Extracellular Vesicles as Theranostic Tools in Kidney Disease

Abstract

Extracellular vesicles are important vectors for cell-cell communication and show potential value for diagnosis and treatment of kidney diseases. The pathologic diagnosis of kidney diseases relies on kidney biopsy, whereas collection of extracellular vesicles from urine or circulating blood may constitute a less invasive diagnostic tool. In particular, urinary extracellular vesicles released mainly from resident kidney cells might provide an alternative tool for detection of kidney injury. Because extracellular vesicles mirror many features of their parent cells, cargoes of several populations of urinary extracellular vesicles are promising biomarkers for disease processes, like diabetic kidney disease, kidney transplant, and lupus nephritis. Contrarily, extracellular vesicles derived from reparative cells, such as mesenchymal stem cells, tubular epithelial progenitor cells, and human umbilical cord blood represent promising regenerative tools for treatment of kidney diseases. Furthermore, induced pluripotent stem cells–derived and engineered extracellular vesicles are being developed for specific applications for the kidney. Nevertheless, some assumptions regarding the specificity and immunogenicity of extracellular vesicles remain to be established. This review focuses on the utility of extracellular vesicles as therapeutic and diagnostic (theranostic) tools in kidney diseases and future directions for studies.

Introduction

Extracellular vesicles are nanoscale membrane vesicles released by cells that carry luminal and membrane-bound cargoes, endowing them with exceptional theranostic (therapeutic and diagnostic) properties. Consequently, since their discovery, interest in their application has been rising progressively, including in kidney applications.

Extracellular vesicles are considered to mainly include exosomes, microvesicles, and apoptotic bodies (1). Exosomes (30–150 nm) are formed primarily from multivesicular bodies, which develop from late endosomes assisted by clusters-of-differentiation 9 (CD9), CD63, endosomal sorting complex required for transport (ESCRT), and Alix proteins. Multivesicular bodies ultimately fuse with the plasma membrane and release exosomes into the extracellular space. Other than this classic ESCRT-dependent pathway, ESCRT-independent paths contribute to exosome formation and sorting of specific molecules, and they seem to involve the tetraspanin CD63 (2) and neutral sphingomyelinase-2 (3). Other ESCRT-independent paths, like amyloid precursor protein–containing extracellular vesicles, are secreted by an Alix- and Syntenin-1–dependent mechanism (4), and the Ras-related protein Rab-31 controls exosome formation via a flotillin proteins–dependent pathway (5).

Microvesicles are generally larger (100–1000 nm) and form directly from the plasma membrane through an interaction between phosphatidylserine redistribution and cytoskeletal protein contraction (6). Apoptotic bodies (500–4000 nm) form during preprogrammed cell death and are characterized by organelle and fragmented DNA content (1). Most apoptotic bodies are recognized and cleared by macrophages (7).

Extracellular Vesicle Isolation and Characterization

Extracellular vesicles are detectable in most bodily fluids and must be isolated in sufficient quantities for analysis. Of available methods, ultracentrifugation is among the most widely used and separates extracellular vesicles into a pellet that dissolves in a buffer (8). Its disadvantages include time consumption, disrupted particle integrity, and protein contamination, which might be ameliorated with one-step sucrose cushion ultracentrifugation (9). Size-exclusion chromatography can isolate size-specific relatively pure extracellular vesicles with higher yield than ultracentrifugation (10). Other technologies used to separate extracellular vesicles include tangential flow filtration (less time consuming and higher yield than ultracentrifugation), ultrafiltration (more efficient), magnetic beads conjugated with antibodies (isolate extracellular vesicles from specific cells), precipitation (low equipment demands, preserves biologic activities of extracellular vesicles, and low protein contamination), microfluidics and lab-on-a-chip technology (produce size-controlled extracellular vesicles), hydrostatic filtration dialysis (urine suitable and inexpensive), and anion exchange chromatography (rapid isolation, high yield, and low protein contamination). Recent studies have promoted ultracentrifugation and hydrostatic filtration dialysis for mRNA sequencing of urinary extracellular vesicles in diabetic kidney disease (DKD) (11) and ultrafiltration in combination with exosomal RNA columns to purify microRNAs (miRNAs) (12).

