of SydneyCamperdownNSWAustralia
| | - Laveniya Satgunaseelan
- Neurosurgery DepartmentChris O'Brien LifehouseCamperdownNSWAustralia
- Department of NeuropathologyRoyal Prince Alfred HospitalCamperdownNSWAustralia
- Sydney Medical School, Faculty of Medicine and Health SciencesThe University of SydneyCamperdownNSWAustralia
| | - Kimberley L. Alexander
- Neurosurgery DepartmentChris O'Brien LifehouseCamperdownNSWAustralia
- Department of NeuropathologyRoyal Prince Alfred HospitalCamperdownNSWAustralia
- School of Medical SciencesThe University of SydneyCamperdownNSWAustralia
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4
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Harding MA, Yavuz H, Gathmann A, Upson S, Swiatecka‐Urban A, Erdbrügger U. Uromodulin and the study of urinary extracellular vesicles. JOURNAL OF EXTRACELLULAR BIOLOGY 2024; 3:e70022. [PMID: 39582686 PMCID: PMC11583080 DOI: 10.1002/jex2.70022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/28/2024] [Revised: 10/05/2024] [Accepted: 10/25/2024] [Indexed: 11/26/2024]
Abstract
Urinary extracellular vesicles (uEVs) are a promising substrate for discovering new biomarkers. In order to investigate the origin of uEVs and the cargo they carry, some types of downstream analysis of uEVs may require concentration and enrichment as well as removal of contaminating substances. Co-isolation of the abundant urinary protein uromodulin with uEVs can be a problem, and may interfere with some techniques, in particular with proteomic analysis tools. Methods of separating out uromodulin and its removal have also not been standardized. This review highlights aspects of uromodulin structure that makes it recalcitrant to separation from uEVs, summarizes frequently used techniques for uEV enrichment and how they affect uromodulin separation, and specific methods for uromodulin removal during preparation of uEVs. The necessity of uromodulin removal for various study endpoints is also examined.
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Affiliation(s)
- Michael A. Harding
- Division of Nephrology, Department of MedicineUniversity of VirginiaCharlottesvilleVirginiaUSA
| | - Hayrettin Yavuz
- Division of Pediatric Nephrology, Department of MedicineUniversity of VirginiaCharlottesvilleVirginiaUSA
| | | | - Samantha Upson
- Division of Nephrology, Department of MedicineUniversity of VirginiaCharlottesvilleVirginiaUSA
| | - Agnieszka Swiatecka‐Urban
- Division of Pediatric Nephrology, Department of MedicineUniversity of VirginiaCharlottesvilleVirginiaUSA
| | - Uta Erdbrügger
- Division of Nephrology, Department of MedicineUniversity of VirginiaCharlottesvilleVirginiaUSA
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5
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Smack C, Johnson B, Nyalwidhe JO, Semmes OJ, Yang L. Small extracellular vesicles: Roles and clinical application in prostate cancer. Adv Cancer Res 2024; 161:119-190. [PMID: 39032949 DOI: 10.1016/bs.acr.2024.05.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/23/2024]
Abstract
Prostate cancer is a significant health problem in the United States. It is remarkably heterogenous, ranging from slow growing disease amenable to active surveillance to highly aggressive forms requiring active treatments. Therefore, being able to precisely determine the nature of disease and appropriately match patients to available and/or novel therapeutics is crucial to improve patients' overall outcome and quality of life. Recently small extracellular vesicles (sEVs), a subset of nanoscale membranous vesicles secreted by various cells, have emerged as important analytes for liquid biopsy and promising vehicles for drug delivery. sEVs contain various biomolecules such as genetic material, proteins, and lipids that recapitulate the characteristics and state of their donor cells. The application of existing and newly developed technologies has resulted in an increased depth of knowledge about biophysical structures, biogenesis, and functions of sEVs. In prostate cancer patients, tumor-derived sEVs can be isolated from biofluids, commonly urine and blood. They mediate intercellular signaling within the tumor microenvironment and distal organ-specific sites, supporting cancer initiation, progression, and metastasis. A mounting body of evidence suggests that sEV components can be potent biomarkers for prostate cancer diagnosis, prognosis, and prediction of disease progression and treatment response. Due to enhanced circulation stability and bio-barrier permeability, sEVs can be also used as effective drug delivery carriers to improve the efficacy and specificity of anti-tumor therapies. This review discusses recent studies on sEVs in prostate cancer and is focused on their role as biomarkers and drug delivery vehicles in the clinical management of prostate cancer.
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Affiliation(s)
- Caleb Smack
- Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, VA, United States; Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA, United States
| | - Benjamin Johnson
- Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, VA, United States; Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA, United States
| | - Julius O Nyalwidhe
- Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, VA, United States; Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA, United States
| | - O John Semmes
- Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, VA, United States; Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA, United States
| | - Lifang Yang
- Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, VA, United States; Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA, United States.
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6
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Zhang G, Ding Y, Zhang H, Wei D, Liu Y, Sun J, Xie Z, Tao WA, Zhu Y. Assessment of urine sample collection and processing variables for extracellular vesicle-based proteomics. Analyst 2024; 149:3416-3424. [PMID: 38716512 DOI: 10.1039/d4an00296b] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/11/2024]
Abstract
Extracellular vesicles (EVs) in urine are a promising source for developing non-invasive biomarkers. However, urine concentration and content are highly variable and dynamic, and actual urine collection and handling often is nonideal. Furthermore, patients such as those with prostate diseases have challenges in sample collection due to difficulties in holding urine at designated time points. Here, we simulated the actual situation of clinical sample collection to examine the stability of EVs in urine under different circumstances, including urine collection time and temporary storage temperature, as well as daily urine sampling under different diet conditions. EVs were isolated using functionalized EVtrap magnetic beads and characterized by nanoparticle tracking analysis (NTA), western blotting, electron microscopy, and mass spectrometry (MS). EVs in urine remained relatively stable during temporary storage for 6 hours at room temperature and for 12 hours at 4 °C, while significant fluctuations were observed in EV amounts from urine samples collected at different time points from the same individuals, especially under certain diets. Sample normalization with creatinine reduced the coefficient of variation (CV) values among EV samples from 17% to approximately 6% and facilitated downstream MS analyses. Finally, based on the results, we applied them to evaluate potential biomarker panels in prostate cancer by data-independent acquisition (DIA) MS, presenting the recommendation that can facilitate biomarker discovery with nonideal handling conditions.
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Affiliation(s)
- Guiyuan Zhang
- School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China.
- Bell Mountain Molecular MedTech Institute, Nanjing 210032, China
- EVLiXiR Biotech, Nanjing 210032, China
| | - Yajie Ding
- School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China.
| | - Hao Zhang
- School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China.
- EVLiXiR Biotech, Nanjing 210032, China
| | - Dong Wei
- School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China.
- Bell Mountain Molecular MedTech Institute, Nanjing 210032, China
| | - Yufeng Liu
- School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China.
- Bell Mountain Molecular MedTech Institute, Nanjing 210032, China
| | - Jie Sun
- School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China.
| | - Zhuoying Xie
- School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China.
| | - W Andy Tao
- Departments of Chemistry and Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA
| | - Yefei Zhu
- School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China.
- Laboratory Medicine Center, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China
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7
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Ma L, Wu Q, You Y, Zhang P, Tan D, Liang M, Huang Y, Gao Y, Ban Y, Chen Y, Yuan J. Neuronal small extracellular vesicles carrying miR-181c-5p contribute to the pathogenesis of epilepsy by regulating the protein kinase C-δ/glutamate transporter-1 axis in astrocytes. Glia 2024; 72:1082-1095. [PMID: 38385571 DOI: 10.1002/glia.24517] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2023] [Revised: 01/17/2024] [Accepted: 02/05/2024] [Indexed: 02/23/2024]
Abstract
Information exchange between neurons and astrocytes mediated by extracellular vesicles (EVs) is known to play a key role in the pathogenesis of central nervous system diseases. A key driver of epilepsy is the dysregulation of intersynaptic excitatory neurotransmitters mediated by astrocytes. Thus, we investigated the potential association between neuronal EV microRNAs (miRNAs) and astrocyte glutamate uptake ability in epilepsy. Here, we showed that astrocytes were able to engulf epileptogenic neuronal EVs, inducing a significant increase in the glutamate concentration in the extracellular fluid of astrocytes, which was linked to a decrease in glutamate transporter-1 (GLT-1) protein expression. Using sequencing and gene ontology (GO) functional analysis, miR-181c-5p was found to be the most significantly upregulated miRNA in epileptogenic neuronal EVs and was linked to glutamate metabolism. Moreover, we found that neuronal EV-derived miR-181c-5p interacted with protein kinase C-delta (PKCδ), downregulated PKCδ and GLT-1 protein expression and increased glutamate concentrations in astrocytes both in vitro and in vivo. Our findings demonstrated that epileptogenic neuronal EVs carrying miR-181c-5p decrease the glutamate uptake ability of astrocytes, thus promoting susceptibility to epilepsy.
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Affiliation(s)
- Limin Ma
- Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
- Department of Neurology, Chongqing University Three Gorges Hospital, Chongqing, China
| | - Qingyuan Wu
- Department of Neurology, Chongqing University Three Gorges Hospital, Chongqing, China
| | - Yu You
- Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Peng Zhang
- Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Dandan Tan
- Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Minxue Liang
- Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Yunyi Huang
- Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Yuan Gao
- Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Yuenan Ban
- Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Yangmei Chen
- Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Jinxian Yuan
- Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
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8
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Bielopolski D, Musante L, Hoorn EJ, Molina H, Barrows D, Carrol TS, Harding MA, Upson S, Qureshi A, Weder MM, Tobin JN, Kost RG, Erdbrügger U. Effect of the DASH diet on the sodium-chloride cotransporter and aquaporin-2 in urinary extracellular vesicles. Am J Physiol Renal Physiol 2024; 326:F971-F980. [PMID: 38634133 PMCID: PMC11386975 DOI: 10.1152/ajprenal.00274.2023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2023] [Revised: 04/05/2024] [Accepted: 04/06/2024] [Indexed: 04/19/2024] Open
Abstract
The dietary approach to stop hypertension (DASH) diet combines the antihypertensive effect of a low sodium and high potassium diet. In particular, the potassium component of the diet acts as a switch in the distal convoluted tubule to reduce sodium reabsorption, similar to a diuretic but without the side effects. Previous trials to understand the mechanism of the DASH diet were based on animal models and did not characterize changes in human ion channel protein abundance. More recently, protein cargo of urinary extracellular vesicles (uEVs) has been shown to mirror tissue content and physiological changes within the kidney. We designed an inpatient open label nutritional study transitioning hypertensive volunteers from an American style diet to DASH diet to examine physiological changes in adults with stage 1 hypertension otherwise untreated (Sacks FM, Svetkey LP, Vollmer WM, Appel LJ, Bray GA, Harsha D, Obarzanek E, Conlin PR, Miller ER 3rd, Simons-Morton DG, Karanja N, Lin PH; DASH-Sodium Collaborative Research Group. N Engl J Med 344: 3-10, 2001). Urine samples from this study were used for proteomic characterization of a large range of pure uEVs (small to large) to reveal kidney epithelium changes in response to the DASH diet. These samples were collected from nine volunteers at three time points, and mass spectrometry identified 1,800 proteins from all 27 samples. We demonstrated an increase in total SLC12A3 [sodium-chloride cotransporter (NCC)] abundance and a decrease in aquaporin-2 (AQP2) in uEVs with this mass spectrometry analysis, immunoblotting revealed a significant increase in the proportion of activated (phosphorylated) NCC to total NCC and a decrease in AQP2 from day 5 to day 11. This data demonstrates that the human kidney's response to nutritional interventions may be captured noninvasively by uEV protein abundance changes. Future studies need to confirm these findings in a larger cohort and focus on which factor drove the changes in NCC and AQP2, to which degree NCC and AQP2 contributed to the antihypertensive effect and address if some uEVs function also as a waste pathway for functionally inactive proteins rather than mirroring protein changes.NEW & NOTEWORTHY Numerous studies link DASH diet to lower blood pressure, but its mechanism is unclear. Urinary extracellular vesicles (uEVs) offer noninvasive insights, potentially replacing tissue sampling. Transitioning to DASH diet alters kidney transporters in our stage 1 hypertension cohort: AQP2 decreases, NCC increases in uEVs. This aligns with increased urine volume, reduced sodium reabsorption, and blood pressure decline. Our data highlight uEV protein changes as diet markers, suggesting some uEVs may function as waste pathways. We analyzed larger EVs alongside small EVs, and NCC in immunoblots across its molecular weight range.
