Isolation and Purification of Exosomes in Urine

Gonzales, Patricia A.; Zhou, Hua; Pisitkun, Trairak; Wang, Nam Sun; Star, Robert A.; Knepper, Mark A.; Yuen, Peter S.T.

doi:10.1007/978-1-60761-711-2_6

Cited by 107 publications

(88 citation statements)

References 5 publications

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“…28 Microparticles in the supernatant can be isolated for further analyses by ultracentrifugation at a later stage, after storage at -80°C. 21,36 Urine preanalytical conditions have been studied in specific proteomic methods including MALDI-TOF-MS, 37 2D-electrophoresis, 4 SELDITOF MS, 31 and LC-MS, 38 showing that collection and processing variables can have a major impact on specific proteins and peptides. In this context, laboratories and biobanks operating under accreditation are advised to track and record preanalytical data systematically using the Standard PREanalytical Code (SPREC), 39,40 while freeze-thaw cycles and other treatments (e.g., use of protease inhibitors, stabilizers) should be reported in the Laboratory Information Management System.…”

Section: Urine Processing Methods 355 Discussionmentioning

confidence: 99%

Method Validation for Preparing Urine Samples for Downstream Proteomic and Metabolomic Applications

Ammerlaan

Trezzi

Mathay

et al. 2014

Biopreservation and Biobanking

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Background: Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints. Methods: Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4°C. The optimal protocol was validated for performance (microparticle counts), and for reproducibility and robustness for centrifugation temperature (4°C vs. RT) and brake speed (soft, medium, hard). Acceptance criteria were based on microparticle counts, cystatin C and creatinine concentrations, and the metabolomic profile. Results: The optimal protocol was a 20-min, 12,000 g centrifugation at 4°C, and was validated for urine collection in terms of microparticle counts. All reproducibility acceptance criteria were met. The protocol was robust for centrifugation at 4°C versus RT for all parameters. The protocol was considered robust overall in terms of brake speeds, although a hard brake gave significantly fewer microparticles than a soft brake. Conclusions: We validated a urine processing method suitable for downstream proteomic and metabolomic applications. Temperature and brake speed can influence analytic results, with 4°C and high brake speed considered optimal. Laboratories and biobanks should ensure these conditions are systematically recorded in the scope of accreditation.

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Section: Urine Processing Methods 355 Discussionmentioning

confidence: 99%

Method Validation for Preparing Urine Samples for Downstream Proteomic and Metabolomic Applications

Ammerlaan

Trezzi

Mathay

et al. 2014

Biopreservation and Biobanking

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“…Urinary exosomes were isolated as reported previously 4,18,19 ; a more detailed protocol is provided as an online-only Data Supplement. Briefly, all urine samples were treated with a protease inhibitor before storage at Ϫ80°C; no phosphatase inhibitors were used.…”

Section: Isolation and Immunoblot Analysis Of Urinary Exosomesmentioning

confidence: 99%

The Phosphorylated Sodium Chloride Cotransporter in Urinary Exosomes Is Superior to Prostasin as a Marker for Aldosteronism

et al. 2012

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Abstract-Urinary exosomes are vesicles derived from renal tubular epithelial cells. Exosomes often contain several disease-associated proteins and are thus useful targets for identifying biomarkers of disease. Here, we hypothesized that the phosphorylated (active) form of the sodium chloride cotransporter (pNCC) or prostasin could serve as biomarkers for aldosteronism. We tested this in 2 animal models of aldosteronism (aldosterone infusion or low-sodium diet) and in patients with primary aldosteronism. Urinary exosomes were isolated from 24-hour urine or spot urine using ultracentrifugation. In rats, a normal or a high dose of aldosterone for 2, 3, or 8 days increased pNCC 3-fold in urinary exosomes (PϽ0.05 for all). A low-sodium diet also increased pNCC in urinary exosomes approximately 1.5-fold after 4 and after 8 days of treatment. The effects of these maneuvers on prostasin in urinary exosomes were less clear, showing a significant 1.5-fold increase only after 2 and 3 days of high-aldosterone infusion. In urinary exosomes of patients with primary aldosteronism, pNCC was 2.6-fold higher (PϽ0.05) while prostasin was 1.5-fold higher (Pϭ0.07) than in patients with essential hypertension. Urinary exosomal pNCC and, to a lesser extent, prostasin are promising markers for aldosteronism in experimental animals and patients. These markers may be used to assess the biological activity of aldosterone and, potentially, as clinical biomarkers for primary aldosteronism. 1 Exosomes are low-density membrane vesicles that originate from multivesicular bodies. Urinary exosomes have sparked interest as potential biomarkers for human disease. 1-3The presence of urinary exosomes and a reproducible method for their isolation were reported in 2004 by Pisitkun and colleagues. 4 Proteomic analysis of these exosomes showed that they contain many disease-related proteins 4,5 ; however, the question remained whether the presence of a given protein in urinary exosomes could provide information on physiological or disease processes in the kidney. Studies addressing this question analyzed urinary exosomes in patients with monogenetic diseases, resulting in inactivity or overactivity of renal sodium transport proteins. For example, in Bartter and Gitelman syndrome, in which the sodium potassium chloride cotransporter and the sodium chloride cotransporter (NCC) are genetically inactivated, these proteins were also found to be absent or reduced in urinary exosomes. 5,6 Conversely, Mayan et al found the abundance of NCC to be increased in patients with familial hyperkalemic hypertension, in which mutations in NCC-regulating kinases cause overactivity of this cotransporter.7 Thus, in these homogeneous groups, the expression of sodium-transport proteins in urinary exosomes correlated with what one would expect from their renal expression. The next step in assessing the potential of exosomes as urinary biomarkers is to analyze their performance in acquired disease. Therefore, in this study, we asked whether various forms of aldosteronism result...

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“…Briefly, exosomes were isolated from frozen urine Following Dithiothreitol (DTT) treatment was applied to increase exosome recovery [22]. Exosomes were depleted from albumin and IgG using ProteoPrep ® Immunoaffinity Albumin and IgG Depletion Kit (Sigma) following manufacturer's protocol and proteins were extracted in lysis buffer (7M urea, 2M thiourea, 4% CHAPS).…”

Section: Rat Urine Collection Exosome Isolation and Protein Extractionmentioning

confidence: 99%

Kidney tissue proteomics reveals regucalcin downregulation in response to diabetic nephropathy with reflection in urinary exosomes

Zubiri

Posada-Ayala

Benito-Martín

et al. 2015

Translational Research

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Isolation and Purification of Exosomes in Urine

Cited by 107 publications

References 5 publications

Method Validation for Preparing Urine Samples for Downstream Proteomic and Metabolomic Applications

Method Validation for Preparing Urine Samples for Downstream Proteomic and Metabolomic Applications

The Phosphorylated Sodium Chloride Cotransporter in Urinary Exosomes Is Superior to Prostasin as a Marker for Aldosteronism

Kidney tissue proteomics reveals regucalcin downregulation in response to diabetic nephropathy with reflection in urinary exosomes

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