Subsequently, extracellular vesicles are identified by markers and morphology. CD9, CD63, CD81, TSG101, and Alix regulate formation and identify exosomes (13); adenosine diphosphate ribosylation factor-6 (14) and vesicle-associated membrane protein-3 (15) characterize microvesicle formation. High-throughput fluorescence correlation spectroscopy facilitates their identification (16). With electron microscopy, extracellular vesicle size can be assessed, and a nanostructure of peripheral limiting membrane can be resolved (17). Moreover, extracellular vesicles shed from cells can be directly observed with high-resolution cryogenic electron microscopy (18). Nanoparticle tracking analysis and dynamic light scattering are useful for determination of size distribution and concentration.

Extracellular vesicles are rich in proteins as well as miRNA, mRNA, long-noncoding, and other types of RNA, and they contain lipids like cholesterol, sphingomyelin, glycosphingolipid, phosphatidylserine, and ceramide. Furthermore, their surface membrane proteins (19) together with cargo act as signal vectors, making extracellular vesicles an important tool for cell-to-cell communication and, potentially, in clinical practice. This review focuses on extracellular vesicles as theranostic tools in kidney disease.

Extracellular Vesicles: Diagnostic Tools in Kidney Disease

Extracellular vesicles can be easily isolated from most body fluids, and their cargo is altered in many diseases, with urinary and circulating extracellular vesicles best characterized. Because their size may prohibit passage of circulating extracellular vesicles across the glomerular filtration barrier, urinary extracellular vesicles are traditionally considered to originate mainly from resident kidney cells (20), and their levels and characteristics are considered suitable to reflect kidney disease. Yet, circulating extracellular vesicles may also be altered in kidney disease (Figure 1A).

Figure 1.

Extracellular vesicles (EVs) are ubiquitous diagnostic and therapeutic tools in kidney diseases. (A) EV-based biomarkers of kidney diseases include urinary and circulating EVs. Urinary EVs have been widely applied to detect kidney diseases or injury, including diabetic kidney disease (DKD), kidney transplantation, lupus nephritis, renal cell carcinoma (RCC), hypertension, polycystic kidney disease, FSGS, IgA nephropathy, obstructive nephropathy, radiation-induced kidney injury, minimal change kidney disease, kidney fibrosis, and basement membrane nephropathy. Circulating EVs have been studied in fewer diseases (including RCC, kidney transplantation, minimal change kidney disease, DKD, and renal injury in dyspnea). (B) EVs harvested from different tissues can serve as therapeutic tools in kidney diseases. Proposed sources for EVs include tissue mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), induced pluripotent stem cells (iPSCs), tubular epithelial cells, urine, and human placenta. B2M, beta-2 microglobulin; BMP, bone morphogenetic protein; CAIX, carbonic anhydrase IX; CD, cluster of differentiation; CEBPA, CCAAT/enhancer-binding protein alpha; CP, ceruloplasmin; CXCL, C-X-C motif chemokine ligand; DDK, dickkopf-related protein; DPEP, dipeptidase; EMMRIN, extracellular matrix metalloproteinase inducer; GGT, γ-glutamyltransferase; GSTA, glutathione S-transferase A; IFNAR, interferon-α/β receptor; IgA, immunoglobulin A; IL, interleukin; L1CAM, L1 cell adhesion molecule; miR, microRNA; MMP, matrix metalloproteinase; NGAL, neutrophil gelatinase-associated lipocalin; PC, polycystin; PCBD, pterin-4-alpha-carbinolamine dehydratase; PCK, phosphoenolpyruvate carboxykinase; PODXL, podocalyxin-like protein; RVD, renovascular disease; TIMPs, tissue inhibitors of metalloproteinases; TGF-β, transforming growth factor β; TMEM, transmembrane protein.

Urinary Extracellular Vesicles

Studies have demonstrated the rich heterogeneity of urinary extracellular vesicles and their suitability for detection of kidney disease (21). For example, proteomics highlighted their resemblance to several kidney proteins and utility in tracking kidney pathology (22); the most promising extracellular vesicle biomarkers are listed in Supplemental Table 1. Typical changes have been described in specific kidney diseases.

Diabetic Kidney Disease.