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Affiliation(s)
- Dana Bielopolski
- The Rockefeller University Center for Clinical and Translational Science, New York, New York, United States
| | - Luca Musante
- School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States
| | - Ewout J Hoorn
- Division of Nephrology and Transplantation, Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands
| | - Henrik Molina
- Proteomics Resource Center, Rockefeller University, New York, New York, United States
| | - Douglas Barrows
- Bioinformatics Resource Center, Rockefeller University, New York, New York, United States
| | - Thomas S Carrol
- Bioinformatics Resource Center, Rockefeller University, New York, New York, United States
| | - Michael A Harding
- Division of Nephrology, Department of Medicine, University of Virginia at Charlottesville, Charlottesville, Virginia, United States
| | - Samantha Upson
- Division of Nephrology, Department of Medicine, University of Virginia at Charlottesville, Charlottesville, Virginia, United States
| | - Adam Qureshi
- The Rockefeller University Center for Clinical and Translational Science, New York, New York, United States
| | - Max M Weder
- Division of Pulmonology, Department of Medicine, University of Virginia at Charlottesville, Charlottesville, Virginia, United States
| | - Jonathan N Tobin
- The Rockefeller University Center for Clinical and Translational Science, New York, New York, United States
- Clinical Directors Network, New York, New York, United States
| | - Rhonda G Kost
- The Rockefeller University Center for Clinical and Translational Science, New York, New York, United States
| | - U Erdbrügger
- Division of Nephrology, Department of Medicine, University of Virginia at Charlottesville, Charlottesville, Virginia, United States
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9
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Le LNH, Munir J, Kim EB, Ryu S. Kidney Cancer and Potential Use of Urinary Extracellular Vesicles. Oncol Rev 2024; 18:1410450. [PMID: 38846051 PMCID: PMC11153667 DOI: 10.3389/or.2024.1410450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2024] [Accepted: 05/08/2024] [Indexed: 06/09/2024] Open
Abstract
Kidney cancer is the 14th most common cancer globally. The 5-year relative survival rate of kidney cancer at a localized stage is 92.9% and it declines to 17.4% in metastatic stage. Currently, the most accurate method of its diagnosis is tissue biopsy. However, the invasive and costly nature of biopsies makes it undesirable in many patients. Therefore, novel biomarkers for diagnosis and prognosis should be explored. Urinary extracellular vesicles (uEVs) are small vesicles (50-200 nm) in urine carrying nucleic acids, proteins and lipids as their cargos. These uEVs' cargos can provide non-invasive alternative to monitor kidney health. In this review, we have summarized recent studies investigating potential use of uEVs' cargos as biomarkers in kidney cancer for diagnosis, prognosis and therapeutic intervention.
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Affiliation(s)
- Linh Nguy-Hoang Le
- Department of Integrated Biomedical Science, Soonchunhyang University, Cheonan, Republic of Korea
- Soonchunhyang Institute of Med-Bio Science (SIMS), Soonchunhyang University, Cheonan, Republic of Korea
| | - Javaria Munir
- Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE, United States
| | - Eun-Bit Kim
- Soonchunhyang Institute of Med-Bio Science (SIMS), Soonchunhyang University, Cheonan, Republic of Korea
| | - Seongho Ryu
- Department of Integrated Biomedical Science, Soonchunhyang University, Cheonan, Republic of Korea
- Soonchunhyang Institute of Med-Bio Science (SIMS), Soonchunhyang University, Cheonan, Republic of Korea
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10
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de Andrade CES, Souza KSCD, Galdino OA, de Lima MAF, de Medeiros PJ, Abbott Galvão Ururahy M, Pereira MG, de Almeida JB, de Rezende AA. New-onset diabetes after kidney transplantation: Assessing urinary Wilm's tumor-1 protein to predict renal allograft dysfunction. Adv Med Sci 2024; 69:153-159. [PMID: 38490331 DOI: 10.1016/j.advms.2024.03.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2023] [Revised: 12/21/2023] [Accepted: 03/12/2024] [Indexed: 03/17/2024]
Abstract
PURPOSE New-onset diabetes after transplantation (NODAT) is a frequent metabolic complication associated with podocyte damage and renal allograft dysfunction. Thus, Wilm's tumor-1 (WT-1) protein, as a podocyte marker, holds promise as an option to evaluate renal allograft dysfunction in NODAT. Therefore, the study aimed to investigate urinary WT-1 levels in NODAT patients during the first year after kidney transplantation (KTx). MATERIALS AND METHODS KTx patients were categorized into non-NODAT and NODAT groups. Fasting blood glucose, glycated hemoglobin (HbA1c), urinary albumin/creatinine ratio (ACR), serum creatinine, estimated glomerular filtration rate (eGFR), and urinary WT-1 were measured at 3, 6, 9, and 12-months post-KTx. RESULTS The NODAT group manifested elevated levels of blood glucose and HbA1c during the first year post-KTx. Also, exhibited elevations in ACR and serum creatinine levels at 6, 9, and 12-months post-KTx when compared to non-NODAT group. Conversely, eGFR values in the NODAT group demonstrated significant declines at 3, 6, and 9-months post-KTx relative to non-NODAT. Furthermore, NODAT group exhibited a median annual eGFR of 47 mL/min/1.73 m2. Urinary WT-1 levels at 3, 6, 9, and 12-months post-KTx were significantly higher in the NODAT group compared to non-NODAT. Additionally, noteworthy positive correlations were identified between urinary WT-1 and HbA1c levels, along with significant negative correlations between urinary WT-1 and eGFR at the 3, 6, 9, and 12-months post-KTx. CONCLUSION The increased urinary WT-1 levels from 3-months post-KTx in NODAT patients may indicate the first sign of podocyte injury, predicting a renal allograft dysfunction in these patients.
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Affiliation(s)
| | - Karla Simone Costa de Souza
- Department of Clinical and Toxicological Analyses, Federal University of Rio Grande do Norte, Natal, RN, Brazil
| | - Ony Araújo Galdino
- Department of Clinical and Toxicological Analyses, Federal University of Rio Grande do Norte, Natal, RN, Brazil
| | | | - Paulo José de Medeiros
- Division of Nephrology, Department of Integrated Medicine, Federal University of Rio Grande do Norte, Natal, RN, Brazil
| | | | - Maurício Galvão Pereira
- Division of Nephrology, Department of Integrated Medicine, Federal University of Rio Grande do Norte, Natal, RN, Brazil
| | - José Bruno de Almeida
- Division of Nephrology, Department of Integrated Medicine, Federal University of Rio Grande do Norte, Natal, RN, Brazil
| | - Adriana Augusto de Rezende
- Department of Clinical and Toxicological Analyses, Federal University of Rio Grande do Norte, Natal, RN, Brazil.
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11
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Rufo J, Zhang P, Wang Z, Gu Y, Yang K, Rich J, Chen C, Zhong R, Jin K, He Y, Xia J, Li K, Wu J, Ouyang Y, Sadovsky Y, Lee LP, Huang TJ. High-yield and rapid isolation of extracellular vesicles by flocculation via orbital acoustic trapping: FLOAT. MICROSYSTEMS & NANOENGINEERING 2024; 10:23. [PMID: 38317693 PMCID: PMC10838941 DOI: 10.1038/s41378-023-00648-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/24/2023] [Revised: 11/01/2023] [Accepted: 11/11/2023] [Indexed: 02/07/2024]
Abstract
Extracellular vesicles (EVs) have been identified as promising biomarkers for the noninvasive diagnosis of various diseases. However, challenges in separating EVs from soluble proteins have resulted in variable EV recovery rates and low purities. Here, we report a high-yield ( > 90%) and rapid ( < 10 min) EV isolation method called FLocculation via Orbital Acoustic Trapping (FLOAT). The FLOAT approach utilizes an acoustofluidic droplet centrifuge to rotate and controllably heat liquid droplets. By adding a thermoresponsive polymer flocculant, nanoparticles as small as 20 nm can be rapidly and selectively concentrated at the center of the droplet. We demonstrate the ability of FLOAT to separate urinary EVs from the highly abundant Tamm-Horsfall protein, addressing a significant obstacle in the development of EV-based liquid biopsies. Due to its high-yield nature, FLOAT reduces biofluid starting volume requirements by a factor of 100 (from 20 mL to 200 µL), demonstrating its promising potential in point-of-care diagnostics.
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Affiliation(s)
- Joseph Rufo
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Peiran Zhang
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Zeyu Wang
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Yuyang Gu
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Kaichun Yang
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Joseph Rich
- Department of Biomedical Engineering, Duke University, Durham, NC USA
| | - Chuyi Chen
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Ruoyu Zhong
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Ke Jin
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Ye He
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Jianping Xia
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Ke Li
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Jiarong Wu
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
| | - Yingshi Ouyang
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, PA USA
| | - Yoel Sadovsky
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, PA USA
- Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, PA USA
| | - Luke P. Lee
- Renal Division and Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA USA
- Department of Bioengineering, Department of Electrical Engineering and Computer Science, University of California, Berkeley, Berkeley, CA USA
- Department of Biophysics, Institute of Quantum Biophysics, Sungkyunkwan University, Suwon, Korea
| | - Tony Jun Huang
- Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC USA
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12
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Ma X, Chen Z, Chen W, Chen Z, Meng X. Exosome subpopulations: The isolation and the functions in diseases. Gene 2024; 893:147905. [PMID: 37844851 DOI: 10.1016/j.gene.2023.147905] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2023] [Revised: 09/26/2023] [Accepted: 10/13/2023] [Indexed: 10/18/2023]
Abstract
Exosomes are nanoscale extracellular vesicles secreted by cells. Exosomes mediate intercellular communication by releasing their bioactive contents (e.g., DNAs, RNAs, lipids, proteins, and metabolites). The components of exosomes are regulated by the producing cells of exosomes. Due to their diverse origins, exosomes are highly heterogeneous in size, content, and function. Depending on these characteristics, exosomes can be divided into multiple subpopulations which have different functions. Efficient enrichment of specific subpopulations of exosomes helps to investigate their biological functions. Accordingly, numerous techniques have been developed to isolate specific subpopulations of exosomes. This review systematically introduces emerging new technologies for the isolation of different exosome subpopulations and summarizes the critical role of specific exosome subpopulations in diseases, especially in tumor occurrence and progression.
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Affiliation(s)
- Xinyi Ma
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China; Zhejiang Provincial Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China; Department of Thoracic Surgery, The First Affiliated Hospital of Ningbo University, Ningbo University, China
| | - Zhenhua Chen
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China; Zhejiang Provincial Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China; Department of Thoracic Surgery, The First Affiliated Hospital of Ningbo University, Ningbo University, China
| | - Wei Chen
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China; Zhejiang Provincial Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China; Department of Thoracic Surgery, The First Affiliated Hospital of Ningbo University, Ningbo University, China
| | - Ziyuan Chen
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China; Zhejiang Provincial Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China; Department of Thoracic Surgery, The First Affiliated Hospital of Ningbo University, Ningbo University, China
| | - Xiaodan Meng
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China; Zhejiang Provincial Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang 315211, China; Department of Thoracic Surgery, The First Affiliated Hospital of Ningbo University, Ningbo University, China.
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13
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Jeanmard N, Bissanum R, Sriplung H, Charoenlappanit S, Roytrakul S, Navakanitworakul R. Proteomic profiling of urinary extracellular vesicles differentiates breast cancer patients from healthy women. PLoS One 2023; 18:e0291574. [PMID: 37922300 PMCID: PMC10624262 DOI: 10.1371/journal.pone.0291574] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2023] [Accepted: 08/31/2023] [Indexed: 11/05/2023] Open
Abstract
Urinary extracellular vesicles (uEVs) reflect the biological conditions of the producing cells. The protein profiling of uEVs allow us to better understand cancer progression in several cancers such as bladder cancer, prostate cancer and kidney cancer but has not been reported in breast cancer. We have, herein, aimed at quantifying the concentration and at generating the proteomic profile of uEVs in patients with breast cancer (BC) as compared to that of healthy controls (CT). Urine samples were collected from 29 CT and 47 patients with BC. uEVs were isolated by using differential ultracentrifugation, and were then characterized by Western blotting and transmission electron microscopy. Moreover, a nanoparticle tracking analysis was used in order to measure the concentration and the size distribution of urine particles and uEVs. The proteomic profiling of the uEVs was facilitated through LC-MS/MS. The uEV concentration was not significantly different between the assessed groups. The undertaken proteomic analysis revealed 15,473 and 11,278 proteins in the BC patients' group and the CT group, respectively. Furthermore, a heat map analysis revealed a differential protein expression, while a principal component analysis highlighted two clusters. The volcano plot indicated 259 differentially expressed proteins (DEPs; 155 up- and 104 down-regulated proteins) in patients with BC compared with CT. The up-regulated proteins from BC-derived uEVs were enriched in pathways related to cancer progression (i.e., cell proliferation, cell survival, cell cycle, cell migration, carbohydrate metabolism, and angiogenesis). Moreover, we verified the expression of the upregulated DEPs using UALCAN for web-based validation. Remarkably, the results indicated that 6 of 155 up-regulated proteins (POSTN, ATAD2, BCAS4, GSK3β, HK1, and Ki-67) were overexpressed in BC compared with normal samples. Since these six proteins often act as markers of cell proliferation and progression, they may be potential biomarkers for BC screening and diagnosis. However, this requires validation in larger cohorts.
