The interest in extracellular vesicles (EVs) has grown exponentially over the past decade,1,2 particularly among researchers in nephrology.3,4 EVs were initially thought as artifacts or cell dust,5 but there is accumulating evidence for their important role as messengers for cell signaling and communication in normal and disease states.1,2,6,7 They may function in the transfer of proteins, lipids, and nucleic acids and represent a novel type of biomarker.8 EVs represent a heterogeneous group of vesicles deriving from activated cells or generated during apoptosis from nearly all mammalian cells.1,9,10 Smaller vesicles or exosomes, 30–100 nm in size, are released from intracellular multivesicular bodies (MVBs).10 In contrast, microparticles (MPs), ranging from 100 to 1000 nm, are thought to be the product of exocytic budding (Figure 1).1 The term EVs in this review is used for both vesicle types. Unfortunately, there is currently no consensus regarding the nomenclature of secreted membrane–enclosed vesicles.11 The term EV has been used by other investigators to describe all forms of secreted vesicles, including exosomes, microvesicles, MPs/ectosomes, apoptotic bodies, and other EV subsets. International societies have started to address this issue and suggested minimal experimental requirements for definition of EVs and their function.12 In this review, we will use the nomenclature used by the investigators of each study discussed.
Figure 1: Biogenesis of EVs. Exosomes are believed to be released from intracellular MVBs when they fuse with the cell membrane. Exosomes, here shown in yellow, ranging from 30 to 100 nm display the same membrane orientation as the cell of origin. In contrast, MPs, shown in blue, ranging from 100 to 1000 nm are thought to be the product of exocytic budding and consist of cytoplasmic components and phospholipids. Exosomes and MPs can be involved in various biologic processes. They can play a role in antigen presentation by transferring MHC molecules and antigen. They can directly activate cell surface receptors, transfer receptors, or deliver factors, such as transcription factors, protein, and various RNAs (including miRNA and mRNA). Internalization pathways have not been clearly delineated. Possibilities include (1) membrane fusion, (2) micropinocytosis, and (3) receptor-dependent endocytosis.
Researchers in nephrology have detected EVs in various body fluids, including urine and blood (Table 1). Because they carry markers of the parent cell, specific EV subpopulations, such as vesicles deriving from endothelial cells, immune cells, or kidney-specific cells, like podocytes, can be determined.1,9,13
Table 1: Overview of EV detection in renal structure and diseases
Analysis of urinary extracellular vesicles (uEVs) may serve as a logical and novel diagnostic approach for the nephrologist, because these vesicles can derive from the kidney and urinary tract. Noninvasive biomarkers are clearly needed in the field of nephrology, particularly for different clinical renal syndromes, including AKI and glomerular and tubular diseases. Compared with other biomarkers, circulating EVs and uEVs are one of a kind, because they not only have a diagnostic or prognostic role but also, are believed to have a functional and therapeutic role.8 For example, they have been found to be antibacterial in urinary tract infections14 or renal protective in models of kidney injury.15
Because of the heterogeneity of these vesicles, their low refractive index, and the inability of most techniques to detect the whole size range, the accurate assessment of these vesicles has remained elusive.16–20 A variety of methods are currently being explored for phenotyping and enumeration of EVs, but the lack of consensus has resulted in disparate results.18,19 Currently there is no single method which can phenotype, size and enumerate the whole range of EVs, therefore posing a challenge to truly understand the biology of EVs.17,21 In this review, we will summarize the current knowledge about the characterization of EVs and the evolving more accurate detection techniques, and provide an overview of the role of EVs in various renal diseases.
Characterization of EVs
Both types of EVs, such as MPs and exosomes, have been examined in urine and/or blood in various renal diseases.3,4 Exosomes are formed by intraluminal budding of endosomal compartments, which create MVBs (Figure 1).10 Exosomes can, therefore, incorporate cargo, such as soluble proteins, mRNAs, microRNAs (miRNAs), noncoding RNA, mitochondrial DNA, transcription factors, and other cytosolic components and molecules.10,22,23 After these vesicles fuse with the plasma membranes of the parent cell, they are released as exosomes into the extracellular environment (Figure 1). Interestingly, the lipid-bilayered exosomes seem to be protected from RNAses,24,25 sheltering the genetic cargo in exosomes. In addition, vesicles can transport biologic cargo to not only neighboring cells but also, relatively long distanced. The fusion of MVB with plasma membranes and release of exosomes is a constitutive process, but it might also be a response to a variety of stimuli.26 Exosomes may be identified by the presence of exosome-specific markers, although evolving evidence supports that only a few membrane proteins, such as CD81 and tumor susceptibility gene 101, might be exosome specific. Other markers—previously thought to be exosome specific, such as membrane proteins CD9 and apoptosis–linked gene 2–interacting protein X—might also be found on larger EVs, like MPs.10 Rigorous studies to identify vesicle-specific markers are crucially needed to further elucidate the specific characteristics of different types of EVs. Furthermore, extensive proteomic analysis has shown that uEVs, including exosomes, can derive from glomerular, tubular, prostate, and bladder cells.13,27 This may also allow the future establishment of EVs as biomarkers for specific disease processes.
In contrast to exosomes, MPs are formed by outward blebbing of the plasma membrane (Figure 1).1 Cytoskeletal reorganizations and alterations in the phospholipid symmetry are likely important parts of the blebbing process, which may differ among cells.28,29 Under normal conditions, aminophospholipids are found solely on the inner part of the plasma membrane. During MP formation, membrane asymmetry is seen as aminophospholipids redistribute to the outer part of the plasma membrane. Outward blebbing may be dependent on various enzymes and calcium influx.28,29 Interestingly, MP formation seems to occur selectively in lipid-rich microdomains (lipid rafts/cavelolae) within the membrane. Like exosomes, MPs carry different surface markers depending on the parent cell and stimulus.1 Ideally, MPs and exosomes should be studied in vivo to understand the true role of EVs in health and disease.