Several urinary extracellular vesicle miRNAs and proteins are altered in DKD (Supplemental Table 1) and linked to its onset. Urinary microvesicle dipeptidyl peptidase IV level correlates with the urinary albumin-creatinine-ratio in participants with DKD (23), and regucalcin is decreased (24). In particular, podocyte markers, like podocalyxin⁺, or a high podocin-nephrin ratio in extracellular vesicles suggest glomerular injury in DKD (25). Additional studies are needed to identify the best markers to detect and reflect DKD severity and how they are affected by glucosuria or protein complexes (26).

Kidney Transplant.

In patients with allograft rejection, the numbers of CD3⁺ urinary extracellular vesicles are elevated (27), and their mRNA and miRNA are useful for diagnosis (Supplemental Table 1). Interestingly, CD133⁺ urinary extracellular vesicles continuously released during homeostatic turnover of the nephron may reflect graft function (28). Moreover, neutrophil gelatinase–associated lipocalin in urinary extracellular vesicles may predict graft recovery (29), as does extracellular vesicle phosphoenolpyruvate carboxykinase (30). Furthermore, acute disturbance of water homeostasis in kidneys post-transplant might be related to decreased urinary extracellular vesicles expression of aquaporin-2 (31). The miRNA bkv-miR-B1–5p secreted by the BK virus that may cause nephropathy in kidney transplant recipients was detected in the blood and urine of patients (32). Therefore, urinary extracellular vesicles in patients after kidney transplant may reflect graft status, function, and complications.

Lupus Nephritis.

Glomerular podocytes have been implicated in glomerular injury in patients with lupus nephritis. Importantly, urinary podocyte microparticles (annexin-V and podocalyxin⁺) track with disease activity (33), and several miRNAs were also proposed as biomarkers of lupus nephritis (Supplemental Table 1). Yet, their selectivity for the pathogenesis of lupus nephritis must be defined before therapeutic targets are selected.

Renal Cell Carcinoma.

Distinct protein content was identified in urinary extracellular vesicles of patients with renal cell carcinoma (RCC) (34), which might serve as biomarker reservoirs (Supplemental Table 1). Specifically, patients with clear-cell RCC have altered urinary extracellular vesicle RNA and miRNAs, and in murine RCC, miR‐204‐5p and miR‐211‐5p are elevated (35). However, it remains unclear whether these changes reflect the pathogenesis of the disease or are secondary to the phenotype.

Hypertension-Related Kidney Injury.

Patients with hypertension release increased numbers of urinary extracellular vesicles bearing markers reflecting kidney injury, including nephrin⁺ and podocalyxin⁺ (podocyte injury) (36), urate-transporter-1⁺/p16⁺ (proximal tubular senescence) (37), and plasmalemmal vesicle–associated protein⁺ (microvascular injury) urinary extracellular vesicles (38). Furthermore, the exosome miR-146a level inversely associates with albuminuria and early kidney injury (39). Therefore, urinary extracellular vesicles might pinpoint injuries to specific kidney compartments. The ability to detect kidney injury preceding albuminuria would be invaluable for early treatment.

Urinary Extracellular Vesicles in Other Kidney Diseases.

Urinary extracellular vesicles have been applied to elucidate other kidney diseases as well. For example, elevated levels of C-C motif chemokine ligand-2 mRNA (40), a-1-antitrypsin, and ceruloplasmin, as well as reduced aminopeptidase and vasorin precursor, were observed in IgA nephropathy, whereas in thin basement membrane nephropathy, vasorin precursor increased (41). Polycystin-transmembrane protein-2 ratio in urinary extracellular vesicles is a good marker in polycystic kidney disease (42), and preeclampsia is associated with an elevated urinary podocin⁺-nephrin⁺ extracellular vesicles ratio (43). Additionally, decreases in the urinary extracellular vesicle CD133 indicate glomerular damage (44), and decreases in vacuolar adenosine triphosphatase B1 identify distal tubular acidosis (45). We found increased levels of podocyte-derived urinary extracellular vesicles in patients with metabolic syndrome–related kidney disease (46). Urinary extracellular vesicles miR-26a (47) and WT1 mRNA (48) may also reflect podocyte injury. Notably, palmitic acid can promote extracellular vesicle production by renal tubular epithelial cells in vitro (49), suggesting that high levels of urinary extracellular vesicles derived from tubular epithelial cells may be a marker of lipotoxicity. Therefore, characterization of urinary extracellular vesicle populations may aid in localizing kidney injury to specific cell types.