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Affiliation(s)
- Nilobon Jeanmard
- Department of Biomedical Sciences and Biomedical Engineering, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand
| | - Rassanee Bissanum
- Department of Biomedical Sciences and Biomedical Engineering, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand
| | - Hutcha Sriplung
- Department of Epidemiology, Faculty of Medicine, Prince of Songkla University, Hat Yai, Thailand
| | - Sawanya Charoenlappanit
- National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand
| | - Sittiruk Roytrakul
- National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand
| | - Raphatphorn Navakanitworakul
- Department of Biomedical Sciences and Biomedical Engineering, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand
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14
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Ding X, Zhang D, Ren Q, Hu Y, Wang J, Hao J, Wang H, Zhao X, Wang X, Song C, Du J, Yang F, Zhu H. Identification of a Non-Invasive Urinary Exosomal Biomarker for Diabetic Nephropathy Using Data-Independent Acquisition Proteomics. Int J Mol Sci 2023; 24:13560. [PMID: 37686366 PMCID: PMC10488032 DOI: 10.3390/ijms241713560] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2023] [Revised: 08/16/2023] [Accepted: 08/28/2023] [Indexed: 09/10/2023] Open
Abstract
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.
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Affiliation(s)
- Xiaonan Ding
- Department of Nephrology, First Medical Center of Chinese People’s Liberation Army General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, National Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China; (X.D.); (D.Z.)
- Medical School of Chinese People’s Liberation Army, Beijing 100853, China
| | - Dong Zhang
- Department of Nephrology, First Medical Center of Chinese People’s Liberation Army General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, National Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China; (X.D.); (D.Z.)
| | - Qinqin Ren
- Department of Nephrology, First Medical Center of Chinese People’s Liberation Army General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, National Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China; (X.D.); (D.Z.)
| | - Yilan Hu
- Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Jifeng Wang
- Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Jing Hao
- Department of Nephrology, First Medical Center of Chinese People’s Liberation Army General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, National Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China; (X.D.); (D.Z.)
| | - Haoran Wang
- Department of Nephrology, First Medical Center of Chinese People’s Liberation Army General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, National Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China; (X.D.); (D.Z.)
| | - Xiaolin Zhao
- Department of Nephrology, First Medical Center of Chinese People’s Liberation Army General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, National Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China; (X.D.); (D.Z.)
| | - Xiaochen Wang
- Department of Nephrology, First Medical Center of Chinese People’s Liberation Army General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, National Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China; (X.D.); (D.Z.)
| | - Chenwen Song
- Department of Nephrology, First Medical Center of Chinese People’s Liberation Army General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, National Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China; (X.D.); (D.Z.)
| | - Junxia Du
- Department of Nephrology, First Medical Center of Chinese People’s Liberation Army General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, National Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China; (X.D.); (D.Z.)
| | - Fuquan Yang
- Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Hanyu Zhu
- Department of Nephrology, First Medical Center of Chinese People’s Liberation Army General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, National Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China; (X.D.); (D.Z.)
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15
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Sinha N, Puri V, Kumar V, Nada R, Rastogi A, Jha V, Puri S. Urinary exosomal miRNA-663a shows variable expression in diabetic kidney disease patients with or without proteinuria. Sci Rep 2023; 13:4516. [PMID: 36934129 PMCID: PMC10024703 DOI: 10.1038/s41598-022-26558-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Accepted: 12/16/2022] [Indexed: 03/20/2023] Open
Abstract
Heterogeneity in the Diabetic Kidney Disease (DKD) diagnosis makes its rational therapeutics challenging. Although albuminuria characterizes DKD, reports also indicate its prevalence among non-proteinuric. Recent understanding of disease progression has thus inclined the focus on proximal tubular cell damage besides the glomeruli. A non-invasive approach exploiting exosomal miRNA derived from human kidney proximal tubular cell line was, hence, targeted. Upon miRNA profiling, three miRNAs, namely, hsa-miR-155-5p, hsa-miR-28-3p, and hsa-miR-425-5p were found to be significantly upregulated, while hsa-miR-663a was downregulated under diabetic conditions. Among these, hsa-miR-663a downregulation was more pronounced in non-proteinuric than proteinuric DKD subjects and was thus selected for the bioinformatics study. Ingenuity Pathway Analysis (IPA) narrowed on to IL-8 signaling and inflammatory response as the most enriched 'canonical pathway' and 'disease pathway' respectively, during DKD. Further, the putative gene network generated from these enriched pathways revealed experimentally induced diabetes, renal tubular injury, and decreased levels of albumin as part of mapping under 'disease and function'. Genes target predictions and annotations by IPA reiterated miR-663a's role in the pathogenesis of DKD following tubular injury. Overall, the observations might offer an indirect reflection of the underlying mechanism between patients who develop proteinuria and non-proteinuria.
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Affiliation(s)
- Nisha Sinha
- Centre for Stem Cell Tissue Engineering and Biomedical Excellence, Panjab University, Chandigarh, India
- Department of Nephrology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Veena Puri
- Centre for Systems Biology and Bioinformatics, Panjab University, Chandigarh, India
| | - Vivek Kumar
- Department of Nephrology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Ritambhra Nada
- Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Ashu Rastogi
- Department of Endocrinology and Metabolism, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Vivekanand Jha
- The George Institute for Global Health, New Delhi, India.
| | - Sanjeev Puri
- Department of Biotechnology, University Institute of Engineering and Technology (UIET), Panjab University, Chandigarh, India.
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16
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A review on comparative studies addressing exosome isolation methods from body fluids. Anal Bioanal Chem 2023; 415:1239-1263. [PMID: 35838769 DOI: 10.1007/s00216-022-04174-5] [Citation(s) in RCA: 59] [Impact Index Per Article: 29.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Revised: 05/17/2022] [Accepted: 06/10/2022] [Indexed: 12/11/2022]
Abstract
Exosomes emerged as valuable sources of disease biomarkers and new therapeutic tools. However, extracellular vesicles isolation with exosome-like characteristics from certain biofluids is still challenging which can limit their potential use in clinical settings. While ultracentrifugation-based procedures are the gold standard for exosome isolation from cell cultures, no unique and standardized method for exosome isolation from distinct body fluids exists. The complexity, specific composition, and physical properties of each biofluid constitute a technical barrier to obtain reproducible and pure exosome preparations, demanding a detailed characterization of both exosome isolation and characterization methods. Moreover, some isolation procedures can affect downstream proteomic or RNA profiling analysis. This review compiles and discussed a set of comparative studies addressing distinct exosome isolation methods from human biofluids, including cerebrospinal fluid, plasma, serum, saliva, and urine, also focusing on body fluid specific challenges, physical properties, and other potential variation sources. This summarized information will facilitate the choice of exosome isolation methods, based on the type of biological samples available, and hopefully encourage the use of exosomes in translational and clinical research.
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Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry. NANOMEDICINE : NANOTECHNOLOGY, BIOLOGY, AND MEDICINE 2023; 48:102638. [PMID: 36549551 DOI: 10.1016/j.nano.2022.102638] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/30/2022] [Revised: 12/01/2022] [Accepted: 12/02/2022] [Indexed: 12/23/2022]
Abstract
Urinary extracellular vesicles (uEVs) are promising biomarkers for various diseases. However, many tools measuring uEVs rely on time-consuming uEV isolation methods, which could induce sample bias. This study demonstrates the detection of single uEVs without isolation using imaging flow cytometry (IFCM). Unstained urine samples contained auto-fluorescent (A-F) particles when characterized with IFCM. Centrifugation successfully removed A-F particles from the unprocessed urine. Based on the disappearance of A-F particles, a gate was defined to distinguish uEVs from A-F particles. The final readouts of IFCM were verified as single EVs based on detergent treatment and serial dilutions. When developing this protocol to measure urine samples with abnormally high protein levels, 25 mg/mL dithiothreitol (DTT) showed improved uEV recovery over 200 mg/mL DTT. This study provides an isolation-free protocol using IFCM to quantify and phenotype single uEVs, eliminating the hindrance and influence of A-F particles, protein aggregates, and coincidence events.
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18
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Extracellular Vesicles' Genetic Cargo as Noninvasive Biomarkers in Cancer: A Pilot Study Using ExoGAG Technology. Biomedicines 2023; 11:biomedicines11020404. [PMID: 36830940 PMCID: PMC9953104 DOI: 10.3390/biomedicines11020404] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2022] [Revised: 01/13/2023] [Accepted: 01/24/2023] [Indexed: 01/31/2023] Open
Abstract
The two most developed biomarkers in liquid biopsy (LB)-circulating tumor cells and circulating tumor DNA-have been joined by the analysis of extracellular vesicles (EVs). EVs are lipid-bilayer enclosed structures released by all cell types containing a variety of molecules, including DNA, mRNA and miRNA. However, fast, efficient and a high degree of purity isolation technologies are necessary for their clinical routine implementation. In this work, the use of ExoGAG, a new easy-to-use EV isolation technology, was validated for the isolation of EVs from plasma and urine samples. After demonstrating its efficiency, an analysis of the genetic material contained in the EVs was carried out. Firstly, the sensitivity of the detection of point mutations in DNA from plasma EVs isolated by ExoGAG was analyzed. Then, a pilot study of mRNA expression using the nCounter NanoString platform in EV-mRNA from a healthy donor, a benign prostate hyperplasia patient and metastatic prostate cancer patient plasma and urine samples was performed, identifying the prostate cancer pathway as one of the main ones. This work provides evidence for the value of using ExoGAG for the isolation of EVs from plasma and urine samples, enabling downstream applications of the analysis of their genetic cargo.
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Importance and implications of exosomes in nephrology and urology. Pflugers Arch 2023; 475:153-166. [PMID: 36399151 PMCID: PMC9849294 DOI: 10.1007/s00424-022-02771-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2022] [Revised: 10/31/2022] [Accepted: 11/07/2022] [Indexed: 11/19/2022]
Abstract
Exosomes are extracellular vesicles that are formed by two invaginations of the plasma membrane and can be released by all eukaryotic cells. Because of their bioactive contents, including nucleic acids and proteins, exosomes can activate a variety of functions in their recipient cells. Due to the plethora of physiological and pathophysiological functions, exosomes have received a lot of attention from researchers over the past few years. However, there is still no consensus regarding isolation and characterization protocols of exosomes and their subtypes. This heterogeneity poses a lot of methodical challenges but also offers new clinical opportunities simultaneously. So far, exosome-based research is still mostly limited to preclinical experiments and early-stage clinical trials since the translation of experimental findings remains difficult. Exosomes could potentially play an important role as future diagnostic and prognostic agents and might also be part of the development of new treatment strategies. Therefore, they have previously been investigated in a variety of nephrological and urological conditions such as acute kidney injury or prostate cancer.
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Yao X, Liao B, Chen F, Liu L, Wu K, Hao Y, Li Y, Wang Y, Fan R, Yin J, Liu L, Guo Y. Comparison of proteomic landscape of extracellular vesicles in pleural effusions isolated by three strategies. Front Bioeng Biotechnol 2023; 11:1108952. [PMID: 37122867 PMCID: PMC10130534 DOI: 10.3389/fbioe.2023.1108952] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2022] [Accepted: 03/28/2023] [Indexed: 05/02/2023] Open
Abstract
Extracellular vesicles (EVs) derived from pleural effusion (PE) is emerging as disease biomarkers. However, the methods for isolation of EVs from PE (pEVs) were rarely studied. In our study, three methods for isolating pEVs of lung cancer patients were compared, including ultracentrifugation (UC), a combination of UC and size exclusion chromatography (UC-SEC) and a combination of UC and density gradient ultracentrifugation (UC-DGU). The subpopulation of pEVs was identified by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western blotting (WB) and nano-flow cytometry (nFCM). Additionally, the proteomic landscape of pEVs was analyzed by Label-free proteomics. The results showed that, compared with UC and UC-DGU, the UC-SEC method separated pEVs with the highest purity. In the proteomic analysis, on average, 1595 proteins were identified in the pEVs isolated by UC-SEC, much more than pEVs isolated by UC (1222) or UC-DGU (807). Furthermore, approximately 90% of identified proteins in each method were found in the EVs public database ExoCarta. Consistent with this, GO annotation indicated that the core proteins identified in each method were mainly enriched in "extracellular exosome." Many of the top 100 proteins with high expression in each method were suggested as protein markers to validate the presence of EVs in the MISEV2018 guidelines. In addition, combined with lung tissue-specific proteins and vesicular membrane proteins, we screened out and validated several novel protein markers (CD11C, HLA DPA1 and HLA DRB1), which were enriched in pEVs rather than in plasma EVs. In conclusion, our study shows that the method of UC-SEC could significantly improve the purity of EVs and the performance of mass spectrometry-based proteomic profiling in analyzing pEVs. The exosomal proteins CD11C, HLA DPA1 and HLA DRB1 may act as potential markers of pEVs. The proteomic analysis of pEVs provides important information and new ideas for studying diseases complicated with PE.