Table 2 summarizes some of the markers used to describe EVs derived from endothelial, immune, or kidney-specific cells (e.g., podocytes). The fact that phospholipids are externalized has also been used for EV detection and characterization.30 This is especially true for MP detection, because Annexin V can bind phosphatydylserine. Nevertheless, it is unclear how much of phosphatidylserine is externalized on exosomes. Elegant cryoelectron microscopy (cryo-EM) studies from platelet-free plasma have shown that >50% of EVs are Annexin V negative; thus, Annexin binding can only be used to detect a minority of EVs.31 In addition, these cryo-EM studies have shown that >75% of circulating EVs are <0.500 μm in size.31 Urinary EVs have also been characterized with EM but not as rigorously as EVs in platelet-free plasma. These studies also indicate a copurification of various vesicle sizes and types.3,4,32 For comprehensive discussion of the biogenesis of EVs, including exosomes and MPs, we refer the reader to other review papers.1,2,8,10
Table 2: Defining origin of EVs
Overview of Detection Techniques of EVS
Various detection techniques for EVs have been used.33,34 The method of detection as well as the sample handling during the preanalytic phase can affect the enumeration of EVs.30,35,36 These limitations should be recognized to design the proper experiments for EV studies. Depending on the scientific question, different techniques might be applied to maximize accuracy. Several studies have looked at the effect of preanalytic steps, including blood and urine collection, anticoagulant use, sample centrifugation, sample freezing, thawing, and storage.30,35–38 Societies for EV research have provided guidelines and recommendations for proper processing of EVs.39,40
There are specific challenging issues regarding the isolation of uEVs. Tamm–Horsfall protein (THP), the most abundant protein in urine, is capable of entrapping uEVs.41 Several studies have, therefore, looked at various methods to isolate and enrich uEVs, which include ultracentrifugation with and without size exclusion chromatography (at 100,000–200,000×g for at least 1 h), ultrafiltration,42 precipitation, and immunoisolation.39,43,44 These different techniques have varying recovery rates of EVs.42,44 Lower recovery rates of EVs have been especially a challenge in patients with heavy or nephrotic-range proteinuria. Changes of urinary concentration represent another challenge for accurate analysis of uEVs. A timed urine collection (e.g., 24-hour urine collection) can overcome this issue, but this is not often available for cross-sectional or longitudinal studies with samples from the same subjects. Normalization to urinary creatinine has been used to overcome the logistic aspect of timed urine collection, because the amount of urinary creatinine excreted is assumed to be constant with regard to time. However, this can be problematic in patients with AKI. Other approaches have included normalization to THP.41 For instance, THP amount has been found to highly correlated with exosomal marker proteins, and therefore, it was used as a housekeeping protein for normalization.41 More detailed discussion of these challenges can be found in other review papers.3,44
Currently, there is no single detection technique that can measure the entire size range of EVs (from 30 to 1000 nm). Exosomes are analyzed using a variety of detection techniques. Nanoparticle tracking analysis (NTA) has recently been used more frequently for exosome quantification and sizing.33,45,46 It measures Brownian motion of vesicles in solution by illuminating them with a laser beam. Size is calculated through the Stokes–Einstein equation. Fluorescence can be added to NTA but is limited to detection of only one surface marker at a time (e.g., aquaporin-2 [AQP-2] has been found on uEVs using this technique).45 Protein aggregates may also be detected by NTA, which is another caveat of this system. Resistive pulse sensing (RPS) is also being explored for exosome detection.17,24 In this system, which is on the basis of the Coulter principle, vesicles in fluid flow through an aperture and electric resistance increase when a vesicle is present.47,48
Although several different techniques for exosomes are used, the most widely used technique for MPs is conventional flow cytometry (FCM).49 Most commonly used FCMs have a detection threshold in the range of 500 nm, newer cytometers can detect sizes as small as 300–500 nm.18,50 FCM provides the possibility of high-throughput analysis and EV phenotyping and the development of EV detection for potential clinical use. In general, a sample with EVs mixed with sheath fluid is running through a channel. A laser crosses the sample stream and generates specific light scattering and fluorescent characteristics of the particles in the stream. Most of the conventional FCMs use scatter for particle detection. More recently, fluorescent intensity of the particles is used as a threshold for particle detection. This approach has the potential to offer better separation from background noise and is less likely affected by aggregation of particles and might detect particles as low as 100–200 nm.51,52 The addition of imaging to FCM can also significantly increase the sensitivity of MP detection.53,54 Imaging flow cytometry (IFCM) can also provide morphologic confirmation and the ability to distinguish true single events from aggregates and cell debris (Figure 2). In addition, IFCM compared with conventional FCM has the ability to detect more Annexin–negative MPs (Figure 3).53 Future studies are needed to determine the effect in differences in sensitivity of FCM and IFCM. Other detection techniques, including RPS and NTA analysis, are also used to enumerate MPs, but these two techniques cannot yet phenotype MPs, as it is done with FCM. Different techniques, including NTA/RPS and FCM, have been now thoroughly compared in two studies.17,21 It is very clear that absolute quantification of EVs substantially differs among the methods described above. A gold standard for quantification of such particles has not yet been established but is being addressed by international societies. In this regard, the International Society for Extracellular Vesicles, the International Society for Advancement of Cytometry, and the International Society of Thrombosis and Hemostasis are working together to develop standards so that data from different studies can be comparable.
Figure 2: Example images of IFCM. Comparison of MPs with other cells/debris. Column 1 shows a bright-field image (BF) of two red blood cells (RBCs) and a white blood cell (WBC) with debris. The size of these RBCs and WBC is about 6–8
µm. Column 2 shows scatter of the laser of these same cells seen in column 1. Column 3 indicates that the debris seen in BF is positive for Annexin V (AV) staining and therefore, could be apoptotic material. Column 4 shows the same WBC (big red circle) and a WBC-derived MP (small red dot). The size of the MP is <1
µm, although it is not measurable with this technique yet. Column 5 shows overlay of all four columns.
53 SSC, side scatter.
Figure 3: Imaging FCM detects more total and Annexin negative MPs compared to conventional FCM. Annexin-negative and -positive EVs detected by FCM and IFCM using the ImageStream X Machine (Amnis, Seattle, WA).
53 Absolute number of MPs per microliter of platelet-free plasma from three different runs by FCM with and without imaging using split samples. Absolute numbers were calculated for both the Annexin V (AV) -positive (green) and -negative (gray) populations. FCM runs 1 (FC1) and FC2 used forward scatter (FSC) as a threshold, and FC3 (arrow) used side scatter (SSC), because recent publications indicated that larger angles of scatter provided better resolution for small particles. In all cases, the ISX detected more total MPs per microliter and AV-negative MPs per microliter than FCM. Although conventional FCM had higher AV positive counts in two runs, this could be explained by the difference in data acquisition by ISX and FCM. Reprinted from reference
53, with permission.
ELISA and Western blot analyses have been used to detect both exosomes and MPs but are limited to analysis in bulk rather than on individual levels.43,55 In addition, these techniques lack the capacity for visual differential confirmation between small cells, debris, and MPs or exosomes but can be used as confirmatory test, such as to show an exosome-specific marker in an EV sample preparation.43 EM (especially cryo-EM) serves currently as the gold standard to characterize the morphology of EVs; however, it is not useful for high-throughput analysis because of costs and time (Figure 4, A and B).31,56Table 3 provides an overview of the most commonly used detection techniques for EVs. The sorting of subpopulations of EVs has yet to be established but is in development in several laboratories.51 Sorting would be highly valuable, because it would enable the ability to more thoroughly characterize the composition and biology of EV subtypes and help to understand their possible role in health and disease.
Figure 4: Electron microscopy images of EVs. (A–C) Cryo-EM images from an EV prep showing EVs of three sizes (approximately 100, approximately 500, and approximately 1000 nm, respectively). (D) Distribution of the range of sizes of EVs detected in platelet-poor plasma by scanning EM. Reprinted from reference
53, with permission.
Table 3: Strengths and weaknesses of various detection techniques for EVs
EVS in Renal Diseases
Because of word limitations, it is not possible to include all studies published thus far on EVs in renal diseases. Here, we will discuss those with major findings. Table 1 provides a more comprehensive summary of studies examining EVs in renal diseases. The major studies are also summarized in Figure 5.
Figure 5: Origin of EVs in renal diseases. Proteomic analysis has identified uEVs, including exosomes from glomerular, tubular, prostate, and bladder cells.
13 , 27 Here, we show a schematic drawing of the glomerulus and kidney tubule. Several markers of podocyte damage have been identified in uEVs, including podocalyxin, podoplanin,
83 and WT-1.
80 , 81 A protective role of EVs derived from MSCs has been described in models of kidney damage. It is suggested that these EVs from MSCs are involved in transfer of mRNA, miRNA, and proteins and reprogramming of their phenotypes.
62 Solute and water transporters have been identified on uEVs: sodium potassium chloride cotransporter (NaKCl) from the thick ascending limb,
27 sodium chloride cotransporter (NCC) from the distal tubule,
89 and AQP-2 deriving from the collecting duct.