Unlike protein and nucleic acid, lipid metabolites in extracellular vesicles as biomarkers for kidney diseases have been studied in less detail. However, a differential lipid composition in urinary exosomes was found in RCC compared with healthy individuals (50). In addition, the presence of phosphatidylserine and other lipid species identified prostate cancer in vivo (51) and in vitro (52). Clearly, lipid metabolites in extracellular vesicles can be potential markers for kidney diseases, and additional research is needed in this area of extracellular vesicles biology.

Changes in urinary extracellular vesicles have also been described in animal models of drug-induced nephrotoxicity (53), ischemia-reperfusion injury (54), AKI (55), and polyarteritis nodosa nephritis (56). Yet, species differences must be considered in application of data obtained from animal models.

Given that extracellular vesicles in the urine originate mainly from kidney cells, it might be feasible to identify their parent cells. Podocyte-derived extracellular vesicles are nephrin, podocalyxin, calyxin, or podocin positive (37,46,57). Extracellular vesicles of proximal tubular cell origin can be identified by megalin, aquaporin-1, osteoprotegerin, and urate transporter-1 (37,57,58), whereas CD144, CD31, or plasmalemmal vesicle–associated protein markers suggest endothelial cell origin (38,57). In addition, uromodulin is a marker for the ascending limb of Henle loop and prominin-2 for distal tubules (37). Therefore, specific markers may assist in localizing cellular alterations to specific nephron segments.

Circulating Extracellular Vesicles

Circulating extracellular vesicles have broader origins than urinary extracellular vesicles and possibly account for a fraction of kidney-derived extracellular vesicles. In particular, they have been linked to diagnosis or severity of RCC (Supplemental Table 2). Furthermore, after kidney transplant, increased circulating extracellular vesicle miR-21 levels distinguish the degree of interstitial fibrosis more accurately than serum creatinine and proteinuria (59). In patients with minimal change nephrotic syndrome, several mRNAs are elevated (Supplemental Table 2), and concentrations of miR-4449 in circulating extracellular vesicles are higher in patients with diabetes with versus without DKD (60). Therefore, the content of circulating extracellular vesicles may reflect CKD subtypes and complications. Nevertheless, the systemic distribution and specificity of these markers for kidney conditions warrant careful verification.

Notably, the perception that circulating extracellular vesicles do not contribute to urinary extracellular vesicles warrants reconsideration. Although usually only small circulating particles (<10 nm) undergo glomerular filtration, larger nanoparticles (300–400 nm) may undergo transcytosis across the peritubular capillary endothelium and accumulate in proximal tubular epithelial cells (61), and they might thus be released into the tubular lumen and appear in the urine. Possibly, a reverse route might be used by kidney-derived extracellular vesicles to reach the systemic circulation. Therefore, the precise source and ultimate destiny of urinary and circulating extracellular vesicles need to be fate mapped. Moreover, circulating extracellular vesicles may more easily cross an injured glomerular filtration barrier, so that increased levels of urinary extracellular vesicles originating from the circulation may indicate glomerular injury.

Extracellular Vesicles as Therapeutic Tools in Kidney Disease

Beyond serving as biomarkers, the extracellular vesicle cargos of proteins, nucleic acids, and lipids may mediate tissue repair, at least when derived from curative cells. Compared with their parent cells, extracellular vesicles are more stable, are traditionally considered to be less immunogenic, and enable longer storage. Because of their small size, they penetrate tissues better, and intra-arterial injections of extracellular vesicles may pose a lower risk of embolization than cell injection. Therefore, since their first therapeutic application in murine AKI (62), extracellular vesicles have been applied widely, including small clinical studies (63). Meta-analyses have shown that extracellular vesicles improve kidney function in experimental AKI and CKD models (64,65). However, their effects depend on their cells of origin.

Mesenchymal Stem Cell–/Stromal Cell–Derived Extracellular Vesicles

Mesenchymal stem/stromal cells used in regenerative medicine can be isolated from bone marrow, adipose tissue, and umbilical cord vein blood and reside in solid organs, such as the liver, kidney, heart, and pancreas. Extracellular vesicles replicate the beneficial therapeutic function of their parent cells but with a lower risk of rejection (66), and they have therefore been widely studied in kidney diseases (Figure 1B).