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Affiliation(s)
- Xue Yao
- School of Medicine, Southwest Jiaotong University, The Affiliated Hospital of Southwest Jiaotong University, The Third People’s Hospital of Chengdu, Chengdu, China
| | - Baixue Liao
- School of Medicine, Southwest Jiaotong University, The Affiliated Hospital of Southwest Jiaotong University, The Third People’s Hospital of Chengdu, Chengdu, China
| | - Feng Chen
- Department of Respiratory, The Third People’s Hospital of Chengdu, The Affiliated Hospital of Southwest Jiaotong University, Chengdu, China
| | - Lüye Liu
- Medical Research Center, The Third People’s Hospital of Chengdu, The Affiliated Hospital of Southwest Jiaotong University, Chengdu, China
| | - Kaiwen Wu
- School of Medicine, Southwest Jiaotong University, The Affiliated Hospital of Southwest Jiaotong University, The Third People’s Hospital of Chengdu, Chengdu, China
| | - Yaying Hao
- Medical Research Center, The Third People’s Hospital of Chengdu, The Affiliated Hospital of Southwest Jiaotong University, Chengdu, China
| | - Yanping Li
- Department of Respiratory, The Third People’s Hospital of Chengdu, The Affiliated Hospital of Southwest Jiaotong University, Chengdu, China
| | - Yuebin Wang
- Department of Respiratory, The Third People’s Hospital of Chengdu, The Affiliated Hospital of Southwest Jiaotong University, Chengdu, China
| | - Ruiling Fan
- School of Pharmacy, North Sichuan Medical College, Nanchong, China
| | - Jun Yin
- Department of Respiratory, The Third People’s Hospital of Chengdu, The Affiliated Hospital of Southwest Jiaotong University, Chengdu, China
| | - Lei Liu
- Medical Research Center, The Third People’s Hospital of Chengdu, The Affiliated Hospital of Southwest Jiaotong University, Chengdu, China
- *Correspondence: Lei Liu, ; Yuanbiao Guo,
| | - Yuanbiao Guo
- Medical Research Center, The Third People’s Hospital of Chengdu, The Affiliated Hospital of Southwest Jiaotong University, Chengdu, China
- *Correspondence: Lei Liu, ; Yuanbiao Guo,
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21
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Lou K, Feng S, Luo H, Zou J, Zhang G, Zou X. Extracellular vesicles derived from macrophages: Current applications and prospects in tumors. Front Bioeng Biotechnol 2022; 10:1097074. [PMID: 36588947 PMCID: PMC9797603 DOI: 10.3389/fbioe.2022.1097074] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2022] [Accepted: 12/07/2022] [Indexed: 12/23/2022] Open
Abstract
Macrophages (Mφs) are significant innate immune cells that perform a variety of tasks in response to different pathogens or stimuli. They are widely engaged in the pathological processes of various diseases and can contribute to tumorigenesis, progression and metastasis by regulating the tumor microenvironment and cancer cells. They are also the basis of chemoresistance. In turn, the tumor microenvironment and the metabolism of cancer cells can limit the differentiation, polarization, mobilization and the ability of Mφs to initiate an effective anti-tumor response. Extracellular vesicles (EVs) are small vesicles released by live cells that serve as crucial mediators of intercellular cell communication as well as a potential promising drug carrier. A growing number of studies have demonstrated that Mφs-EVs are not only important mediators in the pathological processes of various diseases such as inflammatory disorders, fibrosis and cancer, but also show significant potential in immunological modulation, cancer therapy, infectious defense and tissue repair. These natural nanoparticles (NPs) derived from Mφs are believed to be pleiotropic, stable, biocompatible and low immunogenic, providing novel alternatives for cancer treatment. This review provides an update on the pathological and therapeutic roles of Mφs-EVs in cancer, as well as their potential clinical applications and prospects.
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Affiliation(s)
- Kecheng Lou
- The First Clinical College, Gannan Medical University, Ganzhou, Jiangxi, China,Department of Urology, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China
| | - Shangzhi Feng
- The First Clinical College, Gannan Medical University, Ganzhou, Jiangxi, China,Department of Urology, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China
| | - Hui Luo
- The First Clinical College, Gannan Medical University, Ganzhou, Jiangxi, China,Department of Urology, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China
| | - Junrong Zou
- Department of Urology, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China,Institute of Urology, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China,Jiangxi Engineering Technology Research Center of Calculi Prevention, Ganzhou, Jiangxi, China
| | - Guoxi Zhang
- Department of Urology, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China,Institute of Urology, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China,Jiangxi Engineering Technology Research Center of Calculi Prevention, Ganzhou, Jiangxi, China
| | - Xiaofeng Zou
- Department of Urology, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China,Institute of Urology, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China,Jiangxi Engineering Technology Research Center of Calculi Prevention, Ganzhou, Jiangxi, China,*Correspondence: Xiaofeng Zou,
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22
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Alptekin A, Parvin M, Chowdhury HI, Rashid MH, Arbab AS. Engineered exosomes for studies in tumor immunology. Immunol Rev 2022; 312:76-102. [PMID: 35808839 DOI: 10.1111/imr.13107] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2022] [Accepted: 05/27/2022] [Indexed: 12/14/2022]
Abstract
Exosomes are a type of extracellular vesicle (EV) with diameters of 30-150 nm secreted by most of the cells into the extracellular spaces and can alter the microenvironment through cell-to-cell interactions by fusion with the plasma membrane and subsequent endocytosis and release of the cargo. Because of their biocompatibility, low toxicity and immunogenicity, permeability (even through the blood-brain barrier (BBB)), stability in biological fluids, and ability to accumulate in the lesions with higher specificity, investigators have started making designer's exosomes or engineered exosomes to carry biologically active protein on the surface or inside the exosomes as well as using exosomes to carry drugs, micro RNA, and other products to the site of interest. In this review, we have discussed biogenesis, markers, and contents of various exosomes including exosomes of immune cells. We have also discussed the current methods of making engineered and designer's exosomes as well as the use of engineered exosomes targeting different immune cells in the tumors, stroke, as well as at peripheral blood. Genetic engineering and customizing exosomes create an unlimited opportunity to use in diagnosis and treatment. Very little use has been discovered, and we are far away to reach its limits.
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Affiliation(s)
- Ahmet Alptekin
- Georgia Cancer Center, Augusta University, Augusta, Georgia, USA
| | - Mahrima Parvin
- Georgia Cancer Center, Augusta University, Augusta, Georgia, USA
| | | | | | - Ali S Arbab
- Georgia Cancer Center, Augusta University, Augusta, Georgia, USA
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23
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Ruan Z, Liang Y, Chen Z, Yin J, Li C, Pan P, Zhang Q, Wu J, Luo Z. Enterovirus 71 non-structural protein 3A hijacks vacuolar protein sorting 25 to boost exosome biogenesis to facilitate viral replication. Front Microbiol 2022; 13:1024899. [PMID: 36274707 PMCID: PMC9581156 DOI: 10.3389/fmicb.2022.1024899] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2022] [Accepted: 09/21/2022] [Indexed: 11/16/2022] Open
Abstract
Human enterovirus 71 (EV71) is one of the major agents of the hand, foot, and mouth disease (HFMD), and occasionally causes severe neurological complications. There is clinical evidence that EV71 infection increases the exosomes in the serum of severe HFMD patients, suggesting a role of exosomes in EV71 pathogenesis. However, the relationship between exosomes and EV71 replication remains elusive. In this study, we initially found that EV71 infection elevated exosome biogenesis in the cultured cells. Among EV71 non-structural proteins, we identified EV71 3A, but not 3B, constitutively promoted exosome secretion. In detail, EV71 3A protein interacted with vacuolar protein sorting 25 (VPS25), while knock-down of VPS25 reduced EV71 3A protein- and EV71-induced exosome production. Further studies revealed VPS25 located on exosomes and its expression correlated to the exosome production. During EV71 infection, knock-down of VPS25 decreased exosome biogenesis to attenuate viral replication. Consistently, GW4869, an exosome inhibitor, exerted an obviously antiviral activity against EV71 replication companied with the decrease of exosome secretion or formation. These findings suggest the binding of EV71 3A and VPS25 benefited exosome biogenesis, thereby boosting viral replication. This study uncovers a novel mechanism underlying EV71-mediated exosomes in the regulation of viral replication, which provides potential anti-viral strategies against the EV71 infection and transmission in HFMD.
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Affiliation(s)
- Zhihui Ruan
- Guangdong Provincial Key Laboratory of Virology, Institute of Medical Microbiology, Jinan University, Guangzhou, China
| | - Yicong Liang
- Guangdong Provincial Key Laboratory of Virology, Institute of Medical Microbiology, Jinan University, Guangzhou, China
| | - Zicong Chen
- Guangdong Provincial Key Laboratory of Virology, Institute of Medical Microbiology, Jinan University, Guangzhou, China
| | - Jialing Yin
- Guangdong Provincial Key Laboratory of Virology, Institute of Medical Microbiology, Jinan University, Guangzhou, China
| | - Chengcheng Li
- Guangdong Provincial Key Laboratory of Virology, Institute of Medical Microbiology, Jinan University, Guangzhou, China
| | - Pan Pan
- Guangdong Provincial Key Laboratory of Virology, Institute of Medical Microbiology, Jinan University, Guangzhou, China
- Foshan Institute of Medical Microbiology, Foshan, China
| | - Qiwei Zhang
- Guangdong Provincial Key Laboratory of Virology, Institute of Medical Microbiology, Jinan University, Guangzhou, China
- Foshan Institute of Medical Microbiology, Foshan, China
| | - Jianguo Wu
- Guangdong Provincial Key Laboratory of Virology, Institute of Medical Microbiology, Jinan University, Guangzhou, China
- Foshan Institute of Medical Microbiology, Foshan, China
- Jianguo Wu,
| | - Zhen Luo
- Guangdong Provincial Key Laboratory of Virology, Institute of Medical Microbiology, Jinan University, Guangzhou, China
- Foshan Institute of Medical Microbiology, Foshan, China
- *Correspondence: Zhen Luo,
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24
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Identifying stable reference genes in polyethene glycol precipitated urinary extracellular vesicles for RT-qPCR-based gene expression studies in renal graft dysfunction patients. Transpl Immunol 2022; 75:101715. [PMID: 36122652 DOI: 10.1016/j.trim.2022.101715] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2022] [Revised: 09/12/2022] [Accepted: 09/13/2022] [Indexed: 11/22/2022]
Abstract
BACKGROUND Urinary extracellular vesicles (UEVs) hold RNA in their cargo and are potential sources of biomarkers for gene expression studies. The most used technique for gene-expression studies is quantitative polymerase chain reaction (qPCR). It is critical to use stable reference genes (RGs) as internal controls for normalising gene expression data, which aren't currently available for UEVs. METHODS UEVs were precipitated from urine of graft dysfunction patients and healthy controls by Polyethylene glycol, Mn6000 (PEG6K). Vesicular characterisation confirmed the presence of UEVs. Gene expression levels of five commonly used RGs, i.e., Beta-2-Microglobulin (B2M), ribosomal-protein-L13a (RPL13A), Peptidylprolyl-Isomerase-A (PPIA), hydroxymethylbilane synthase (HMBS), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were quantified, and their stability was established through the RefFinder. The stability of identified RGs was validated by quantification of Perforin and granzyme B, signature molecules of renal graft dysfunction. RESULTS Urine precipitated with 12% 6 K PEG yielded round and double-membraned UEVs of size ranging from 30 to 100 nm, as confirmed through transmission electron microscopy. Nanoparticle tracking analysis (59 ± 22 nm) and Dynamic-light-scattering (78 ± 56.5 nm) confirmed their size profile. Semi-quantitative Exocheck antibody array demonstrated the presence of EV protein markers in UEV. Using the comparative ΔCт method and RefFinder analysis, B2M (1.6) and RPL13A (1.8) genes emerged as the most stable reference genes. Validation of target gene expression in renal graft dysfunction patients confirmed the efficiency of B2M and RPL13A through significant upregulation compared to other RGs. CONCLUSIONS Our study identified and validated B2M and RPL13A as optimal RGs for mRNA quantification studies in the UEVs of patients with renal graft dysfunction.