43 Figure adapted from previous publications.
3 , 4 , 44AKI
Noninvasive biomarkers are clearly needed to diagnose and predict the outcome of AKI. Although validation is still needed, studies of uEVs in different animal models of AKI and some human cohorts have shown promise. In this regard, increased levels of Na+/H+ exchanger type 3 in urinary membrane fractions, representing exosomes pellets, were found in acute tubular necrosis but not in prerenal azotemia and other causes of acute renal failure in 68 patients admitted to the intensive care unit.57 This finding suggests a diagnostic potential of Na+/H+ exchanger type 3–containing exosomes in AKI of acute tubular necrosis origin.57 In a rat model of cisplatin-induced AKI, Fetuin-A, a filtered protein metabolized in proximal tubular cells, was suggested to be a predictive marker of AKI. Only exosomal Fetuin-A but not free Fetuin-A was detectable in the urine before morphologic injury developed.58 Thus, urinary exosomal biomarkers might be more enriched in the urine than its free form and therefore, have a potential to be a more sensitive biomarker in AKI. This notion is supported by another study showing that, although there were no changes in urinary–free neutrophil gelatinase–associated lipocalin neutrophil gelatinase–associated lipocalin levels, neutrophil gelatinase–associated lipocalin levels in urinary exosomes (uEXs) were increased in patients with delayed graft function after kidney transplantation.59 Additional investigation is needed to determine if other vesicular subtypes, such as MPs, and not only exosomes might carry the markers described above and whether these EVs have a functional role.
A potential therapeutic role of EVs in AKI has been examined extensively in animal models. Several studies have shown that mesenchymal stem cells (MSCs) have the potential to reverse AKI and chronic kidney injury in different experimental models by a paracrine mechanism.60,61 EVs derived from these MSC (MSC EVs) might cause horizontal transfer of mRNA, miRNA and proteins and reprogramming of their phenotype.62 EVs likely mimic the effect of MSC by inhibiting apoptosis and stimulating cell proliferation. Bruno et al demonstrated the protective effect of EVs derived from human bone marrow MSCs in tubular epithelial cells (TEC) of immunodeficient mice (SCID) with glycerol induced AKI.15 They showed that MSC EVs labeled with PKH26 dye were incorporated by tubular epithelial cells. RNAse treatment abrogated the protective effects of MSC EVs, suggesting an RNA dependent mechanism and the occurrence of horizontal transfer of human mRNA in MSC EVs to target cells, which was proven by the detection of human-specific mRNA in MSC EV–treated TECs.15 Since these initial studies, the therapeutic role of MSC EVs has been tested in other models of AKI. In a lethal cisplatin –induced AKI model, a single injection of MSC EVs ameliorated renal dysfunction and histopathology, but only multiple injections decreased mortality and restored normal histology and renal function at day 21 in surviving mice.63 A similar finding was described in a gentamicin-induced model of AKI.64 The therapeutic effect of EVs from other types of stem cells has also been described.65 Injection of EVs derived from endothelial progenitor cells in a rat model of AKI (ischemia reperfusion injury) protected against progression of CKD by inhibiting capillary rarefaction, glomerulosclerosis, and tubulointerstitial fibrosis.65 It remains to be determined which subpopulations of EVs confer these protective effects and how EVs get to the damaged tubular cells and interact with their target(s). More recent data indicate that downregulation of miRNAs in MSCs reduced the proregenerative effect of EVs derived from these cells in a model of AKI.66 Future studies need to elucidate if specific genetic or epigenetic material transferred by EVs directly influences the regenerative capacity of these tubular cells.
CKD/Uremia/ESRD
Circulating MPs have been the most studied EVs in humans with CKD and ESRD, conditions in which MPs have been associated with endothelial dysfunction,67 cardiovascular mortality,68,69 increased risk for calcification,70,71 anemia,72,73 and disease-specific alteration in coagulation.74 In a study of a heterogeneous group of patients with chronic renal failure and those on peritoneal dialysis and hemodialysis, all patients compared with healthy controls had higher levels of endothelial-derived microparticles (EMPs) and MPs of leukocyte origin.75 This is in line with other studies.76,77 In particular, elevated EMPs were associated with endothelial damage in renal failure. The effect of the uremic milieu on MPs is supported by in vitro experiments showing that uremic toxins (such as indoxyl sulfate and p-cresyl sulfate) directly induced release of EMPs.78 In addition, MPs from patients with ESRD can impair endothelial-dependent vasorelaxation, suggesting a functional role of MPs in modulating endothelial function.67 In this regard, EMPs alone decreased endothelial nitric oxide release by 60%.67 However, no significant link could be found between endothelial dysfunction, anemia, arterial stiffness, and endothelial MPs in 46 patients with GFR<30 ml/min.79 This negative study shows the complexity and emphasizes the need for additional in vivo studies, such as animal models of CKD. The predictive role of EMP was examined in a prospective study of 81 stable patients on hemodialysis.68 Baseline EMP levels were found to independently predict all-cause and cardiovascular mortality. Such predictive markers might enable the identification of patients who need more aggressive or intensive treatment.
Glomerular Disease
The role of EVs in glomerular disease has been illustrated in a number of studies. In FSGS,80 a disease with significant podocyte damage, Wilm’s tumor-1 (WT-1), a transcription factor required for kidney development, was examined in uEXs as a marker for podocytes damage. Increased expression of WT-1 in uEXs was found 1 week earlier than urinary albumin excretion in a mouse model of collapsing glomerulopathy. The same group also examined this in humans and found that exosomal WT-1 levels were significantly higher in children with active nephrotic syndrome caused by FSGS compared with those with steroid–sensitive nephrotic syndrome (SSNS) not yet in remission.80 However, another group detected exosomal WT-1 only in 60% of 40 children with FSGS and SSNS,81 suggesting that WT-1 in uEXs may not be a sensitive marker for FSGS and SSNS. Alternatively, the patient groups studied might have been too heterogeneous. Larger studies of patients, ideally more homogenous and with biopsy-proven glomerulopathy, are needed to clarify these incongruent results. In addition, WT-1 in uEXs might not be as specific as a podocyte marker, because it is expressed in different parts of the urinary tract, including the ureter and bladder.82 This marker should also be tested in larger vesicles. Podocalyxin and podoplanin were detected in urinary MPs in diabetic nephropathy (DN), another disease process with podocyte damage.83 Podocalyxin and podoplanin might, therefore, be more fitting biomarkers for podocyte damage, because they are podocyte surface markers. Other evidence supports that some of these podocyte-derived EVs seem to originate from tip vesiculation of podocytes and are not coming from shed podocytes in the urine, which was shown by histologic examination of patients with nephrotic syndrome.32 In addition to the study of known markers for kidney structure and/or damage, such as podocalyxin, proteomic analysis of uEX has been used to identify other unknown proteins that might play a significant role in glomerular disease. One example is a proteomic analysis from patients of early IgA nephropathy and thin basement membrane nephropathy that identified four candidate urinary exosomal proteins to differentiate early IgA nephropathy from thin basement membrane nephropathy. These candidates are aminopeptidase N, vasorin precursor, α-1-antitrypsin, and ceruloplasmin.84 Validation studies are needed, especially to define the predictive role of these potential novel markers. It would also be important to determine whether these proteins within exosomes have causal or protective roles in disease.