Bone Marrow Mesenchymal Stem Cell–Derived Extracellular Vesicles.

Bone marrow mesenchymal stem cell–derived extracellular vesicles are beneficial in AKI, possibly by their miRNA load (67), but they are not all equally effective. Extracellular vesicles with medium flotation density enriched with exosomal markers were the most effective for tubular epithelial cell protection (68). Similarly, kidney function improved with exosomal-enriched but not with microvesicle-enriched secretome of bone marrow mesenchymal stem cells (69).

In animal models of diabetic nephropathy, these extracellular vesicles blunted albuminuria and relieved inflammation and fibrosis (70). They protected kidney function in some respects better than their parent mesenchymal stem cells (71), attenuated kidney fibrosis in unilateral ureteral obstruction (72), and ameliorated kidney injury in nephropathy (73). However, in rats with kidney transplant rejection, bone marrow mesenchymal stem cell extracellular vesicles did not prolong graft and recipient survival (74). Clearly, their utility might be dictated by the underlying disease. In addition, given the invasiveness of bone marrow collection, an allogeneic source might be warranted for clinical applications.

Adipose Mesenchymal Stem Cell–Derived Extracellular Vesicles.

In our studies, adipose mesenchymal stem cell–derived extracellular vesicles improved intrarenal microvascular density in pigs with renovascular disease, possibly by attenuating kidney inflammation and fibrosis (75–77). Like bone marrow mesenchymal stem cell extracellular vesicles, adipose mesenchymal stem cell extracellular vesicles ameliorated diabetic nephropathy. In db/db mice, adipose mesenchymal stem cell extracellular vesicles decreased podocyte apoptosis and enhanced autophagy, possibly by regulating miR-486/Smad1/mammalian target of rapamycin signaling (78).

However, the adipose tissue niche dictates the renoprotective function of these extracellular vesicles, as obesity or metabolic syndrome changes their cargoes and attenuates their therapeutic potency (79,80). Consequently, in pigs with renovascular disease, lean but not metabolic syndrome extracellular vesicles restored kidney function (81). Therefore, the ambient microenvironment of donor mesenchymal stem cells and extracellular vesicles should be taken into account when planning autologous transplantation (Figure 2). Future developments may need to improve mesenchymal stem cell function before autologous delivery.

Figure 2.

Obesity blunts the efficacy of adipose MSC-derived EVs as a therapeutic tool in kidney diseases. Lean adipose MSC-derived EVs cover curative effects, whereas obesity or metabolic syndrome changes their cargoes and blunts their ability to alleviate cell or organ injury. HTN, hypertensive nephropathy; MAPK, mitogen-activated protein kinase; miRNA, microRNA; mTOR, mammalian target of rapamycin; RVD, renovascular disease.

Umbilical Cord Vein Blood Mesenchymal Stem Cell–Derived Extracellular Vesicles.

The first clinical trial of extracellular vesicles used umbilical cord vein blood mesenchymal stem cell extracellular vesicles in 20 patients with CKD, in whom kidney function improved with no adverse effects (63). In animal models, such extracellular vesicles improved kidney function in ischemia-reperfusion injury and restarted the cell cycle in murine diabetic nephropathy (82). Notably, umbilical cord extracts, including growth factors, extracellular matrices, and exosomes, also increased the therapeutic effect of bone marrow mesenchymal stem cells in diabetic nephropathy (83).

Induced Pluripotent Stem Cell–Derived Extracellular Vesicles.

Induced pluripotent stem cells (iPSCs) are produced by reprogramming adult mesenchymal cells that can differentiate into various lineages, including mesenchymal stem cells. Importantly, although tissue-specific mesenchymal stem cells can be safely passaged to few generations, iPSCs can renew numerous times (84). Therefore, iPSC mesenchymal stem cell extracellular vesicles are receiving escalating attention in regenerative therapy. Like adipose mesenchymal stem cell–derived extracellular vesicles, iPSC mesenchymal stem cell extracellular vesicles exert antifibrotic, antioxidative stress, anti-inflammatory, and antiapoptosis functions (85,86).

