Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers? : Journal of the American Society of Nephrology

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Extracellular Vesicles in Renal Diseases

More than Novel Biomarkers?

Erdbrügger, Uta; Le, Thu H.

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Journal of the American Society of Nephrology 27(1):p 12-26, January 2016. | DOI: 10.1681/ASN.2015010074
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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.1620 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.30,3538 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 , 44


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.


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.


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.



The authors thank Paula Timm for her excellent help with graphic design.

Published online ahead of print. Publication date available at www.jasn.org.


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extracellular vesicles; kidney disease; biomarker; exosomes; microparticles

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