There is accumulating data about the role of EVs in DN. Burger et al.83 detected elevated levels of podocalyxin– and podoplanin–positive urinary MPs in diabetic mice before the onset of albuminuria. Again, analysis of the whole range of vesicles, including exosomes and Annexin-negative populations, might provide additional information. WT-1 expression in uEXs was also found to be increased in patients with diabetes.85 Exosomal WT-1 was detected in all patients with diabetes and proteinuria but only one half of those without proteinuria and only 1 of 25 healthy controls. Expression of WT-1 in uEXs also correlated significantly with decline in kidney function. These studies might have significant implications for the clinical application of podocycte-derived EVs, including exosomes and MPs, as early markers for glomerular damage in DN. In addition, EVs have been described as carriers for genetic material. Differential miRNA profiling in uEXs was used to identify miRNAs that can differentiate patients with type 1 diabetes with and without DN.86 Of 226 miRNAs, 22 miRNAs differed in matched pairs of patients with microalbuminuria and patients with normoalbuminuria. miRNA145 and miRNA130 were enriched in patients with microalbuminuria. Similarly, in an animal model of streptozosin-induced DN, increased urinary exosomal levels of miRNA145 were found. Interestingly, miRNA145 was also overexpressed within the glomeruli. It remains to be established whether the genetic material transported by exosomes has specific downstream effects or if their detection can be used as potential new biomarkers.
Tubular Disease
Kidney solute and water transporters have been described in uEVs and studied as potential biomarker for tubular damage.13,27,87 In 1995, Kanno et al.43 first detected AQP-2, a vasopressin–sensitive water channel, in the collecting duct in the urine. Immunogold labeling by EM clearly showed submicrometer vesicles being positive for this water channel. At that time, it was not known how these vesicles were generated. In a later study, murine kidney collecting duct cells were treated with desmopressin, a synthetic of vasopressin.88 These treated cells displayed increased AQP-2 expression in uEXs. Transfer of these uEXs from treated to untreated cells caused an increase in functional AQP-2 expression in untreated cells, supporting the hypothesis that exosomes contribute to cell to cell communication within the kidney.88
Solute transporters, such as Na-K-Cl cotransporter, are normally found in uEX fractions but were found to be absent in uEX in Bartter syndrome type 1, a genetic disorder caused by a mutation of the gene encoding for sodium potassium chloride cotransporter 2.27 Similarly, Na-Cl cotransporter was absent in uEX in Gittelman’s syndrome.89 Additional studies are needed to address whether these uEVs, including exosomes and MPs, can be used as a preliminary screening tool in patients suspected to have these genetic disorders.
Polycystic kidney disease (PKD) is the most common inherited kidney disease. PKD is caused by mutations in genes encoding for proteins involved in function of primary cilia, including polycystin-1 (PC1), PC2, and fibrocystin.90 These proteins and others, such as Cystin and ADP ribosylation factor–like 6, have been found in uEX of patients with PKD.13,27 Another approach to find a novel diagnostic/monitoring tool for PKD was the study of lectin profiles of uEVs,91 showing that lectin microarray profiles of uEVs of patients with PKD and healthy controls have different patterns. This suggests possible disease–specific modifications. Recently, Hogan et al.92 showed that transmembrane protein-2 (TMEM2) in uEX was 2-fold higher in patients with PKD1 compared with controls. PC to TMEM ratio correlated inversely with kidney volume. Urine exosomal PC1 to TMEM2 or PC2 to TMEM2 ratio could, therefore, provide a nonimaging technique to monitor kidney volume and therefore, disease progression in patients with PKD.
Summary
Compared with other biomarkers, circulating EVs and uEVs are one of a kind, because they not only have a diagnostic or prognostic role but also, are believed to have functional and therapeutic roles. Key challenges remain. It is unclear if their functions described in vitro have physiologic significance in vivo. Translational studies are clearly needed. Furthermore, there is no single method that can enumerate and phenotype such heterogeneous groups of vesicles of different sizes and characteristics. Newer detection techniques are in development, and older ones are in refinement. Establishment of a methodologic consensus and a clearer understanding of the composition of these vesicles will enable the comparison of scientific data and collaboration. Until a consensus nomenclature is developed, it is crucial that researchers provide detailed information about their methodology and use several techniques to ensure that the particles that they are studying are truly EVs and not non-EV components, such as lipids or protein complexes. In addition, a standard and uniform language should be used among all scientific communications. Studies using more rigor to identify and characterize EVs will also broaden our perspectives on their various roles, including new insight into the pathophysiology and possible diagnostic and treatment options for renal diseases.
Disclosures
None.
The authors thank Paula Timm for her excellent help with graphic design.
Published online ahead of print. Publication date available at www.jasn.org.
References
1. Burger D, Schock S, Thompson CS, Montezano AC, Hakim AM, Touyz RM:
Microparticles: Biomarkers and beyond. Clin Sci (Lond) 124: 423–441, 2013
2. Barteneva NS, Fasler-Kan E, Bernimoulin M, Stern JN, Ponomarev ED, Duckett L, Vorobjev IA: Circulating
microparticles: Square the circle. BMC Cell Biol 14: 23, 2013
3. Dear JW, Street JM, Bailey MA: Urinary exosomes: A reservoir for biomarker discovery and potential mediators of intrarenal signalling. Proteomics 13: 1572–1580, 2013
4. Salih M, Zietse R, Hoorn EJ: Urinary extracellular vesicles and the kidney: Biomarkers and beyond. Am J Physiol Renal Physiol 306: F1251–F1259, 2014
5. Wolf P: The nature and significance of platelet products in human plasma. Br J Haematol 13: 269–288, 1967
6. Xiao X, Ma X, Liu L, Wang J, Bi K, Liu Y, Fan R, Zhao B, Chen Y, Chen J: Cellular membrane
microparticles: Potential targets of combinational therapy for vascular disease [published online ahead of print October 14, 2014]. Curr Vasc Pharmaco
7. Kourembanas S: Exosomes: Vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Annu Rev Physiol 77: 13–27, 2015