Endothelial Progenitor Cell–Derived Extracellular Vesicles

Endothelial progenitor cells originate from bone marrow and bear some stem cell characteristics, but they are lineage committed. Extracellular vesicles released by these cells stimulate proliferation, differentiation, and angiogenesis, which make them a potential therapeutic tool. In rats, endothelial progenitor cell extracellular vesicles ameliorate kidney dysfunction and damage by enhancing tubular epithelial cell proliferation and reducing inflammation, possibly through their miRNA content (87). In rat GN, endothelial progenitor cell extracellular vesicles also ameliorated kidney function through immune/inflammatory regulation and antiapoptosis (88).

Tubular Epithelial Cell–Derived Extracellular Vesicles

Extracellular vesicles released by tubular epithelial cells mediate their crosstalk with other kidney resident and inflammatory cells (89,90). Notably, tubular epithelial cell extracellular vesicles have been implicated in the pathophysiology of several kidney diseases, such as diabetic nephropathy (91). Nonetheless, extracellular vesicles derived from healthy tubular epithelial cells might be effective regenerative tools. Tubular epithelial cell extracellular vesicles accelerate kidney recovery in ischemia-reperfusion injury by alleviating tissue damage, partly by activating transcription factor-3 and CD26 (92,93). We identified renoprotective effects of scattered tubular-like cell–derived extracellular vesicles. Scattered tubular-like cell extracellular vesicles improved mitochondrial function of injured tubular epithelial cells in vitro and stenotic kidney perfusion in vivo (94). However, unlike mesenchymal stem cells, tubular epithelial cells are generally not immune privileged, limiting the application of their extracellular vesicles to autologous formulations (95).

Extracellular vesicles derived from other sources that were studied in kidney disease include liver stem cells, urine, and immune cells.

Engineered Extracellular Vesicles in Kidney Disease

Clearly, extracellular vesicles show potential for kidney damage repair. However, variable stability and retention of certain extracellular vesicle populations affect their efficacy, and an unwholesome ancestry might yield suboptimal extracellular vesicles. The easiest and most widely studied method to engineer superior extracellular vesicles applies hypoxia (a form of stress), which induces the release of large numbers of extracellular vesicles with enhanced properties (96). Their efficacy has also been augmented by transfection with miRNA, specific miRNA mimics, or antagomirs (97,98). Protein overexpression of the pluripotency factor oct-4 in mesenchymal stem cell extracellular vesicles also enhanced their antiapoptotic effects in AKI (99). Intriguingly, kidney-targeting macrophage-derived microvesicles containing dexamethasone suppressed kidney inflammation and fibrosis (100), leveraging extracellular vesicles as a drug delivery vehicle. Also, extracellular vesicles harvested from parent cells engineered to release erythropoietin or neurotrophic factor improved anemia and peritubular capillary loss, respectively (101,102).

In addition to their cargo, the extracellular vesicle surface can be modified to harness their potential. Encapsulation in a collagen matrix (103) or hydrogel (104) augmented their kidney retention and repair thanks to continuous and steady release. Importantly, extracellular vesicles coated with anti-KIM1 antibodies showed better homing and restoration of kidney function and blunting of inflammation (105). Selective targeting is an exciting approach that may allow for the decrease of the dose and the minimization of off-target accumulation of extracellular vesicles. Of particular interest are strategies that leverage the known pathways of extracellular vesicles physiology (production, trafficking, release, etc.). Recently, two endogenous “scaffold” proteins of extracellular vesicles, PTGFRN and BASP1, have been identified. PTGFRN enables high-density surface display, and BASP1 promotes luminal loading of various molecules (106), providing a potential strategy for extracellular vesicle engineering. For example, antibodies against kidney cell proteins can be fused to PTGFRN and glucocorticoids loaded with BASP1 protein to specifically target kidney cells and deliver glucocorticoids to improve the efficacy and reduce side effects. Alternatively, lysosome-associated membrane protein-2, glycosylphosphatidylinositol, and C1C2 domain of lactadherin binding with phosphatidylserine can be exploited as anchors for kidney cell marker proteins. Also, because the source, size, and composition of extracellular vesicles can affect their preferential target (107), it is important to ensure which extracellular vesicles preferentially target the kidney.