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25
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Wu A, Wolley MJ, Fenton RA, Stowasser M. Using human urinary extracellular vesicles to study physiological and pathophysiological states and regulation of the sodium chloride cotransporter. Front Endocrinol (Lausanne) 2022; 13:981317. [PMID: 36105401 PMCID: PMC9465297 DOI: 10.3389/fendo.2022.981317] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2022] [Accepted: 08/09/2022] [Indexed: 11/29/2022] Open
Abstract
The thiazide-sensitive sodium chloride cotransporter (NCC), expressed in the renal distal convoluted tubule, plays a major role in Na+, Cl- and K+ homeostasis and blood pressure as exemplified by the symptoms of patients with non-functional NCC and Gitelman syndrome. NCC activity is modulated by a variety of hormones, but is also influenced by the extracellular K+ concentration. The putative "renal-K+ switch" mechanism is a relatively cohesive model that links dietary K+ intake to NCC activity, and may offer new targets for blood pressure control. However, a remaining hurdle for full acceptance of this model is the lack of human data to confirm molecular findings from animal models. Extracellular vesicles (EVs) have attracted attention from the scientific community due to their potential roles in intercellular communication, disease pathogenesis, drug delivery and as possible reservoirs of biomarkers. Urinary EVs (uEVs) are an excellent sample source for the study of physiology and pathology of renal, urothelial and prostate tissues, but the diverse origins of uEVs and their dynamic molecular composition present both methodological and data interpretation challenges. This review provides a brief overview of the state-of-the-art, challenges and knowledge gaps in current uEV-based analyses, with a focus on the application of uEVs to study the "renal-K+ switch" and NCC regulation. We also provide recommendations regarding biospecimen handling, processing and reporting requirements to improve experimental reproducibility and interoperability towards the realisation of the potential of uEV-derived biomarkers in hypertension and clinical practice.
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Affiliation(s)
- Aihua Wu
- Endocrine Hypertension Research Centre, University of Queensland Diamantina Institute, Greenslopes and Princess Alexandra Hospitals, Brisbane, QLD, Australia
| | - Martin J. Wolley
- Endocrine Hypertension Research Centre, University of Queensland Diamantina Institute, Greenslopes and Princess Alexandra Hospitals, Brisbane, QLD, Australia
- Department of Nephrology, Royal Brisbane and Women’s Hospital, Brisbane, QLD, Australia
| | | | - Michael Stowasser
- Endocrine Hypertension Research Centre, University of Queensland Diamantina Institute, Greenslopes and Princess Alexandra Hospitals, Brisbane, QLD, Australia
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26
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Yao C, Chen X, Xu Y, Wang F, Ji J, Xu H, He J, Wang L, Li Y. Comparing pretreatment strategies to increase the yield and purity of human urinary extracellular vesicles. J Chromatogr B Analyt Technol Biomed Life Sci 2022; 1206:123359. [PMID: 35785645 DOI: 10.1016/j.jchromb.2022.123359] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2022] [Revised: 06/27/2022] [Accepted: 06/28/2022] [Indexed: 11/30/2022]
Abstract
Extracellular vesicles (EVs) are membranous vesicles released by various cells, and are involved in intercellular communication and disease progression. EVs that are isolated from urine are good indicators of urinary system diseases and help certain urological studies. During isolation of urine EVs, Dithiothreitol (DTT) is widely used to reduce the contamination of the major contaminant Tamm-Horsefall protein (THP),which is the most abundant protein in the human urine and the most difficult contaminant to remove in the isolation of urine EVs. Unfortunately, DTT can interfere with subsequent analysis due to its strong reducing ability and cannot completely remove THP. To optimize the urine EV isolation strategy, we compared two pretreatment protocols: incubating urine with NaCl and DTT before centrifugation. After a series of analyses by nanoparticle tracking analysis (NTA), western blotting (WB), and transmission electron microscopy (TEM), we found that NaCl removed more THP than DTT in a low-speed centrifugation step and that the residual EVs also had lower THP contamination post NaCl treatment. Remarkably, the yield of EVs obtained via the salting-out method was significantly higher than those obtained by the other methods (P = 0.001). Our study is the first to demonstrate that the salting-out method is better than the traditional DTT method in terms of efficiency in removing THP and EV yields.
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Affiliation(s)
- Chungen Yao
- Department of Urology, Haining Branch of the First Affiliated Hospital, Zhejiang University, Haining, Zhejiang Province, China.
| | - Xi Chen
- Department of Urology, NO. 971 Hospital, Qingdao, Shandong Province, China.
| | - Yalong Xu
- Department of Urology, General Hospital of Central Theater Command, Wuhan, Hubei Province, China.
| | - Fubo Wang
- Center for Genomic and Personalized Medicine, Guangxi key Laboratory for Genomic and Personalized Medicine, Guangxi Collaborative Innovation Center for Genomic and Personalized Medicine, Guangxi Medical University, Nanning, China.
| | - Jin Ji
- Department of Urology, Changhai Hospital, Navy Medical University, Shanghai, China.
| | - Huan Xu
- Department of Urology, Changhai Hospital, Navy Medical University, Shanghai, China.
| | - Jingyi He
- Department of Urology, Changhai Hospital, Navy Medical University, Shanghai, China.
| | - Lei Wang
- Department of Urology, Changhai Hospital, Navy Medical University, Shanghai, China.
| | - Yun Li
- Shanghai Shibei Hospital of Jingan District, Shanghai, China.
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27
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Isolation and Characterization of Urinary Extracellular Vesicles from Healthy Donors and Patients with Castration-Resistant Prostate Cancer. Int J Mol Sci 2022; 23:ijms23137134. [PMID: 35806139 PMCID: PMC9266865 DOI: 10.3390/ijms23137134] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2022] [Revised: 06/22/2022] [Accepted: 06/22/2022] [Indexed: 02/04/2023] Open
Abstract
Prostate cancer (PCa) is the most commonly diagnosed malignancy among men in developed countries. The five-year survival rate for men diagnosed with early-stage PCa is approximately 100%, while it is less than 30% for castration-resistant PCa (CRPC). Currently, the detection of prostate-specific antigens as biomarkers for the prognosis of CRPC is criticized because of its low accuracy, high invasiveness, and high false-positive rate. Therefore, it is important to identify new biomarkers for prediction of CRPC progression. Extracellular vesicles (EVs) derived from tumors have been highlighted as potential markers for cancer diagnosis and prognosis. Specifically, urinary EVs directly reflect changes in the pathophysiological conditions of the urogenital system because it is exposed to prostatic secretions. Thus, detecting biomarkers in urinary EVs provides a promising approach for performing an accurate and non-invasive liquid biopsy for CPRC. In this study, we effectively isolated urinary EVs with low protein impurities using size-exclusion chromatography combined with ultrafiltration. After EV isolation and characterization, we evaluated the miRNAs in urinary EVs from healthy donors and patients with CRPC. The results indicated that miRNAs (miR-21-5p, miR-574-3p, and miR-6880-5p) could be used as potential biomarkers for the prognosis of CRPC. This analysis of urinary EVs contributes to the fast and convenient prognosis of diseases, including CRPC, in the clinical setting.
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28
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Yakubovich EI, Polischouk AG, Evtushenko VI. Principles and Problems of Exosome Isolation from Biological Fluids. BIOCHEMISTRY (MOSCOW), SUPPLEMENT SERIES A: MEMBRANE AND CELL BIOLOGY 2022; 16:115-126. [PMID: 35730027 PMCID: PMC9202659 DOI: 10.1134/s1990747822030096] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 01/14/2022] [Revised: 01/20/2022] [Accepted: 01/21/2022] [Indexed: 12/03/2022]
Abstract
Exosomes, the subclass of small membrane extracellular vesicles, have great diagnostic and therapeutic potential, but the lack of standardized methods for their efficient isolation and analysis limits the introduction of exosomal technologies into clinical practice. This review discusses the problems associated with the isolation of exosomes from biological fluids, as well as the principles of traditional and alternative methods of isolation. The aim of the presented review is to illustrate the variety of approaches based on the physical and biochemical properties of exosomes that can be used for exosome isolation. The advantages and disadvantages of different methods are discussed.
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Affiliation(s)
- E. I. Yakubovich
- Granov Russian Research Center for Radiology and Surgical Technologies, Ministry of Health of the Russian Federation, 197758 St. Petersburg, Russia
| | - A. G. Polischouk
- Granov Russian Research Center for Radiology and Surgical Technologies, Ministry of Health of the Russian Federation, 197758 St. Petersburg, Russia
| | - V. I. Evtushenko
- Granov Russian Research Center for Radiology and Surgical Technologies, Ministry of Health of the Russian Federation, 197758 St. Petersburg, Russia
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29
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Lin CM, Sung CC, Yang SS, Chen YC, Huang SM, Lin SH. Generation and analysis of pseudohypoaldosteronism type II knock-in mice caused by a nonsense KLHL3 mutation in the Kelch domain. FASEB J 2022; 36:e22363. [PMID: 35621709 DOI: 10.1096/fj.202101827rr] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2021] [Revised: 05/05/2022] [Accepted: 05/10/2022] [Indexed: 11/11/2022]
Abstract
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 KLHL3 mutations in the same domain that cause PHAII have not been fully investigated in vivo. We generated and analyzed Klhl3 KI mice harboring a nonsense W523X mutation (corresponding to the human KLHL3 W470X 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 Klhl3 mRNA 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 KLHL3 W470X 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 KLHL3 mutations in PHAII.
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Affiliation(s)
- Chien-Ming Lin
- Department of Pediatrics, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Chih-Chien Sung
- Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Sung-Sen Yang
- Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.,Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan
| | - Ying-Chuan Chen
- Department of Physiology & Biophysics, National Defense Medical Center, Taipei, Taiwan
| | - Shih-Ming Huang
- Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan
| | - Shih-Hua Lin
- Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.,Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan
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30
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Clos-Sansalvador M, Monguió-Tortajada M, Roura S, Franquesa M, Borràs FE. Commonly used methods for extracellular vesicles’ enrichment: implications in downstream analyses and use. Eur J Cell Biol 2022; 101:151227. [DOI: 10.1016/j.ejcb.2022.151227] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2022] [Revised: 04/12/2022] [Accepted: 04/12/2022] [Indexed: 02/08/2023] Open
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31
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Abreu CM, Costa-Silva B, Reis RL, Kundu SC, Caballero D. Microfluidic platforms for extracellular vesicle isolation, analysis and therapy in cancer. LAB ON A CHIP 2022; 22:1093-1125. [PMID: 35253032 DOI: 10.1039/d2lc00006g] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Extracellular vesicles (EVs) are small lipidic particles packed with proteins, DNA, messenger RNA and microRNAs of their cell of origin that act as critical players in cell-cell communication. These vesicles have been identified as pivotal mediators in cancer progression and the formation of metastatic niches. Hence, their isolation and analysis from circulating biofluids is envisioned as the next big thing in the field of liquid biopsies for early non-invasive diagnosis and patient follow-up. Despite the promise, current benchtop isolation strategies are not compatible with point-of-care testing in a clinical setting. Microfluidic platforms are disruptive technologies capable of recovering, analyzing, and quantifying EVs within clinical samples with limited volume, in a high-throughput manner with elevated sensitivity and multiplexing capabilities. Moreover, they can also be employed for the controlled production of synthetic EVs and effective drug loading to produce EV-based therapies. In this review, we explore the use of microfluidic platforms for the isolation, characterization, and quantification of EVs in cancer, and compare these platforms with the conventional methodologies. We also highlight the state-of-the-art in microfluidic approaches for EV-based cancer therapeutics. Finally, we analyze the currently active or recently completed clinical trials involving EVs for cancer diagnosis, treatment or therapy monitoring and examine the future of EV-based point-of-care testing platforms in the clinic and EV-based therapy production by the industry.
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Affiliation(s)
- Catarina M Abreu
- 3B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ciência e Tecnologia, Barco, 4805-017, Guimarães, Portugal.
- ICVS/3B's-PT Government Associate Laboratory, Braga, Guimarães, Portugal
| | - Bruno Costa-Silva
- Champalimaud Physiology and Cancer Programme, Champalimaud Foundation, Av. Brasília, 1400-038, Lisbon, Portugal
| | - Rui L Reis
- 3B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ciência e Tecnologia, Barco, 4805-017, Guimarães, Portugal.
- ICVS/3B's-PT Government Associate Laboratory, Braga, Guimarães, Portugal
| | - Subhas C Kundu
- 3B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ciência e Tecnologia, Barco, 4805-017, Guimarães, Portugal.
- ICVS/3B's-PT Government Associate Laboratory, Braga, Guimarães, Portugal
| | - David Caballero
- 3B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ciência e Tecnologia, Barco, 4805-017, Guimarães, Portugal.