8. EL Andaloussi S, Mäger I, Breakefield XO, Wood MJ: Extracellular vesicles: Biology and emerging therapeutic opportunities. Nat Rev Drug Discov 12: 347–357, 2013
9. van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R: Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 64: 676–705, 2012
10. Colombo M, Raposo G, Théry C: Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30: 255–289, 2014
11. Gould SJ, Raposo G: As we wait: Coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles 2: 10.3402/jev.v2i0.20389, 2013
12. Lötvall J, Hill AF, Hochberg F, Buzás EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, Sahoo S, Tahara H, Wauben MH, Witwer KW, Théry C: Minimal experimental requirements for definition of extracellular vesicles and their functions: A position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 3: 26913, 2014
13. Pisitkun T, Shen RF, Knepper MA: Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A 101: 13368–13373, 2004
14. Hiemstra TF, Charles PD, Gracia T, Hester SS, Gatto L, Al-Lamki R, Floto RA, Su Y, Skepper JN, Lilley KS, Karet Frankl FE: Human urinary exosomes as innate immune effectors. J Am Soc Nephrol 25: 2017–2027, 2014
15. Bruno S, Grange C, Deregibus MC, Calogero RA, Saviozzi S, Collino F, Morando L, Busca A, Falda M, Bussolati B, Tetta C, Camussi G: Mesenchymal stem cell-derived microvesicles protect against acute tubular injury. J Am Soc Nephrol 20: 1053–1067, 2009
16. Dragovic RA, Gardiner C, Brooks AS, Tannetta DS, Ferguson DJ, Hole P, Carr B, Redman CW, Harris AL, Dobson PJ, Harrison P, Sargent IL: Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine (Lond) 7: 780–788, 2011
17. van der Pol E, Coumans FA, Grootemaat AE, Gardiner C, Sargent IL, Harrison P, Sturk A, van Leeuwen TG, Nieuwland R: Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost 12: 1182–1192, 2014
18. Chandler WL, Yeung W, Tait JF: A new microparticle size calibration standard for use in measuring smaller
microparticles using a new flow cytometer. J Thromb Haemost 9: 1216–1224, 2011
19. Robert S, Poncelet P, Lacroix R, Raoult D, Dignat-George F: More on: Calibration for the measurement of
microparticles: Value of calibrated polystyrene beads for flow cytometry-based sizing of biological
microparticles. J Thromb Haemost 9: 1676–1678, 2011
20. van der Pol E, van Gemert MJ, Sturk A, Nieuwland R, van Leeuwen TG: Single vs. swarm detection of
microparticles and exosomes by flow cytometry. J Thromb Haemost 10: 919–930, 2012
21. Maas SL, de Vrij J, van der Vlist EJ, Geragousian B, van Bloois L, Mastrobattista E, Schiffelers RM, Wauben MH, Broekman ML, Nolte-’t Hoen EN: Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics. J Control Release 200: 87–96, 2015
22. Zhou H, Cheruvanky A, Hu X, Matsumoto T, Hiramatsu N, Cho ME, Berger A, Leelahavanichkul A, Doi K, Chawla LS, Illei GG, Kopp JB, Balow JE, Austin HA 3rd, Yuen PS, Star RA: Urinary exosomal transcription factors, a new class of biomarkers for renal disease. Kidney Int 74: 613–621, 2008
23. Miranda KC, Bond DT, Levin JZ, Adiconis X, Sivachenko A, Russ C, Brown D, Nusbaum C, Russo LM: Massively parallel sequencing of human urinary exosome/microvesicle RNA reveals a predominance of non-coding RNA. PLoS One 9: e96094, 2014
24. Cheng L, Sun X, Scicluna BJ, Coleman BM, Hill AF: Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine. Kidney Int 86: 433–444, 2014
25. Miranda KC, Bond DT, McKee M, Skog J, Păunescu TG, Da Silva N, Brown D, Russo LM: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease. Kidney Int 78: 191–199, 2010
26. Mathivanan S, Ji H, Simpson RJ: Exosomes: Extracellular organelles important in intercellular communication. J Proteomics 73: 1907–1920, 2010
27. Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, Wang NS, Knepper MA: Large-scale proteomics and phosphoproteomics of urinary exosomes. J Am Soc Nephrol 20: 363–379, 2009
28. Piccin A, Murphy WG, Smith OP: Circulating
microparticles: Pathophysiology and clinical implications. Blood Rev 21: 157–171, 2007
29. Morel O, Jesel L, Freyssinet JM, Toti F: Cellular mechanisms underlying the formation of circulating
microparticles. Arterioscler Thromb Vasc Biol 31: 15–26, 2011
30. Dey-Hazra E, Hertel B, Kirsch T, Woywodt A, Lovric S, Haller H, Haubitz M, Erdbruegger U: Detection of circulating
microparticles by flow cytometry: Influence of centrifugation, filtration of buffer, and freezing. Vasc Health Risk Manag 6: 1125–1133, 2010
31. Arraud N, Linares R, Tan S, Gounou C, Pasquet JM, Mornet S, Brisson AR: Extracellular vesicles from blood plasma: Determination of their morphology, size, phenotype and concentration. J Thromb Haemost 12: 614–627, 2014
32. Hara M, Yanagihara T, Hirayama Y, Ogasawara S, Kurosawa H, Sekine S, Kihara I: Podocyte membrane vesicles in urine originate from tip vesiculation of podocyte microvilli. Hum Pathol 41: 1265–1275, 2010
33. van der Pol E, Hoekstra AG, Sturk A, Otto C, van Leeuwen TG, Nieuwland R: Optical and non-optical methods for detection and characterization of
microparticles and exosomes. J Thromb Haemost 8: 2596–2607, 2010
34. van der Pol E, Coumans F, Varga Z, Krumrey M, Nieuwland R: Innovation in detection of
microparticles and exosomes. J Thromb Haemost 11[Suppl 1]: 36–45, 2013
35. Shet AS: Characterizing blood
microparticles: Technical aspects and challenges. Vasc Health Risk Manag 4: 769–774, 2008
36. Yuana Y, Bertina RM, Osanto S: Pre-analytical and analytical issues in the analysis of blood
microparticles. Thromb Haemost 105: 396–408, 2011
37. Jayachandran M, Miller VM, Heit JA, Owen WG: Methodology for isolation, identification and characterization of microvesicles in peripheral blood. J Immunol Methods 375: 207–214, 2012
38. Trummer A, De Rop C, Tiede A, Ganser A, Eisert R: Recovery and composition of
microparticles after snap-freezing depends on thawing temperature. Blood Coagul Fibrinolysis 20: 52–56, 2009
39. Witwer KW, Buzas EI, Bemis LT, Bora A, Lasser C, Lotvall J, Nolte-'t Hoen EN, Piper MG, Sivaraman S, Skog J, Thery C, Wauben MH, Hochberg F: Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2: 10.3402/jev.v2i0.20360, 2013
40. Lacroix R, Judicone C, Mooberry M, Boucekine M, Key NS, Dignat-George FThe ISTH SSC Workshop: Standardization of pre-analytical variables in plasma microparticle determination: Results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop [published online ahead of print April 2, 2013]. J Thromb Haemost 10.1111/jth.12207
41. Fernández-Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, Knepper MA: Tamm-Horsfall protein and urinary exosome isolation. Kidney Int 77: 736–742, 2010
