Management of Tamm–Horsfall Protein for Reliable Urinary Analytics

Xu, Xiaomeng; Barreiro, Karina; Musante, Luca; Kretz, Oliver; Hu, Lin; Zou, Hequn; Huber, Tobias B.; Holthöfer, Harry

doi:10.1002/prca.201900018

Cited by 29 publications

(35 citation statements)

References 41 publications

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“…Clinical biomarker studies should, therefore, attempt to validate biomarker detection in urine collected without prior DRE, given this does not negatively influence detection sensitivity [67]. The treatment of urine or uEV with the reducing agent dithiothreitol (DTT), zwitterionic detergent CHAPS or urea to denature and remove abundant THP has previously been proposed [68–70]. However, this study demonstrated no significant advantage of DTT treatment on reducing THP contamination or increasing uEV yield when applying a BU ODG.…”

Section: Discussionmentioning

confidence: 97%

Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density‐based fractionation of urine

Dhondt

Geeurickx

Tulkens

et al. 2020

J of Extracellular Vesicle

127

164

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Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometrybased proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers.

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Section: Discussionmentioning

confidence: 97%

Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density‐based fractionation of urine

Dhondt

Geeurickx

Tulkens

et al. 2020

J of Extracellular Vesicle

127

164

View full text Add to dashboard Cite

show abstract

“…However, as our focus was on RNA sequencing, we did not include any further purification step. As a reference, HFD in combination with protocols to reduce THP interference has been successfully used for uEV proteomics (Hu et al., 2018; Xu et al., 2019). The UC protocol presented in our study has not been used for proteomics, but UC in combination with reducing agents such as DTT (Rood et al., 2010; Wolley et al., 2017), or sucrose gradient centrifugation (Hogan et al., 2014), or size exclusion chromatography (Rood et al., 2010) have been used for uEV proteomics with good results.…”

Section: Discussionmentioning

confidence: 99%

Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease

Barreiro

Dwivedi

Leparc

et al. 2020

J of Extracellular Vesicle

Self Cite

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Urinary Extracellular Vesicles (uEV) have emerged as a source for biomarkers of kidney damage, holding potential to replace the conventional invasive techniques including kidney biopsy. However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compared performance of three established uEV isolation workflows for their subsequent use in transcriptomics analysis for biomarker discovery in diabetic kidney disease. We collected urine samples from individuals with type 1 diabetes with macroalbuminuria and healthy controls. We isolated uEV by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit‐ based isolation method (NG), each with different established urine clearing steps. Purified EVs were analysed by electron microscopy, nanoparticle tracking analysis, and Western blotting. Isolated RNAs were subjected to miRNA and RNA sequencing. HFD and UC samples showed close similarities based on mRNA sequencing data. NG samples had a lower number of reads and different mRNA content compared to HFD or UC. For miRNA sequencing data, satisfactory miRNA counts were obtained by all methods, but miRNA contents differed slightly. This suggests that the isolation workflows enrich specific subpopulations of miRNA‐rich uEV preparation components. Our data shows that HFD,UC and the kit‐based method are suitable methods to isolate uEV for miRNA‐seq. However, only HFD and UC were suitable for mRNA‐seq in our settings.

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“…Additionally, the high variation of EV recovery due to inter-day urine collection seems to be hampered by the addition of protease inhibitors [ 148 ]. Unfortunately, the optimal inhibitor cocktail is still an open debate with several authors showing that different protease inhibitor cocktails had different impacts on the ratio of THP polymerization and consequently on the yield of urinary EVs in the UC pellets [ 148 , 151 , 152 , 153 ].…”

Section: Extracellular Vesicles From Liquid Biopsies As a Source Omentioning

confidence: 99%

Urinary Biomarkers in Bladder Cancer: Where Do We Stand and Potential Role of Extracellular Vesicles

Oliveira

Caires

Oliveira

et al. 2020

Cancers

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Extracellular vesicles (EVs) are small membrane vesicles released by all cells and involved in intercellular communication. Importantly, EVs cargo includes nucleic acids, lipids, and proteins constantly transferred between different cell types, contributing to autocrine and paracrine signaling. In recent years, they have been shown to play vital roles, not only in normal biological functions, but also in pathological conditions, such as cancer. In the multistep process of cancer progression, EVs act at different levels, from stimulation of neoplastic transformation, proliferation, promotion of angiogenesis, migration, invasion, and formation of metastatic niches in distant organs, to immune escape and therapy resistance. Moreover, as products of their parental cells, reflecting their genetic signatures and phenotypes, EVs hold great promise as diagnostic and prognostic biomarkers. Importantly, their potential to overcome the current limitations or the present diagnostic procedures has created interest in bladder cancer (BCa). Indeed, cystoscopy is an invasive and costly technique, whereas cytology has poor sensitivity for early staged and low-grade disease. Several urine-based biomarkers for BCa were found to overcome these limitations. Here, we review their potential advantages and downfalls. In addition, recent literature on the potential of EVs to improve BCa management was reviewed and discussed.

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Management of Tamm–Horsfall Protein for Reliable Urinary Analytics

Cited by 29 publications

References 41 publications

Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density‐based fractionation of urine

Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density‐based fractionation of urine

Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease

Urinary Biomarkers in Bladder Cancer: Where Do We Stand and Potential Role of Extracellular Vesicles

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