- ICVS/3B's-PT Government Associate Laboratory, Braga, Guimarães, Portugal
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da Silva Lira Filho A, Fajardo EF, Chang KP, Clément P, Olivier M. Leishmania Exosomes/Extracellular Vesicles Containing GP63 Are Essential for Enhance Cutaneous Leishmaniasis Development Upon Co-Inoculation of Leishmania amazonensis and Its Exosomes. Front Cell Infect Microbiol 2022; 11:709258. [PMID: 35186777 PMCID: PMC8851419 DOI: 10.3389/fcimb.2021.709258] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2021] [Accepted: 12/17/2021] [Indexed: 12/22/2022] Open
Abstract
Protozoan parasites of the genus Leishmania are transmitted by the bite of infected sand flies leading to a wide range of diseases called leishmaniasis. Recently, we demonstrated that Leishmania spp.-derived exosomes/extracellular vesicles (EVs/LeishEXO) were released in the lumen of the sand fly midgut and to be co-egested with the parasite during the blood meal and that LeishEXO were found to stimulate an inflammatory response conducting to an exacerbated cutaneous leishmaniasis, also it was shown that these vesicles cargo important virulence factors like GP63. Thus, this study aimed to confirm through morphological and proteomic analysis a novel model specificity utilizing another set of GP63-altered Leishmania amazonensis parasite strains. Consequently, we proposed to further study the impact of different GP63 vesicle expression levels on their ability to modulate innate inflammatory cell responses, and finally to determine the importance of GP63 vesicle content on the exacerbation of the cutaneous Leishmania spp. pathology after their host co-inoculation. Our results revealed that the protein composition of extracted extracellular vesicles were similar to each other and that GP63 was the sole virulence factor changed in the exosomes composition confirming the specificity of the chosen novel model. We further demonstrated that vesicles with different GP63 EVs cargo displayed distinctive macrophage immunomodulatory capabilities at both gene and protein expression in vitro. Finally, we showed their diverse impact on the Leishmania spp. cutaneous pathology in an in vivo setting and confirmed GP63 as a primordial component of the ability of these EVs in augmenting the inflammatory cutaneous response in Leishmania spp. infection. Our findings provide new insight on the immune response happening in cutaneous leishmaniasis, shade light on the mechanism behind the host-pathogen interaction occurring in the initial moments of infection, thus creating the opportunity of using them as the target of new pharmacological treatments and vaccinations.
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Affiliation(s)
- Alonso da Silva Lira Filho
- Department of Microbiology and Immunology, McGill University, Montréal, QC, Canada
- Infectious Diseases and Immunity in Global Health Program, The Research Institute of the McGill University Health Centre, Montréal, QC, Canada
| | - Emanuella Francisco Fajardo
- Department of Microbiology and Immunology, McGill University, Montréal, QC, Canada
- Infectious Diseases and Immunity in Global Health Program, The Research Institute of the McGill University Health Centre, Montréal, QC, Canada
| | - Kwang Poo Chang
- Department of Microbiology/Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, United States
| | - Pauline Clément
- Infectious Diseases and Immunity in Global Health Program, The Research Institute of the McGill University Health Centre, Montréal, QC, Canada
| | - Martin Olivier
- Department of Microbiology and Immunology, McGill University, Montréal, QC, Canada
- Infectious Diseases and Immunity in Global Health Program, The Research Institute of the McGill University Health Centre, Montréal, QC, Canada
- *Correspondence: Martin Olivier,
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Ramirez-Garrastacho M, Bajo-Santos C, Line A, Martens-Uzunova ES, de la Fuente JM, Moros M, Soekmadji C, Tasken KA, Llorente A. Extracellular vesicles as a source of prostate cancer biomarkers in liquid biopsies: a decade of research. Br J Cancer 2022; 126:331-350. [PMID: 34811504 PMCID: PMC8810769 DOI: 10.1038/s41416-021-01610-8] [Citation(s) in RCA: 54] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2021] [Revised: 10/19/2021] [Accepted: 10/21/2021] [Indexed: 01/02/2023] Open
Abstract
Prostate cancer is a global cancer burden and considerable effort has been made through the years to identify biomarkers for the disease. Approximately a decade ago, the potential of analysing extracellular vesicles in liquid biopsies started to be envisaged. This was the beginning of a new exciting area of research investigating the rich molecular treasure found in extracellular vesicles to identify biomarkers for a variety of diseases. Vesicles released from prostate cancer cells and cells of the tumour microenvironment carry molecular information about the disease that can be analysed in several biological fluids. Numerous studies document the interest of researchers in this field of research. However, methodological issues such as the isolation of vesicles have been challenging. Remarkably, novel technologies, including those based on nanotechnology, show promise for the further development and clinical use of extracellular vesicles as liquid biomarkers. Development of biomarkers is a long and complicated process, and there are still not many biomarkers based on extracellular vesicles in clinical use. However, the knowledge acquired during the last decade constitutes a solid basis for the future development of liquid biopsy tests for prostate cancer. These are urgently needed to bring prostate cancer treatment to the next level in precision medicine.
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Affiliation(s)
- Manuel Ramirez-Garrastacho
- Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway
| | | | - Aija Line
- Latvian Biomedical Research and Study Centre, Riga, Latvia
| | - Elena S Martens-Uzunova
- Erasmus MC Cancer Institute, University Medical Center Rotterdam, Department of Urology, Laboratory of Experimental Urology, Erasmus MC, Rotterdam, The Netherlands
| | - Jesus Martinez de la Fuente
- Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC-Universidad de Zaragoza, Zaragoza, Spain
- Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain
| | - Maria Moros
- Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC-Universidad de Zaragoza, Zaragoza, Spain
- Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain
| | - Carolina Soekmadji
- Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia
- School of Biomedical Sciences, Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia
| | - Kristin Austlid Tasken
- Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway
- Institute of Clinical Medicine, University of Oslo, Oslo, Norway
| | - Alicia Llorente
- Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.
- Department for Mechanical, Electronics and Chemical Engineering, Oslo Metropolitan University, Oslo, Norway.
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Wang N, Yuan S, Fang C, Hu X, Zhang YS, Zhang LL, Zeng XT. Nanomaterials-Based Urinary Extracellular Vesicles Isolation and Detection for Non-invasive Auxiliary Diagnosis of Prostate Cancer. Front Med (Lausanne) 2022; 8:800889. [PMID: 35096890 PMCID: PMC8795515 DOI: 10.3389/fmed.2021.800889] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2021] [Accepted: 12/13/2021] [Indexed: 12/12/2022] Open
Abstract
Extracellular vesicles (EVs) are natural nanoparticles secreted by cells in the body and released into the extracellular environment. They are associated with various physiological or pathological processes, and considered as carriers in intercellular information transmission, so that EVs can be used as an important marker of liquid biopsy for disease diagnosis and prognosis. EVs are widely present in various body fluids, among which, urine is easy to obtain in large amount through non-invasive methods and has a small dynamic range of proteins, so it is a good object for studying EVs. However, most of the current isolation and detection of EVs still use traditional methods, which are of low purity, time consuming, and poor efficiency; therefore, more efficient and highly selective techniques are urgently needed. Recently, inspired by the nanoscale of EVs, platforms based on nanomaterials have been innovatively explored for isolation and detection of EVs from body fluids. These newly developed nanotechnologies, with higher selectivity and sensitivity, greatly improve the precision of isolation target EVs from urine. This review focuses on the nanomaterials used in isolation and detection of urinary EVs, discusses the advantages and disadvantages between traditional methods and nanomaterials-based platforms, and presents urinary EV-derived biomarkers for prostate cancer (PCa) diagnosis. We aim to provide a reference for researchers who want to carry out studies about nanomaterial-based platforms to identify urinary EVs, and we hope to summarize the biomarkers in downstream analysis of urinary EVs for auxiliary diagnosis of PCa disease in detail.
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Affiliation(s)
- Na Wang
- Center for Evidence-Based and Translational Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Shuai Yuan
- Center for Evidence-Based and Translational Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Cheng Fang
- Center for Evidence-Based and Translational Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Xiao Hu
- Center for Evidence-Based and Translational Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China.,Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Yu-Sen Zhang
- Center for Evidence-Based and Translational Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China.,Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Ling-Ling Zhang
- Center for Evidence-Based and Translational Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Xian-Tao Zeng
- Center for Evidence-Based and Translational Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China.,Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China
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Correll VL, Otto JJ, Risi CM, Main BP, Boutros PC, Kislinger T, Galkin VE, Nyalwidhe JO, Semmes OJ, Yang L. Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis. J Extracell Vesicles 2022; 11:e12184. [PMID: 35119778 PMCID: PMC8815402 DOI: 10.1002/jev2.12184] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 11/22/2021] [Accepted: 12/22/2021] [Indexed: 01/23/2023] Open
Abstract
The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co-isolate protein contaminants that mask lower-abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm-Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co-isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer-associated EV-resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs.
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Affiliation(s)
- Vanessa L. Correll
- Leroy T. Canoles Jr. Cancer Research CenterEastern Virginia Medical SchoolNorfolkVirginiaUSA
| | - Joseph J. Otto
- Leroy T. Canoles Jr. Cancer Research CenterEastern Virginia Medical SchoolNorfolkVirginiaUSA
| | - Cristina M. Risi
- Department of Physiological SciencesEastern Virginia Medical SchoolNorfolkVirginiaUSA
| | - Brian P. Main
- Leroy T. Canoles Jr. Cancer Research CenterEastern Virginia Medical SchoolNorfolkVirginiaUSA
| | - Paul C. Boutros
- Department of Medical BiophysicsUniversity of TorontoTorontoCanada
- Department of Pharmacology and ToxicologyUniversity of TorontoTorontoCanada
- Department of Human GeneticsUniversity of CaliforniaLos AngelesCaliforniaUSA
- Department of UrologyUniversity of CaliforniaLos AngelesCaliforniaUSA
- Institute for Precision HealthUniversity of CaliforniaLos AngelesCaliforniaUSA
- Jonsson Comprehensive Cancer CenterUniversity of CaliforniaLos AngelesCaliforniaUSA
| | - Thomas Kislinger
- Department of Medical BiophysicsUniversity of TorontoTorontoCanada
- Princess Margaret Cancer CentreUniversity Health NetworkTorontoCanada
| | - Vitold E. Galkin
- Department of Physiological SciencesEastern Virginia Medical SchoolNorfolkVirginiaUSA
| | - Julius O. Nyalwidhe
- Leroy T. Canoles Jr. Cancer Research CenterEastern Virginia Medical SchoolNorfolkVirginiaUSA
- Department of Microbiology and Molecular Cell BiologyEastern Virginia Medical SchoolNorfolkVirginiaUSA
| | - O. John Semmes
- Leroy T. Canoles Jr. Cancer Research CenterEastern Virginia Medical SchoolNorfolkVirginiaUSA
- Department of Microbiology and Molecular Cell BiologyEastern Virginia Medical SchoolNorfolkVirginiaUSA
| | - Lifang Yang
- Leroy T. Canoles Jr. Cancer Research CenterEastern Virginia Medical SchoolNorfolkVirginiaUSA
- Department of Microbiology and Molecular Cell BiologyEastern Virginia Medical SchoolNorfolkVirginiaUSA
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Ding X, Wang X, Du J, Han Q, Zhang D, Zhu H. A systematic review and Meta-analysis of urinary extracellular vesicles proteome in diabetic nephropathy. Front Endocrinol (Lausanne) 2022; 13:866252. [PMID: 36034457 PMCID: PMC9405893 DOI: 10.3389/fendo.2022.866252] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Accepted: 07/04/2022] [Indexed: 11/16/2022] Open
Abstract
Diabetic nephropathy (DN) is a major microvascular complication of both type 1 and type 2 diabetes mellitus and is the most frequent cause of end-stage renal disease with an increasing prevalence. Presently there is no non-invasive method for differential diagnosis, and an efficient target therapy is lacking. Extracellular vesicles (EV), including exosomes, microvesicles, and apoptotic bodies, are present in various body fluids such as blood, cerebrospinal fluid, and urine. Proteins in EV are speculated to be involved in various processes of disease and reflect the original cells' physiological states and pathological conditions. This systematic review is based on urinary extracellular vesicles studies, which enrolled patients with DN and investigated the proteins in urinary EV. We systematically reviewed articles from the PubMed, Embase, Web of Science databases, and China National Knowledge Infrastructure (CNKI) database until January 4, 2022. The article quality was appraised according to the Newcastle-Ottawa Quality Assessment Scale (NOS). The methodology of samples, isolation and purification techniques of urinary EV, and characterization methods are summarized. Molecular functions, biological processes, and pathways were enriched in all retrievable urinary EV proteins. Protein-protein interaction analysis (PPI) revealed pathways of potential biomarkers. A total of 539 articles were retrieved, and 13 eligible records were enrolled in this systematic review and meta-analysis. And two studies performed mass spectrometry to obtain the proteome profile. Two of them enrolled only T1DM patients, two studies enrolled both patients with T1DM and T2DM, and other the nine studies focused on T2DM patients. In total 988 participants were enrolled, and DN was diagnosed according to UACR, UAER, or decreased GFR. Totally 579 urinary EV proteins were detected and 28 of them showed a potential value to be biomarkers. The results of bioinformatics analysis revealed that urinary EV may participate in DN through various pathways such as angiogenesis, biogenesis of EV, renin-angiotensin system, fluid shear stress and atherosclerosis, collagen degradation, and immune system. Besides that, it is necessary to report results compliant with the guideline of ISEV, in orderto assure repeatability and help for further studies. This systematic review concordance with previous studies and the results of meta-analysis may help to value the methodology details when urinary EV proteins were reported, and also help to deepen the understanding of urinary EV proteins in DN.