42. Rood IM, Deegens JK, Merchant ML, Tamboer WP, Wilkey DW, Wetzels JF, Klein JB: Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome. Kidney Int 78: 810–816, 2010
43. Kanno K, Sasaki S, Hirata Y, Ishikawa S, Fushimi K, Nakanishi S, Bichet DG, Marumo F: Urinary excretion of aquaporin-2 in patients with diabetes insipidus. N Engl J Med 332: 1540–1545, 1995
44. Musante L, Tataruch DE, Holthofer H: Use and isolation of urinary exosomes as biomarkers for diabetic nephropathy. Front Endocrinol (Lausanne) 5: 149, 2014
45. Oosthuyzen W, Sime NE, Ivy JR, Turtle EJ, Street JM, Pound J, Bath LE, Webb DJ, Gregory CD, Bailey MA, Dear JW: Quantification of human urinary exosomes by nanoparticle tracking analysis. J Physiol 591: 5833–5842, 2013
46. Filipe V, Hawe A, Jiskoot W: Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm Res 27: 796–810, 2010
47. Vogel R, Willmott G, Kozak D, Roberts GS, Anderson W, Groenewegen L, Glossop B, Barnett A, Turner A, Trau M: Quantitative sizing of nano/
microparticles with a tunable elastomeric pore sensor. Anal Chem 83: 3499–3506, 2011
48. Coulter WH: Means for counting particles suspended in a fluid. US Patent 2,656,508. 1953
49. Dignat-Goerge F: Questionnaire on Microparticle Detection and Characterization: A Retrospective Analysis. Vascular Biology Working Group. Standardization Sub Committee International Society of Thrombosis and Hemostasis; 2005, Sydney, Australia. Vascular Biology Working Group, Sydney, Australia, Standatization Sub Committee International Society of Thrombosis and Hemostasis, 2005
50. Robert S, Lacroix R, Poncelet P, Harhouri K, Bouriche T, Judicone C, Wischhusen J, Arnaud L, Dignat-George F: High-sensitivity flow cytometry provides access to standardized measurement of small-size
microparticles--brief report. Arterioscler Thromb Vasc Biol 32: 1054–1058, 2012
51. Kormelink TG, Arkesteijn GJ, Nauwelaers FA, van den Engh G, Nolte-’t Hoen EN, Wauben MH: Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry [published online ahead of print February 16, 2015]. Cytometry A 10.1002/cyto.a.22644
52. Arraud N, Gounou C, Turpin D, Brisson AR: Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry [published online ahead of print April 9, 2015]. Cytometry A 10.1002/cyto.a.22669
53. Erdbrügger U, Rudy CK, E Etter M, Dryden KA, Yeager M, Klibanov AL, Lannigan J: Imaging flow cytometry elucidates limitations of microparticle analysis by conventional flow cytometry. Cytometry A 85: 756–770, 2014
54. Headland SE, Jones HR, D’Sa AS, Perretti M, Norling LV: Cutting-edge analysis of extracellular
microparticles using ImageStream(X) imaging flow cytometry. Sci Rep 4: 5237, 2014
55. Osumi K, Ozeki Y, Saito S, Nagamura Y, Ito H, Kimura Y, Ogura H, Nomura S: Development and assessment of enzyme immunoassay for platelet-derived
microparticles. Thromb Haemost 85: 326–330, 2001
56. Yuana Y, Koning RI, Kuil ME, Rensen PC, Koster AJ, Bertina RM, Osanto S: Cryo-electron microscopy of extracellular vesicles in fresh plasma. J Extracell Vesicles 2: 10.3402/jev.v2i0.21494, 2013
57. du Cheyron D, Daubin C, Poggioli J, Ramakers M, Houillier P, Charbonneau P, Paillard M: Urinary measurement of Na+/H+ exchanger isoform 3 (NHE3) protein as new marker of tubule injury in critically ill patients with ARF. Am J Kidney Dis 42: 497–506, 2003
58. Zhou H, Pisitkun T, Aponte A, Yuen PS, Hoffert JD, Yasuda H, Hu X, Chawla L, Shen RF, Knepper MA, Star RA: Exosomal Fetuin-A identified by proteomics: A novel urinary biomarker for detecting acute kidney injury. Kidney Int 70: 1847–1857, 2006
59. Alvarez S, Suazo C, Boltansky A, Ursu M, Carvajal D, Innocenti G, Vukusich A, Hurtado M, Villanueva S, Carreño JE, Rogelio A, Irarrazabal CE: Urinary exosomes as a source of kidney dysfunction biomarker in renal transplantation. Transplant Proc 45: 3719–3723, 2013
60. Bianchi F, Sala E, Donadei C, Capelli I, La Manna G: Potential advantages of acute kidney injury management by mesenchymal stem cells. World J Stem Cells 6: 644–650, 2014
61. Erpicum P, Detry O, Weekers L, Bonvoisin C, Lechanteur C, Briquet A, Beguin Y, Krzesinski JM, Jouret F: Mesenchymal stromal cell therapy in conditions of renal ischaemia/reperfusion. Nephrol Dial Transplant 29: 1487–1493, 2014
62. Camussi G, Deregibus MC, Tetta C: Paracrine/endocrine mechanism of stem cells on kidney repair: Role of microvesicle-mediated transfer of genetic information. Curr Opin Nephrol Hypertens 19: 7–12, 2010
63. Bruno S, Grange C, Collino F, Deregibus MC, Cantaluppi V, Biancone L, Tetta C, Camussi G: Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury. PLoS One 7: e33115, 2012
64. Reis LA, Borges FT, Simões MJ, Borges AA, Sinigaglia-Coimbra R, Schor N: Bone marrow-derived mesenchymal stem cells repaired but did not prevent gentamicin-induced acute kidney injury through paracrine effects in rats. PLoS One 7: e44092, 2012
65. Cantaluppi V, Gatti S, Medica D, Figliolini F, Bruno S, Deregibus MC, Sordi A, Biancone L, Tetta C, Camussi G: Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia-reperfusion injury by microRNA-dependent reprogramming of resident renal cells. Kidney Int 82: 412–427, 2012
66. Collino F, Bruno S, Incarnato D, Dettori D, Neri F, Provero P, Pomatto M, Oliviero S, Tetta C, Quesenberry PJ, Camussi G: AKI recovery induced by mesenchymal stromal cell-derived extracellular vesicles carrying MicroRNAs [published online ahead of print April 21, 2015]. J Am Soc Nephrol doi:ASN.2014070710
67. Amabile N, Guérin AP, Leroyer A, Mallat Z, Nguyen C, Boddaert J, London GM, Tedgui A, Boulanger CM: Circulating endothelial
microparticles are associated with vascular dysfunction in patients with end-stage renal failure. J Am Soc Nephrol 16: 3381–3388, 2005
68. Amabile N, Guérin AP, Tedgui A, Boulanger CM, London GM: Predictive value of circulating endothelial
microparticles for cardiovascular mortality in end-stage renal failure: A pilot study. Nephrol Dial Transplant 27: 1873–1880, 2012
69. Chen YL, Chen CH, Wallace CG, Wang HT, Yang CC, Yip HK: Levels of circulating
microparticles in patients with chronic cardiorenal disease. J Atheroscler Thromb 22: 247–256, 2015
70. Soriano S, Carmona A, Triviño F, Rodriguez M, Alvarez-Benito M, Martín-Malo A, Alvarez-Lara MA, Ramírez R, Aljama P, Carracedo J: Endothelial damage and vascular calcification in patients with chronic kidney disease. Am J Physiol Renal Physiol 307: F1302–F1311, 2014
71. Buendía P, Montes de Oca A, Madueño JA, Merino A, Martín-Malo A, Aljama P, Ramírez R, Rodríguez M, Carracedo J: Endothelial
microparticles mediate inflammation-induced vascular calcification. FASEB J 29: 173–181, 2015
