Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

Gonzales, Patricia A.; Pisitkun, Trairak; Hoffert, Jason D.; Tchapyjnikov, Dmitry; Star, Robert A.; Kleta, Robert; Wang, Nam Sun; Knepper, Mark A.

doi:10.1681/asn.2008040406

Cited by 653 publications

(657 citation statements)

References 28 publications

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“…The KEGG pathway analysis showed the prevalence of these proteins in metabolic (12.0%), endocytosis (6.2%), biosynthesis (4.3%), phagosome (3.9%), regulation of actin cytoskeleton (3.7%), and lysosome (2.5%) pathways. These compositions were very similar to the previously deposited database of human urine exosomes (a total of 1160 proteins, Gonzales et al [18], https ://hpcwe bapps .cit.nih.gov/ESBL/ Datab ase/Exoso me/). Thus, our starting materials were human urine so-called "exosome", although this fraction is known to contain other EVs subclasses.…”

Section: Proteomic Analysis Of Evs Immunoprecipitated With Aqp2 Antibodysupporting

confidence: 85%

“…Among AQP family members, only AQP1, AQP2 and AQP7 have been reported in urine exosomes [18,25]. As the Pf values of EVs were correlated with the amount of AQP2 in the EVs (Fig.…”

Section: Discussionmentioning

confidence: 91%

“…Several membrane transport proteins (channels and transporters; such as NCC, NKCC2, SGLT1,2 and ENaC) have been reported to be present in EVs [18,27]. However, none of them have been examined for their functions directly.…”

Section: Discussionmentioning

confidence: 99%

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AQP2 in human urine is predominantly localized to exosomes with preserved water channel activities

et al. 2018

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Background AQP2 water channel is critical for urinary concentration in the kidney. Interestingly, AQP2 is abundantly excreted in the urine as extracellular vesicles (EVs), which is known to be a useful biomarker for water-balance disorders although the character of AQP2-enriched EVs is poorly understood including water channel function. Methods Human urine EVs were obtained by a differential centrifugation method. AQP2-bearing EVs were isolated by immunoprecipitation with an AQP2-specific antibody, and the proteins in the EVs were analyzed by LC-MS/MS proteomic analysis. Osmotic water permeability (Pf) of the AQP2-rich EVs was measured by a stopped-flow method monitoring scattered light intensity in response to outwardly directed osmotic gradient. Results Sequential centrifugation of human urine showed that AQP2 was present predominantly (80%) in low-density EVs (160,000 g), whereas negligible amount in high-density EVs (17,000 g). Proteomic analysis of the AQP2-bearing EVs identified 137 proteins, mostly in the endosome pathway, including the components of ESCRT (endosomal sorting complex required transporter)-I, II, III. Pf value of the 160,000 g EVs was 4.75 ± 0.38 × 10 −4 cm s −1 (mean ± SE) with the activation energy of 3.51 kcal mol −1 which was inhibited with 0.3 mM HgCl 2 by 63%, suggesting a channel-mediated water transport. Moreover, Pf value showed a significant correlation with the abundance of AQP2 protein in EVs. Conclusion Taken together, AQP2 is localized predominantly to urinary exosomes with preserved water channel activities.

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Section: Proteomic Analysis Of Evs Immunoprecipitated With Aqp2 Antibodysupporting

confidence: 85%

“…Among AQP family members, only AQP1, AQP2 and AQP7 have been reported in urine exosomes [18,25]. As the Pf values of EVs were correlated with the amount of AQP2 in the EVs (Fig.…”

Section: Discussionmentioning

confidence: 91%

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AQP2 in human urine is predominantly localized to exosomes with preserved water channel activities

et al. 2018

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“…Adachi et al have used high accuracy MS to detect more than 1500 proteins in human urine, 16 including a large proportion of membrane proteins, making urine indeed an interesting body fluid for biomarker research 79 . Similarly, Knepper and colleagues detected nearly 1100 proteins in human urinary exosomes and also determined phosphorylation sites 80 . Major achievements of urine proteomics have been reviewed elsewhere 3,4,81 .…”

Section: Application Of Proteomics To Normal and Diseased Kidney Funcmentioning

confidence: 94%

Toward Quantitative Proteomics of Organ Substructures: Implications for Renal Physiology

Velić

Maček

Wagner

2010

Seminars in Nephrology

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AcknowledgementsResearch in the laboratories of the authors has been supported by grants from the Minstry of Science, Baden-Wuerttenberg (to BM) and the Swiss National Science Foundation, the 6 th and 7 th EU Framework projects (EuReGene and EUNEFRON) (to CAW). We thank MatthiasMann for critically reading and discussing the manuscript. Summary 2Organs are complex structures that consist of multiple tissues with different levels of gene expression. To achieve a comprehensive coverage and accurate quantitation data, organs should ideally be separated into morphological and/or functional substructures prior to gene or protein expression analysis. However, due to complex morphology and elaborate isolation protocols, this was so far often difficult to achieve. Kidneys are organs where functional and morphological subdivision is especially important. Each subunit of the kidney, the nephron, itself consists of more than 10 subsegments with distinct morphological and functional characteristics. For a full understanding of kidney physiology, global gene and protein expression analyses have to be performed at the level of the nephron subsegments; however, such studies have so far been extremely rare.Here we describe the latest approaches in quantitative high accuracy mass spectrometrybased proteomics and their application to quantitative proteomics studies of the whole kidney and nephron subsegments, both in humans and in animal models. We compare these studies with similar studies performed on other organ substructures. We argue that the newest technologies used for preparation, processing and measurement of small amounts of starting material are finally enabling global and subsegment-specific quantitative measurement of protein levels in the kidney and other organs. These new technologies and approaches are making a decisive impact on our understanding of the (patho)physiological processes at the molecular level.3

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“…Recently, a new phosphorylation site uniquely located at S811 in NCC SV was identified [46]. Tutakhel et al .…”

Section: Functional Role Of Nccsvmentioning

confidence: 99%

Role of the alternative splice variant of NCC in blood pressure control

2018

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The renal thiazide-sensitive sodium-chloride cotransporter (NCC), located in the distal convoluted tubule (DCT) of the kidney, plays an important role in blood pressure regulation by fine-tuning sodium excretion. The human SLC12A3 gene, encoding NCC, gives rise to three isoforms, of which only the third isoform (NCC3) has been extensively investigated so far. However, recent studies unraveled the importance of the isoforms 1 and 2, collectively referred to as NCC splice variant (NCCSV), in several (patho)physiological conditions. In the human kidney, NCCSV localizes to the apical membrane of the DCT and could constitute a functional route for renal sodium-chloride reabsorption. Analysis of urinary extracellular vesicles (uEVs), a non-invasive method for measuring renal responses, demonstrated that NCCSV abundance changes in response to acute water loading and correlates with patients’ thiazide responsiveness. Furthermore, a novel phosphorylation site at serine 811 (S811), exclusively present in NCCSV, was shown to play an instrumental role in NCCSV as well as NCC3 function. This review aims to summarize these new insights of NCCSV function in humans that broadens the understanding on NCC regulation in blood pressure control.

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Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

Cited by 653 publications

References 28 publications

AQP2 in human urine is predominantly localized to exosomes with preserved water channel activities

AQP2 in human urine is predominantly localized to exosomes with preserved water channel activities

Toward Quantitative Proteomics of Organ Substructures: Implications for Renal Physiology

Role of the alternative splice variant of NCC in blood pressure control

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