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Affiliation(s)
- Xiaonan Ding
- Medical School of Chinese People’s Liberation Army (PLA), Beijing, China
- Department of Nephrology, The First Medical Center, Chinese People’s Liberation Army (PLA) General Hospital, Chinese People’s Liberation Army (PLA) Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, China
| | - Xiaochen Wang
- Medical School of Chinese People’s Liberation Army (PLA), Beijing, China
- Department of Nephrology, The First Medical Center, Chinese People’s Liberation Army (PLA) General Hospital, Chinese People’s Liberation Army (PLA) Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, China
| | - Junxia Du
- Medical School of Chinese People’s Liberation Army (PLA), Beijing, China
- Department of Nephrology, The First Medical Center, Chinese People’s Liberation Army (PLA) General Hospital, Chinese People’s Liberation Army (PLA) Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, China
| | - Qiuxia Han
- Department of Nephrology, The First Medical Center, Chinese People’s Liberation Army (PLA) General Hospital, Chinese People’s Liberation Army (PLA) Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, China
| | - Dong Zhang
- Department of Nephrology, The First Medical Center, Chinese People’s Liberation Army (PLA) General Hospital, Chinese People’s Liberation Army (PLA) Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, China
- *Correspondence: Hanyu Zhu, ; Dong Zhang,
| | - Hanyu Zhu
- Department of Nephrology, The First Medical Center, Chinese People’s Liberation Army (PLA) General Hospital, Chinese People’s Liberation Army (PLA) Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, China
- *Correspondence: Hanyu Zhu, ; Dong Zhang,
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Wang Y, Amdanee N, Zhang X. Exosomes in schizophrenia: Pathophysiological mechanisms, biomarkers, and therapeutic targets. Eur Psychiatry 2022; 65:e61. [PMID: 36082534 PMCID: PMC9532215 DOI: 10.1192/j.eurpsy.2022.2319] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
While schizophrenia (SCZ) is a devastating psychiatric disorder that detrimentally affects a significant portion of the worldwide population, its diagnosis is traditionally based on a relatively subjective assessment of current symptoms and medical history, devoid of an objective diagnostic modality. Antipsychotic medications are commonly used in the treatment of SCZ; however, some patients have low remission rates or forsake treatment due to the associated multiple side effects, resulting in recurrent episodes of the disease and poor prognosis. These situations imply that the diagnosis, treatment, and prognosis of SCZ need to be improved to increase the odds of a better outcome. Mounting studies have found that extracellular vesicles (EVs) play essential roles in the central nervous system. They are implicated in several mechanisms closely associated with SCZ such as cellular communication and synaptic plasticity. They can additionally exhibit neuroprotective and therapeutic effects. Since they possess distinct constituents, are readily available, easily detectable, and dependent on the internal environment, they can potentially serve as reliable biomarkers for disease diagnosis. Moreover, their biological configuration along with their ability to increase the bioavailability of their constituents and modulate intricate intracellular reactions in target cells, propel EVs as new targets for treatment. This review paper summarizes relevant research pertaining to the roles of EVs in SCZ, with the aim of improving insights into SCZ pathogenesis and evaluating EVs as potential biomarkers in the diagnosis and treatment of SCZ.
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Perpetuo L, Ferreira R, Thongboonkerd V, Guedes S, Amado F, Vitorino R. Urinary exosomes: Diagnostic impact with a bioinformatic approach. Adv Clin Chem 2022; 111:69-99. [DOI: 10.1016/bs.acc.2022.07.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
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Saad MG, Beyenal H, Dong WJ. Exosomes as Powerful Engines in Cancer: Isolation, Characterization and Detection Techniques. BIOSENSORS 2021; 11:518. [PMID: 34940275 PMCID: PMC8699402 DOI: 10.3390/bios11120518] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/17/2021] [Revised: 11/28/2021] [Accepted: 12/02/2021] [Indexed: 06/01/2023]
Abstract
Exosomes, powerful extracellular nanovesicles released from almost all types of living cells, are considered the communication engines (messengers) that control and reprogram physiological pathways inside target cells within a community or between different communities. The cell-like structure of these extracellular vesicles provides a protective environment for their proteins and DNA/RNA cargos, which serve as biomarkers for many malicious diseases, including infectious diseases and cancers. Cancer-derived exosomes control cancer metastasis, prognosis, and development. In addition to the unique structure of exosomes, their nanometer size and tendency of interacting with cells makes them a viable novel drug delivery solution. In recent years, numerous research efforts have been made to quantify and characterize disease-derived exosomes for diagnosis, monitoring, and therapeutic purposes. This review aims to (1) relate exosome biomarkers to their origins, (2) focus on current isolation and detection methods, (3) discuss and evaluate the proposed technologies deriving from exosome research for cancer treatment, and (4) form a conclusion about the prospects of the current exosome research.
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Affiliation(s)
| | | | - Wen-Ji Dong
- The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA; (M.G.S.); (H.B.)
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Kumar A, Dhadi SR, Mai N, Taylor C, Roy JW, Barnett DA, Lewis SM, Ghosh A, Ouellette RJ. The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids. J Extracell Vesicles 2021; 10:e12138. [PMID: 34478244 PMCID: PMC8409086 DOI: 10.1002/jev2.12138] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Revised: 07/08/2021] [Accepted: 08/12/2021] [Indexed: 12/17/2022] Open
Abstract
Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy-based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently-available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non-toxic, and/or clinically-amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non-immunogenicity, biodegradability, and low toxicity. Chitosan-precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic-based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture-conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method.
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Affiliation(s)
- Awanit Kumar
- Atlantic Cancer Research InstituteMonctonNew BrunswickCanada
| | | | - Ngoc‐Nu Mai
- Atlantic Cancer Research InstituteMonctonNew BrunswickCanada
| | | | - Jeremy W. Roy
- Atlantic Cancer Research InstituteMonctonNew BrunswickCanada
- Beatrice Hunter Cancer Research InstituteHalifaxNova ScotiaCanada
| | - David A. Barnett
- Atlantic Cancer Research InstituteMonctonNew BrunswickCanada
- Department of Chemistry and BiochemistryMount Allison UniversitySackvilleNew BrunswickCanada
| | - Stephen M. Lewis
- Atlantic Cancer Research InstituteMonctonNew BrunswickCanada
- Department of Chemistry and BiochemistryUniversité de MonctonMonctonNew BrunswickCanada
- Beatrice Hunter Cancer Research InstituteHalifaxNova ScotiaCanada
| | - Anirban Ghosh
- Atlantic Cancer Research InstituteMonctonNew BrunswickCanada
| | - Rodney J. Ouellette
- Atlantic Cancer Research InstituteMonctonNew BrunswickCanada
- Department of Chemistry and BiochemistryUniversité de MonctonMonctonNew BrunswickCanada
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Oshikawa‐Hori S, Yokota‐Ikeda N, Sonoda H, Sasaki Y, Ikeda M. Reduced urinary release of AQP1- and AQP2-bearing extracellular vesicles in patients with advanced chronic kidney disease. Physiol Rep 2021; 9:e15005. [PMID: 34435473 PMCID: PMC8387789 DOI: 10.14814/phy2.15005] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2021] [Revised: 07/26/2021] [Accepted: 07/29/2021] [Indexed: 11/24/2022] Open
Abstract
Although several studies have shown that release of water channel proteins, aquaporin 1 (AQP1) and AQP2 in urinary extracellular vesicles (uEV-AQP1 and -AQP2), were altered in experimental kidney injury models, their release in human chronic kidney disease (CKD) has been largely unexplored. The aim of the present study was to clarify whether the release of uEV-AQP1 and -AQP2 is altered in patients with CKD. Urine samples were collected from 15 healthy volunteers (normal group) and 62 CKD patients who were categorized into six glomerular filtration rate (GFR) categories (G1, G2, G3a, G3b, G4, and G5) in between 2005 and 2016 at Miyazaki Prefectural Miyazaki Hospital, Japan. uEV-proteins were evaluated by immunoblot analysis. The release of AQP1 and AQP2 were significantly decreased in patients with both CKD G4 and G5, in comparison with the normal group. The area under the receiver operating characteristic (ROC) curve (AUC) values for AQP1 and AQP2 in patients with CKD G4 and G5 were 0.926 and 0.881, respectively. On the other hand, the AUC values in patients with CKD G1-G3 were 0.512 for AQP1 and 0.680 for AQP2. Multiple logistic regression analysis showed that AQP1 and AQP2 in combination were useful for detecting CKD G4 and G5, with a higher AUC value of 0.945. These results suggest that the release of uEV-AQP1 and -AQP2 was decreased in patients with CKD G4 and G5, and these proteins might be helpful to detect advanced CKD.
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Affiliation(s)
- Sayaka Oshikawa‐Hori
- Department of Veterinary PharmacologyFaculty of AgricultureUniversity of MiyazakiMiyazakiJapan
| | - Naoko Yokota‐Ikeda
- Department of NephrologyMiyazaki Prefectural Miyazaki HospitalMiyazakiJapan
| | - Hiroko Sonoda
- Department of Veterinary PharmacologyFaculty of AgricultureUniversity of MiyazakiMiyazakiJapan
| | - Yosuke Sasaki
- Department of Animal and Grassland SciencesFaculty of AgricultureUniversity of MiyazakiMiyazakiJapan
| | - Masahiro Ikeda
- Department of Veterinary PharmacologyFaculty of AgricultureUniversity of MiyazakiMiyazakiJapan
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42
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Zhang Q, Yang X, Liu H. Extracellular Vesicles in Cancer Metabolism: Implications for Cancer Diagnosis and Treatment. Technol Cancer Res Treat 2021; 20:15330338211037821. [PMID: 34427131 PMCID: PMC8388228 DOI: 10.1177/15330338211037821] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Metabolic reprogramming is one of the most common characteristics of cancer cells. The metabolic alterations of glucose, amino acids and lipids can support the aggressive phenotype of cancer cells. Exosomes, a kind of extracellular vesicles, participate in the intercellular communication through transferring bioactive molecules. Increasing evidence has demonstrated that enzymes, metabolites and non-coding RNAs in exosomes are responsible for the metabolic alteration of cancer cells. In this review, we summarize the past and recent findings of exosomes in altering cancer metabolism and elaborate on the role of the specific enzymes, metabolites and non-coding RNAs transferred by exosomes. Moreover, we give evidence of the role of exosomes in cancer diagnosis and treatment. Finally, we discuss the existing problems in the study and application of exosomes in cancer diagnosis and treatment.
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Affiliation(s)
- Qian Zhang
- Department of Clinical Laboratory, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Xiangling Yang
- Department of Clinical Laboratory, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.,Guangdong Institute of Gastroenterology, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.,Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Huanliang Liu
- Department of Clinical Laboratory, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.,Guangdong Institute of Gastroenterology, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.,Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China
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43
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Elgamal S, Cocucci E, Sass EJ, Mo XM, Blissett AR, Calomeni EP, Rogers KA, Woyach JA, Bhat SA, Muthusamy N, Johnson AJ, Larkin KT, Byrd JC. Optimizing extracellular vesicles' isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant. JCI Insight 2021; 6:e137937. [PMID: 34369387 PMCID: PMC8410027 DOI: 10.1172/jci.insight.137937] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture-conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.