72. Farag YM, Keithy-Reddy SR, Mittal BV, Bansal V, Fareed J, Singh AK: Modulation of platelet activation in chronic kidney disease patients on erythropoiesis-stimulating agents. Clin Appl Thromb Hemost 18: 453–461, 2012
73. Daniel L, Fakhouri F, Joly D, Mouthon L, Nusbaum P, Grunfeld JP, Schifferli J, Guillevin L, Lesavre P, Halbwachs-Mecarelli L: Increase of circulating neutrophil and platelet
microparticles during acute vasculitis and hemodialysis. Kidney Int 69: 1416–1423, 2006
74. Gao C, Xie R, Yu C, Wang Q, Shi F, Yao C, Xie R, Zhou J, Gilbert GE, Shi J: Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and
microparticles release in patients with nephrotic syndrome. Thromb Haemost 107: 681–689, 2012
75. Merino A, Portolés J, Selgas R, Ojeda R, Buendia P, Ocaña J, Bajo MA, del Peso G, Carracedo J, Ramírez R, Martín-Malo A, Aljama P: Effect of different dialysis modalities on microinflammatory status and endothelial damage. Clin J Am Soc Nephrol 5: 227–234, 2010
76. Faure V, Dou L, Sabatier F, Cerini C, Sampol J, Berland Y, Brunet P, Dignat-George F: Elevation of circulating endothelial
microparticles in patients with chronic renal failure. J Thromb Haemost 4: 566–573, 2006
77. Dursun I, Poyrazoglu HM, Gunduz Z, Ulger H, Yykylmaz A, Dusunsel R, Patyroglu T, Gurgoze M: The relationship between circulating endothelial
microparticles and arterial stiffness and atherosclerosis in children with chronic kidney disease. Nephrol Dial Transplant 24: 2511–2518, 2009
78. Meijers BK, Van Kerckhoven S, Verbeke K, Dehaen W, Vanrenterghem Y, Hoylaerts MF, Evenepoel P: The uremic retention solute p-cresyl sulfate and markers of endothelial damage. Am J Kidney Dis 54: 891–901, 2009
79. Weber C, Sigrist M, Romann A, Whalen BA, Levin A: Exploring the relationships between hemoglobin, the endothelium and vascular health in patients with chronic kidney disease. Nephron Extra 1: 190–200, 2011
80. Zhou H, Kajiyama H, Tsuji T, Hu X, Leelahavanichkul A, Vento S, Frank R, Kopp JB, Trachtman H, Star RA, Yuen PS: Urinary exosomal Wilms’ tumor-1 as a potential biomarker for podocyte injury. Am J Physiol Renal Physiol 305: F553–F559, 2013
81. Lee H, Han KH, Lee SE, Kim SH, Kang HG, Cheong HI: Urinary exosomal WT1 in childhood nephrotic syndrome. Pediatr Nephrol 27: 317–320, 2012
82. Ramani P, Cowell JK: The expression pattern of Wilms’ tumour gene (WT1) product in normal tissues and paediatric renal tumours. J Pathol 179: 162–168, 1996
83. Burger D, Thibodeau JF, Holterman CE, Burns KD, Touyz RM, Kennedy CR: Urinary podocyte
microparticles identify prealbuminuric diabetic glomerular injury. J Am Soc Nephrol 25: 1401–1407, 2014
84. Moon PG, Lee JE, You S, Kim TK, Cho JH, Kim IS, Kwon TH, Kim CD, Park SH, Hwang D, Kim YL, Baek MC: Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy. Proteomics 11: 2459–2475, 2011
85. Kalani A, Mohan A, Godbole MM, Bhatia E, Gupta A, Sharma RK, Tiwari S: Wilm’s tumor-1 protein levels in urinary exosomes from diabetic patients with or without proteinuria. PLoS One 8: e60177, 2013
86. Barutta F, Tricarico M, Corbelli A, Annaratone L, Pinach S, Grimaldi S, Bruno G, Cimino D, Taverna D, Deregibus MC, Rastaldi MP, Perin PC, Gruden G: Urinary exosomal microRNAs in incipient diabetic nephropathy. PLoS One 8: e73798, 2013
87. Hoorn EJ, Pisitkun T, Zietse R, Gross P, Frokiaer J, Wang NS, Gonzales PA, Star RA, Knepper MA: Prospects for urinary proteomics: Exosomes as a source of urinary biomarkers. Nephrology (Carlton) 10: 283–290, 2005
88. Street JM, Birkhoff W, Menzies RI, Webb DJ, Bailey MA, Dear JW: Exosomal transmission of functional aquaporin 2 in kidney cortical collecting duct cells. J Physiol 589: 6119–6127, 2011
89. Joo KW, Lee JW, Jang HR, Heo NJ, Jeon US, Oh YK, Lim CS, Na KY, Kim J, Cheong HI, Han JS: Reduced urinary excretion of thiazide-sensitive Na-Cl cotransporter in Gitelman syndrome: Preliminary data. Am J Kidney Dis 50: 765–773, 2007
90. Yoder BK, Hou X, Guay-Woodford LM: The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia. J Am Soc Nephrol 13: 2508–2516, 2002
91. Gerlach JQ, Krüger A, Gallogly S, Hanley SA, Hogan MC, Ward CJ, Joshi L, Griffin MD: Surface glycosylation profiles of urine extracellular vesicles. PLoS One 8: e74801, 2013
92. Hogan MC, Bakeberg JL, Gainullin VG, Irazabal MV, Harmon AJ, Lieske JC, Charlesworth MC, Johnson KL, Madden BJ, Zenka RM, McCormick DJ, Sundsbak JL, Heyer CM, Torres VE, Harris PC, Ward CJ: Identification of biomarkers for PKD1 using urinary exosomes [published online ahead of print December 4, 2014]. J Am Soc Nephrol doi:ASN.2014040354
93. Sonoda H, Yokota-Iked N, Oshikawa S, Kanno Y, Yoshinaga K, Uchida K, Ueda Y, Kimiya K, Uezono S, Ueda A, Ito K, Ikeda M: Decreased abundance of urinary exosomal aquaporin-1 in renal ischemia-reperfusion injury. Am J Physiol Renal Physiol 297: F1006–F1016, 2009
94. Tőkés-Füzesi M, Woth G, Ernyey B, Vermes I, Mühl D, Bogár L, Kovács GL:
Microparticles and acute renal dysfunction in septic patients. J Crit Care 28(2): 141–147, 2013
95. Zhou Y, Xu H, Xu W, Wang B, Wu H, Tao Y, Zhang B, Wang M, Mao F, Yan Y, Gao S, Gu H, Zhu W, Qian H: Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro. Stem Cell Res Ther 4: 34, 2013
96. Tomasoni S, Longaretti L, Rota C, Morigi M, Conti S, Gotti E, Capelli C, Introna M, Remuzzi G, Benigni A: Transfer of growth factor receptor mRNA via exosomes unravels the regenerative effect of mesenchymal stem cells. Stem Cells Dev 22: 772–780, 2013
97. Choi HY, Moon SJ, Ratliff BB, Ahn SH, Jung A, Lee M, Lee S, Lim BJ, Kim BS, Plotkin MD, Ha SK, Park HC:
Microparticles from kidney-derived mesenchymal stem cells act as carriers of proangiogenic signals and contribute to recovery from acute kidney injury. PLoS One 9: e87853, 2014
98. Ando M, Iwata A, Ozeki Y, Tsuchiya K, Akiba T, Nihei H: Circulating platelet-derived
microparticles with procoagulant activity may be a potential cause of thrombosis in uremic patients. Kidney Int 62: 1757–1763, 2002
99. Boulanger CM, Amabile N, Guérin AP, Pannier B, Leroyer AS, Mallat CN, Tedgui A, London GM: In vivo shear stress determines circulating levels of endothelial
microparticles in end-stage renal disease. Hypertension 49: 902–908, 2007
100. Trappenburg MC, van Schilfgaarde M, Frerichs FC, Spronk HM, ten Cate H, de Fijter CW, Terpstra WE, Leyte A: Chronic renal failure is accompanied by endothelial activation and a large increase in microparticle numbers with reduced procoagulant capacity. Nephrol Dial Transplant 27: 1446–1453, 2012
101. Sakurai K, Saito T, Yamauchi F, Asahi D, Hosoya H: Comparison of the effects of predilution and postdilution hemodiafiltration on neutrophils, lymphocytes and platelets. J Artif Organs 16: 316–321, 2013
