Urinary Microvesicle-Bound Uromodulin: A Potential Molecular Biomarker in Diabetic Kidney Disease

Lou, Neng-jun; Ni, Yicheng; Jia, Hongying; Deng, Jingjing; Jiang, Lu; Zheng, Fengjie; Sun, Aili

doi:10.1155/2017/3918681

Cited by 9 publications

(6 citation statements)

References 30 publications

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“…This was most likely due to limitations in the exosome extraction method from plasma and the high lipid content in the exosomal preparation that interfered with the HPLC separation. In addition, large vesicles were isolated from plasma and urine by immunoprecipitation using an antibody against AD-1, an extracellular vesicle marker (25)(26)(27)(28). Immunoprecipitated extracts analyzed by Western blotting confirmed that HSP27 was present in extracellular vesicles (Supplemental Fig.…”

Section: Ms Detection Of Hsp27 Isolated From Human Plasma Extracellulmentioning

confidence: 91%

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Characterization of heat shock protein 27 in extracellular vesicles: a potential anti‐inflammatory therapy

et al. 2018

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Previously, we reported that elevated serum levels of heat shock protein 27 (HSP27) are predictive of a lower risk of having a heart attack, stroke, or death from cardiovascular disease. Moreover, augmenting HSP27 (or the murine ortholog, HSP25) attenuated experimental atherogenesis, reduced inflammation, and lowered cholesterol levels. Recently, we noted that HSP27 activates NF-κB via TLR-4, resulting in attenuation of plaque inflammation; however, the precise anti-atherosclerosis mechanisms mediated by extracellular HSP27 are incompletely understood. Our purpose in this study was to investigate the existence of HSP27 in extracellular vesicles (EVs) and whether HSP27 elicited atheroprotective effects on target cells. Here, we provide evidence that HSP27 localizes to EVs derived from THP-1 cells using transmission electron microscopy (TEM) and immunogold labeling, Western blotting, ELISA, and fluorescence-activated cell sorting. TEM imaging indicated that HSP27 is found at the exosomal membrane. Multiple reactor monitor-mass spectrometric analysis of large vesicles, which included microparticles and exosomes, isolated from human plasma, also led to detection of HSP27 using the unique signature peptide, R.LFDQAFGLPR.L. Studies using THP-1 and human embryonic kidney cells show that HSP27-laden exosomes significantly stimulated NF-κB activation ( P < 0.001) and release of IL-10 ( P < 0.0001), suggesting that HSP27 may be important exosomal cargo with beneficial anti-inflammatory effects.-Shi, C., Ulke-Lemée, A., Deng, J., Batulan, Z., O'Brien, E. R. Characterization of heat shock protein 27 in extracellular vesicles: a potential anti-inflammatory therapy.

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Section: Ms Detection Of Hsp27 Isolated From Human Plasma Extracellulmentioning

confidence: 91%

“…An antibody against AD1, a protein found on hepatic plasma membranes, has been shown (25)(26)(27)(28) to be useful as a microvesicle marker. Hence, we employed the AD1 antibody to isolate microvesicles from plasma and urine taken from healthy human participants using immunoprecipitation.…”

Section: Immunoprecipitation Of Extracellular Vesicles From Human Plamentioning

confidence: 99%

Characterization of heat shock protein 27 in extracellular vesicles: a potential anti‐inflammatory therapy

et al. 2018

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“…In particular, we applied several controls for each downstream analysis; this is especially important for flow cytometry analysis as many particles can mimic EVs. One of the major drawbacks of enriching uEVs is the co-sedimentation of THP, which can sediment readily at very low speed 25 and entrap uEVs in its filaments 18 or bind EVs 26 . Thus, one of our main goals was to reduce THP interference for MS analysis, RNA extraction and miRNA quantification, and NTA enumeration.…”