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Affiliation(s)
- Sara Elgamal
- Division of Hematology, Department of Internal Medicine, College of Medicine.,Comprehensive Cancer Center
| | - Emanuele Cocucci
- Comprehensive Cancer Center.,Division of Pharmaceutics, College of Pharmacy
| | - Ellen J Sass
- Division of Hematology, Department of Internal Medicine, College of Medicine.,Comprehensive Cancer Center
| | - Xiaokui M Mo
- Comprehensive Cancer Center.,Department of Biomedical Informatics, College of Medicine
| | | | | | - Kerry A Rogers
- Division of Hematology, Department of Internal Medicine, College of Medicine.,Comprehensive Cancer Center
| | - Jennifer A Woyach
- Division of Hematology, Department of Internal Medicine, College of Medicine.,Comprehensive Cancer Center
| | - Seema A Bhat
- Division of Hematology, Department of Internal Medicine, College of Medicine.,Comprehensive Cancer Center
| | - Natarajan Muthusamy
- Division of Hematology, Department of Internal Medicine, College of Medicine.,Comprehensive Cancer Center.,College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA
| | - Amy J Johnson
- Division of Hematology, Department of Internal Medicine, College of Medicine.,Comprehensive Cancer Center.,Division of Pharmaceutics, College of Pharmacy
| | - Karilyn T Larkin
- Division of Hematology, Department of Internal Medicine, College of Medicine.,Comprehensive Cancer Center
| | - John C Byrd
- Division of Hematology, Department of Internal Medicine, College of Medicine.,Comprehensive Cancer Center.,Division of Pharmaceutics, College of Pharmacy.,College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA
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44
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Extracellular Vesicles: New Tools for Early Diagnosis of Breast and Genitourinary Cancers. Int J Mol Sci 2021; 22:ijms22168430. [PMID: 34445131 PMCID: PMC8395117 DOI: 10.3390/ijms22168430] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2021] [Revised: 07/29/2021] [Accepted: 08/03/2021] [Indexed: 12/12/2022] Open
Abstract
Breast cancers and cancers of the genitourinary tract are the most common malignancies among men and women and are still characterized by high mortality rates. In order to improve the outcomes, early diagnosis is crucial, ideally by applying non-invasive and specific biomarkers. A key role in this field is played by extracellular vesicles (EVs), lipid bilayer-delimited structures shed from the surface of almost all cell types, including cancer cells. Subcellular structures contained in EVs such as nucleic acids, proteins, and lipids can be isolated and exploited as biomarkers, since they directly stem from parental cells. Furthermore, it is becoming even more evident that different body fluids can also serve as sources of EVs for diagnostic purposes. In this review, EV isolation and characterization methods are described. Moreover, the potential contribution of EV cargo for diagnostic discovery purposes is described for each tumor.
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45
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Bao C, He C. The role and therapeutic potential of MSC-derived exosomes in osteoarthritis. Arch Biochem Biophys 2021; 710:109002. [PMID: 34352243 DOI: 10.1016/j.abb.2021.109002] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2021] [Revised: 07/27/2021] [Accepted: 07/31/2021] [Indexed: 02/08/2023]
Abstract
Osteoarthritis (OA) is the most common painful disease with chronic articular cartilage degeneration. The pathological process of OA is complex and characterized by the imbalance between the synthesis and catabolism of chondrocytes and extracellular matrix, leading to the progressive destruction of articular cartilage damage. Because of the self-renewal and differentiation of mesenchymal stem cells (MSCs), various exogenous MSC-based cell therapies have been developed to treat OA. Moreover, the efficacy of MSC- based therapy is mainly attributed to the paracrine of cytokines, growth factors, and exosomes. Exosomes derived from MSCs can deliver various DNAs, RNAs, proteins and lipids, thus promoting MSCs migration and cartilage repair. Therefore, MSC-derived exosomes are considered as a promising alternative therapy for OA. In this review, we summarized properties of MSC-derived exosomes and the new role of MSC-derived exosomes in the treatment of OA. We also proposed possible perspectives of MSC-derived exosomes as cell-free regenerative reagents in the treatment of OA.
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Affiliation(s)
- Chuncha Bao
- Department of Rehabilitation Medicine, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; Key Laboratory of Rehabilitation Medicine, West China Hospital, Sichuan University, Chengdu, China
| | - Chengqi He
- Department of Rehabilitation Medicine, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; Key Laboratory of Rehabilitation Medicine, West China Hospital, Sichuan University, Chengdu, China.
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46
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Wu Q, Fenton RA. Urinary proteomics for kidney dysfunction: insights and trends. Expert Rev Proteomics 2021; 18:437-452. [PMID: 34187288 DOI: 10.1080/14789450.2021.1950535] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Introduction: Kidney dysfunction poses a high burden on patients and health care systems. Early detection and accurate prediction of kidney disease progression remains a major challenge. Compared to existing clinical parameters, urinary proteomics has the potential to reveal molecular alterations within the kidney that may alter its function before the onset of clinical symptoms. Thus, urinary proteomics has greater prognostic potential for assessment of kidney dysfunction progression.Areas covered: Advances in urinary proteomics for major causes of kidney dysfunction are discussed. The application of urinary extracellular vesicles for studying kidney dysfunction are discussed. Technological advances in urinary proteomics are discussed. The literature was identified using a database search for titles containing 'proteom*' and 'urin*' and published within the past 5 years. Retrieved literature was manually filtered to retain kidney dysfunctions-related studies.Expert opinion: Despite major advances, diagnosis by urinary proteomics has not been fully applied in any clinical settings. This could be attributed to the complex nature of kidney diseases, in addition to the constraints on study power and feasibility of incorporating mass spectrometry techniques in daily routine analysis. Nevertheless, we are confident that advances in urinary proteomics will soon provide superior insights into kidney disease beyond existing clinical parameters.
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Affiliation(s)
- Qi Wu
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
| | - Robert A Fenton
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
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Huda MN, Nafiujjaman M, Deaguero IG, Okonkwo J, Hill ML, Kim T, Nurunnabi M. Potential Use of Exosomes as Diagnostic Biomarkers and in Targeted Drug Delivery: Progress in Clinical and Preclinical Applications. ACS Biomater Sci Eng 2021; 7:2106-2149. [PMID: 33988964 PMCID: PMC8147457 DOI: 10.1021/acsbiomaterials.1c00217] [Citation(s) in RCA: 113] [Impact Index Per Article: 28.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2021] [Accepted: 05/03/2021] [Indexed: 12/13/2022]
Abstract
Exosomes are cell-derived vesicles containing heterogeneous active biomolecules such as proteins, lipids, mRNAs, receptors, immune regulatory molecules, and nucleic acids. They typically range in size from 30 to 150 nm in diameter. An exosome's surfaces can be bioengineered with antibodies, fluorescent dye, peptides, and tailored for small molecule and large active biologics. Exosomes have enormous potential as a drug delivery vehicle due to enhanced biocompatibility, excellent payload capability, and reduced immunogenicity compared to alternative polymeric-based carriers. Because of active targeting and specificity, exosomes are capable of delivering their cargo to exosome-recipient cells. Additionally, exosomes can potentially act as early stage disease diagnostic tools as the exosome carries various protein biomarkers associated with a specific disease. In this review, we summarize recent progress on exosome composition, biological characterization, and isolation techniques. Finally, we outline the exosome's clinical applications and preclinical advancement to provide an outlook on the importance of exosomes for use in targeted drug delivery, biomarker study, and vaccine development.
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Affiliation(s)
- Md Nurul Huda
- Environmental Science & Engineering, University of Texas at El Paso, El Paso, TX 79968
| | - Md Nafiujjaman
- Department of Biomedical Engineering, Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, MI 48824
| | - Isaac G Deaguero
- Biomedical Engineering, University of Texas at El Paso, El Paso, TX 79968
| | - Jude Okonkwo
- John A Paulson School of Engineering, Harvard University, Cambridge, MA 02138
| | - Meghan L. Hill
- Department of Biomedical Engineering, Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, MI 48824
| | - Taeho Kim
- Department of Biomedical Engineering, Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, MI 48824
| | - Md Nurunnabi
- Environmental Science & Engineering, University of Texas at El Paso, El Paso, TX 79968
- Biomedical Engineering, University of Texas at El Paso, El Paso, TX 79968
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Texas at El Paso, El Paso, TX 79902
- Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX 79968
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48
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Saenz-Pipaon G, Echeverria S, Orbe J, Roncal C. Urinary Extracellular Vesicles for Diabetic Kidney Disease Diagnosis. J Clin Med 2021; 10:jcm10102046. [PMID: 34064661 PMCID: PMC8151759 DOI: 10.3390/jcm10102046] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2021] [Revised: 04/30/2021] [Accepted: 05/07/2021] [Indexed: 12/12/2022] Open
Abstract
Diabetic kidney disease (DKD) is the leading cause of end stage renal disease (ESRD) in developed countries, affecting more than 40% of diabetes mellitus (DM) patients. DKD pathogenesis is multifactorial leading to a clinical presentation characterized by proteinuria, hypertension, and a gradual reduction in kidney function, accompanied by a high incidence of cardiovascular (CV) events and mortality. Unlike other diabetes-related complications, DKD prevalence has failed to decline over the past 30 years, becoming a growing socioeconomic burden. Treatments controlling glucose levels, albuminuria and blood pressure may slow down DKD evolution and reduce CV events, but are not able to completely halt its progression. Moreover, one in five patients with diabetes develop DKD in the absence of albuminuria, and in others nephropathy goes unrecognized at the time of diagnosis, urging to find novel noninvasive and more precise early diagnosis and prognosis biomarkers and therapeutic targets for these patient subgroups. Extracellular vesicles (EVs), especially urinary (u)EVs, have emerged as an alternative for this purpose, as changes in their numbers and composition have been reported in clinical conditions involving DM and renal diseases. In this review, we will summarize the current knowledge on the role of (u)EVs in DKD.
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Affiliation(s)
- Goren Saenz-Pipaon
- Laboratory of Atherothrombosis, Program of Cardiovascular Diseases, Cima Universidad de Navarra, 31008 Pamplona, Spain; (G.S.-P.); (J.O.)
- IdiSNA, Instituto de Investigación Sanitaria de Navarra, 31008 Pamplona, Spain
| | - Saioa Echeverria
- Endocrinology Service, Clínica Universidad de Navarra, 31008 Pamplona, Spain;
| | - Josune Orbe
- Laboratory of Atherothrombosis, Program of Cardiovascular Diseases, Cima Universidad de Navarra, 31008 Pamplona, Spain; (G.S.-P.); (J.O.)
- IdiSNA, Instituto de Investigación Sanitaria de Navarra, 31008 Pamplona, Spain
- CIBERCV, Instituto de Salud Carlos III, 28029 Madrid, Spain
| | - Carmen Roncal
- Laboratory of Atherothrombosis, Program of Cardiovascular Diseases, Cima Universidad de Navarra, 31008 Pamplona, Spain; (G.S.-P.); (J.O.)
- IdiSNA, Instituto de Investigación Sanitaria de Navarra, 31008 Pamplona, Spain
- CIBERCV, Instituto de Salud Carlos III, 28029 Madrid, Spain
- Correspondence: ; Tel.: +34-948194700
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Karttunen J, Stewart SE, Kalmar L, Grant AJ, Karet Frankl FE, Williams TL. Size-Exclusion Chromatography Separation Reveals That Vesicular and Non-Vesicular Small RNA Profiles Differ in Cell Free Urine. Int J Mol Sci 2021; 22:ijms22094881. [PMID: 34063036 PMCID: PMC8124894 DOI: 10.3390/ijms22094881] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2021] [Revised: 04/22/2021] [Accepted: 04/30/2021] [Indexed: 12/26/2022] Open
Abstract
Urinary extracellular vesicles (EVs) and their RNA cargo are a novel source of biomarkers for various diseases. We aimed to identify the optimal method for isolating small (<200 nm) EVs from human urine prior to small RNA analysis. EVs from filtered healthy volunteer urine were concentrated using three methods: ultracentrifugation (UC); a precipitation-based kit (PR); and ultrafiltration (UF). EVs were further purified by size-exclusion chromatography (SEC). EV preparations were analysed with transmission electron microscopy (TEM), Western blotting, nanoparticle tracking analysis (NTA) and an Agilent Bioanalyzer Small RNA kit. UF yielded the highest number of particles both before and after SEC. Small RNA analysis from UF-concentrated urine identified two major peaks at 10–40 nucleotides (nt) and 40–80 nt. In contrast, EV preparations obtained after UC, PR or SEC combined with any concentrating method, contained predominantly 40–80 nt sized small RNA. Protein fractions from UF+SEC contained small RNA of 10–40 nt in size (consistent with miRNAs). These data indicate that most of the microRNA-sized RNAs in filtered urine are not associated with small-sized EVs, and highlights the importance of removing non-vesicular proteins and RNA from urine EV preparations prior to small RNA analysis.
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Affiliation(s)
- Jenni Karttunen
- Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK; (J.K.); (L.K.); (A.J.G.)
| | - Sarah E. Stewart
- Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge CB2 0QQ, UK;
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC 3086, Australia
| | - Lajos Kalmar
- Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK; (J.K.); (L.K.); (A.J.G.)
| | - Andrew J. Grant
- Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK; (J.K.); (L.K.); (A.J.G.)
| | | | - Tim L. Williams
- Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK; (J.K.); (L.K.); (A.J.G.)
- Correspondence:
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50
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Affiliation(s)
- Robert W. Hunter
- Centre for Cardiovascular Science, Queen’s Medical Research Institute, Edinburgh Bioquarter, University of Edinburgh, Edinburgh, United Kingdom,Department of Renal Medicine, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom
| | - James W. Dear
- Centre for Cardiovascular Science, Queen’s Medical Research Institute, Edinburgh Bioquarter, University of Edinburgh, Edinburgh, United Kingdom,Department of Toxicology, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom
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