102. Di Marco GS, König M, Stock C, Wiesinger A, Hillebrand U, Reiermann S, Reuter S, Amler S, Köhler G, Buck F, Fobker M, Kümpers P, Oberleithner H, Hausberg M, Lang D, Pavenstädt H, Brand M: High phosphate directly affects endothelial function by downregulating annexin II. Kidney Int 83: 213–222, 2013
103. van Koppen A, Joles JA, van Balkom BW, Lim SK, de Kleijn D, Giles RH, Verhaar MC: Human embryonic mesenchymal stem cell-derived conditioned medium rescues kidney function in rats with established chronic kidney disease. PLoS One 7: e38746, 2012
104. Sun AL, Deng JT, Guan GJ, Chen SH, Liu YT, Cheng J, Li ZW, Zhuang XH, Sun FD, Deng HP: Dipeptidyl peptidase-IV is a potential molecular biomarker in diabetic kidney disease. Diab Vasc Dis Res 9: 301–308, 2012
105. Zubiri I, Posada-Ayala M, Sanz-Maroto A, Calvo E, Martin-Lorenzo M, Gonzalez-Calero L, de la Cuesta F, Lopez JA, Fernandez-Fernandez B, Ortiz A, Vivanco F, Alvarez-Llamas G: Diabetic nephropathy induces changes in the proteome of human urinary exosomes as revealed by label-free comparative analysis. J Proteomics 96: 92–102, 2014
106. Hara M, Yanagihara T, Kihara I, Higashi K, Fujimoto K, Kajita T: Apical cell membranes are shed into urine from injured podocytes: A novel phenomenon of podocyte injury. J Am Soc Nephrol 16: 408–416, 2005
107. Tkaczyk M, Baj Z: Surface markers of platelet function in idiopathic nephrotic syndrome in children. Pediatr Nephrol 17: 673–677, 2002
108. Brogan PA, Shah V, Brachet C, Harnden A, Mant D, Klein N, Dillon MJ: Endothelial and platelet
microparticles in vasculitis of the young. Arthritis Rheum 50: 927–936, 2004
109. Erdbruegger U, Grossheim M, Hertel B, Wyss K, Kirsch T, Woywodt A, Haller H, Haubitz M: Diagnostic role of endothelial
microparticles in vasculitis. Rheumatology (Oxford) 47: 1820–1825, 2008
110. Kümpers P, Erdbrügger U, Grossheim M, Meyer GP, Hiss M, Gwinner W, Haller H, Haubitz M: Endothelial
microparticles as a diagnostic aid in Churg-Strauss vasculitis-induced cardiomyopathy. Clin Exp Rheumatol 26[Suppl 49]: S86–S89, 2008
111. Eleftheriou D, Hong Y, Klein NJ, Brogan PA: Thromboembolic disease in systemic vasculitis is associated with enhanced microparticle-mediated thrombin generation. J Thromb Haemost 9: 1864–1867, 2011
112. Hong Y, Eleftheriou D, Hussain AA, Price-Kuehne FE, Savage CO, Jayne D, Little MA, Salama AD, Klein NJ, Brogan PA: Anti-neutrophil cytoplasmic antibodies stimulate release of neutrophil
microparticles. J Am Soc Nephrol 23: 49–62, 2012
113. Yahata T, Suzuki C, Yoshioka A, Hamaoka A, Ikeda K: Platelet activation dynamics evaluated using platelet-derived
microparticles in Kawasaki disease. Circ J 78: 188–193, 2014
114. Ding YY, Ren Y, Feng X, Xu QQ, Sun L, Zhang JM, Dou JJ, Lv HT, Yan WH: Correlation between brachial artery flow-mediated dilation and endothelial microparticle levels for identifying endothelial dysfunction in children with Kawasaki disease. Pediatr Res 75: 453–458, 2014
115. Pitanga TN, de Aragao Franca L, Rocha VC, Meirelles T, Borges VM, Goncalves MS, Pontes-de-Carvalho LC, Noronha-Dutra AA, dos-Santos WL: Neutrophil-derived
microparticles induce myeloperoxidase-mediated damage of vascular endothelial cells. BMC Cell Biol 15: 21, 2014
116. Jimenez JJ, Jy W, Mauro LM, Horstman LL, Ahn YS: Elevated endothelial
microparticles in thrombotic thrombocytopenic purpura: Findings from brain and renal microvascular cell culture and patients with active disease. Br J Haematol 112: 81–90, 2001
117. Camus SM, Gausserès B, Bonnin P, Loufrani L, Grimaud L, Charue D, De Moraes JA, Renard JM, Tedgui A, Boulanger CM, Tharaux PL, Blanc-Brude OP: Erythrocyte
microparticles can induce kidney vaso-occlusions in a murine model of sickle cell disease. Blood 120: 5050–5058, 2012
118. Isobe K, Mori T, Asano T, Kawaguchi H, Nonoyama S, Kumagai N, Kamada F, Morimoto T, Hayashi M, Sohara E, Rai T, Sasaki S, Uchida S: Development of enzyme-linked immunosorbent assays for urinary thiazide-sensitive Na-Cl cotransporter measurement. Am J Physiol Renal Physiol 305: F1374–F1381, 2013
119. van der Lubbe N, Jansen PM, Salih M, Fenton RA, van den Meiracker AH, Danser AH, Zietse R, Hoorn EJ: The phosphorylated sodium chloride cotransporter in urinary exosomes is superior to prostasin as a marker for aldosteronism. Hypertension 60: 741–748, 2012
120. Hogan MC, Manganelli L, Woollard JR, Masyuk AI, Masyuk TV, Tammachote R, Huang BQ, Leontovich AA, Beito TG, Madden BJ, Charlesworth MC, Torres VE, LaRusso NF, Harris PC, Ward CJ: Characterization of PKD protein-positive exosome-like vesicles. J Am Soc Nephrol 20: 278–288, 2009
121. Al-Massarani G, Vacher-Coponat H, Paul P, Widemann A, Arnaud L, Loundou A, Robert S, Berland Y, Dignat-George F, Camoin-Jau L: Impact of immunosuppressive treatment on endothelial biomarkers after kidney transplantation. Am J Transplant 8: 2360–2367, 2008
122. Al-Massarani G, Vacher-Coponat H, Paul P, Arnaud L, Loundou A, Robert S, Moal V, Berland Y, Dignat-George F, Camoin-Jau L: Kidney transplantation decreases the level and procoagulant activity of circulating
microparticles. Am J Transplant 9: 550–557, 2009
123. Pisitkun T, Gandolfo MT, Das S, Knepper MA, Bagnasco SM: Application of systems biology principles to protein biomarker discovery: Urinary exosomal proteome in renal transplantation. Proteomics Clin Appl 6: 268–278, 2012
124. Peake PW, Pianta TJ, Succar L, Fernando M, Pugh DJ, McNamara K, Endre ZH: A comparison of the ability of levels of urinary biomarker proteins and exosomal mRNA to predict outcomes after renal transplantation. PLoS One 9: e98644, 2014
125. Esteva-Font C, Guillén-Gómez E, Diaz JM, Guirado L, Facundo C, Ars E, Ballarin JA, Fernández-Llama P: Renal sodium transporters are increased in urinary exosomes of cyclosporine-treated kidney transplant patients. Am J Nephrol 39: 528–535, 2014
126. Chatelet F, Brianti E, Ronco P, Roland J, Verroust P: Ultrastructural localization by monoclonal antibodies of brush border antigens expressed by glomeruli. I. Renal distribution. Am J Pathol 122: 500–511, 1986
127. Lacroix R, Robert S, Poncelet P, Dignat-George F: Overcoming limitations of microparticle measurement by flow cytometry. Semin Thromb Hemost 36: 807–818, 2010
128. Yuana Y, Oosterkamp TH, Bahatyrova S, Ashcroft B, Garcia Rodriguez P, Bertina RM, Osanto S: Atomic force microscopy: A novel approach to the detection of nanosized blood
microparticles. J Thromb Haemost 8: 315–323, 2010
129. Ashcroft BA, de Sonneville J, Yuana Y, Osanto S, Bertina R, Kuil ME, Oosterkamp TH: Determination of the size distribution of blood
microparticles directly in plasma using atomic force microscopy and microfluidics. Biomed Microdevices 14: 641–649, 2012