Section: Discussionmentioning

confidence: 99%

Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs

Musante

Bontha

Salvia

et al. 2020

Sci Rep

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Urinary extracellular vesicles (ueVs) provide bio-markers for kidney and urogenital diseases.centrifugation is the most common method used to enrich ueVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (≥9.0 mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. cryo-transmission electron Microscopy (≥3.0 mL urine) provided evidence of a heterogeneous population of eVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (≥4.5 mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5 mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single eV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0 mL of urine respectively. this work characterizes a neglected source of ueVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of ueV analysis such as autofluorescence of podocyte origin.Urinary extracellular vesicles (uEVs) are a medley of exosomes, exosome-like vesicles and microparticles/ microvesicles 1-4 . Confusing nomenclature aside 5,6 , all uEVs secreted in urine transport proteins, nucleic acid and small metabolites from all epithelial cells forming the nephron and lower urinary tract 7,8 . Thus, uEVs have become a valuable source of biomarkers for identifying any changes in the physio-pathological state of their parental cell. Moreover, uEVs are also bio-activators in renal diseases 9,10 . The most common method in use to enrich uEVs is a 2 or 3 step centrifugation protocol [11][12][13] . While it has been commonly discarded, the pellet obtained at relative low centrifugation force has proved to be an additional source of uEVs 14,15 . However this pellet has not been thoroughly characterized.In addition, the concomitant presence of multiple biomarker in uEVs offers the possibility to integrate multi-omic data analysis to better understand mechanism and possibly identify key role molecules implicated in the onset and progression of the disease 16 . However, no study has reported the amount of volume of urine that is necessary to collect to support multiple analyses. Hence, this study aims to: (1) estimate the minimum volume of urine necessary to yield uEVs for characterization according to both minimal information for studies of extracellular vesicles (MISEV) 17 and downstream analysis applying a very rigorous approach using several control sets for each analysis; (2) test the limit of detection of the techniques employed for downstream analysis and EV characterization before and after eliminati...

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“…Urine samples were collected at the earliest visit during the above observation period and stored at À80 to À85 ℃ until measurement. Next, 30 urinary biomarkers that could be measured at the internal or external laboratory were selected from urinary biomarkers reported by previous studies [1][2][3][4][5][8][9][10][11] to be involved in diabetic nephropathy (Table 1). These included novel biomarkers which were classified according to their modes of action as follows: inflammation, fibrosis, angiogenesis, mineral metabolism, glomerular injury, tubular injury and thick ascending limb injury.…”

Section: Biomarker Assaysmentioning

confidence: 99%

Panel of novel urine biomarkers for incident microalbuminuria in people with type 2 diabetes mellitus

et al. 2020

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Aim The need to identify novel biomarkers for early diagnosis and treatment of diabetic nephropathy is widely recognized. However, only a few studies have investigated the association between biomarkers and the onset of albuminuria. In this study, we aimed to investigate a panel of biomarkers suitable for predicting microalbuminuria. Methods Some 346 Japanese people with type 2 diabetes exhibiting normoalbuminuria were studied. The endpoint was defined as the onset of microalbuminuria. Thirty biomarkers were selected from among urinary biomarkers described in previous studies. A panel of biomarkers was selected using least absolute shrinkage and selection operator (LASSO). The prognostic performance of the developed panel was evaluated. Results During a mean follow-up of 6.2 years, 45 people progressed to microalbuminuria. A composite panel of six biomarkers (monocyte chemoattractant protein-1, osteopontin, soluble human tumour necrosis factor receptor-1, tenascin C, vascular endothelial growth factor-A and kidney injury molecule-1) was developed using LASSO. Compared with the basal model comprising estimated GFR and urinary albumin-to-creatinine ratio, addition of these six biomarkers significantly increased the overall C index from 0.773 to 0.824 (P = 0.019). Net reclassification improvement and integrated discrimination improvement were estimated to be 0.412 (P = 0.049) and 0.040 (P = 0.040), respectively. Decision curve analysis also showed that the model with the six novel biomarkers had a better prognostic value for predicting the onset of microalbuminuria. The optimism was moderate or negligible according to measures. Conclusions The panel consisting of six novel urinary biomarkers effectively predicted incident microalbuminuria in people with type 2 diabetes.

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Urinary Microvesicle-Bound Uromodulin: A Potential Molecular Biomarker in Diabetic Kidney Disease

Cited by 9 publications

References 30 publications

Characterization of heat shock protein 27 in extracellular vesicles: a potential anti‐inflammatory therapy

Characterization of heat shock protein 27 in extracellular vesicles: a potential anti‐inflammatory therapy

Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs

Panel of novel urine biomarkers for incident microalbuminuria in people with type 2 diabetes mellitus

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