Extracellular vesicle uptake is a low-yield process (108), and modification with a virus-derived fusogenic protein can significantly improve their cargo delivery efficiency (109). In addition, clathrin-mediated endocytosis increases extracellular vesicle uptake, whereas caveolin-dependent endocytosis is a negative regulator (110). Theoretically, given that LDL is taken up by cells through clathrin-mediated endocytosis, LDL coating may increase extracellular vesicle uptake through the same route and, in turn, magnify cargo delivery. Following uptake, some contents of extracellular vesicles are degraded by the lysosome, whereas some escape degradation by an unclear mechanism (111). Enabling extracellular vesicles to escape postuptake degradation can increase the efficiency of cargo delivery. Increasing extracellular vesicle circulation time can also improve the efficiency of cargo delivery and might be achievable by upregulating CD47 (112).

Challenges and Future Directions in the Study of Extracellular Vesicles

Despite comprehensive studies, extracellular vesicle application as a therapeutic tool has mostly been confined to animal models, with few small clinical trials published, possibly because their production cost, sensitivity, and specificity have yet to be validated against benchmarks, like biopsies or imaging modalities, in large clinical studies. Standardization of extracellular vesicle production and characterization is paramount to establish irrefutable confidence in their ability to deliver consistent regenerative effects (113). Improving methods of extracellular vesicle, isolation, and cargo detection is critical because they remain relatively expensive and complex. Randomized controlled clinical trials are needed to verify their efficacy and safety in kidney diseases and compare extracellular vesicles harvested from different sources. The administration route and dose of extracellular vesicles also affect their distribution. Intra-arterial or intrarenal injection has been the main route of extracellular vesicle delivery to decrease lung and liver uptake, but specific targeting may permit systemic delivery in kidney diseases. A dose of 100 μg/kg (protein per body weight) is effective for umbilical cord mesenchymal stem cell–derived extracellular vesicles to ameliorate CKD in patients (63). Similarly, delivery of 10¹¹ particles of mesenchymal stem cell–derived extracellular vesicles reduces pig ischemic kidney damage (114), and the amount obtained from 10⁶ to 10⁷ mesenchymal stem cells per kilogram of body weight reduce graft-versus-host disease in patients (115). However, their dose-effect relationships should be determined in uniform units, and the source, scalability, and regimen of extracellular vesicles should be optimized. Moreover, although large-scale cell culture systems (e.g., stirred tank bioreactors) can be used for mass production of extracellular vesicles, stem cells cannot expand indefinitely, confounding their clinical value as sources for extracellular vesicles. iPSCs characterized by continuous renewal might be an ideal source for extracellular vesicles, but supporting animal and clinical studies are needed. Engineered retrofitting may further improve extracellular vesicle retention and decrease off-target effects, decreasing their dose and cost (116). Such new promising directions to enhance efficacy and stability may paint a brighter future for engineered extracellular vesicles. Confidence in the diagnostic validity of their cargo would be enhanced by linking it mechanistically to pathophysiologic processes in specific diseases and by reproducing findings at independent laboratories. This would also require strict standardization of harvesting, characterization, and delivery regimens.

Furthermore, although extracellular vesicles are conventionally assumed to provoke little immune response (66) relative to their parent cells, this notion has not been rigorously tested. In particular, extracellular vesicles derived from nonimmune-privileged cells, like tubular epithelial cells, might well maintain their immunogenicity (117), warranting caution during in vivo applications.

In conclusion, extracellular vesicles bear an exciting promise and might evolve to constitute efficacious and versatile off-the-shelf biologic products. Additional studies are needed to standardize and increase the production; validate efficacy; and optimize targeted homing, local retention, and therapeutic cargo of these novel particles. Hopefully, such studies would establish the feasibility of these particles as a useful theranostic tool in kidney disease.

Disclosures

W. Huang is supported by China scholarship council and reports research funding from the National Natural Science Foundation of China and honoraria from Dongzhimen Hospital. A. Lerman reports consultancy agreements with AstraZeneca, Itamar Medical, Phillips, and Shahal Telecommunication and serves as an advisor to CureSpec. L.O. Lerman reports consultancy agreements with AstraZeneca, Butterfly Biosciences, and CureSpec; research funding from AstraZeneca; honoraria from AstraZeneca, Butterfly Biosciences, and CureSpec; patents or royalties from Cohbar and Stealth Biopharmaceuticals; serving in an advisory or leadership role with AstraZeneca and CureSpec; and other interests or relationships with the American Heart Association and the National Institutes of Health. The remaining author has nothing to disclose.

Funding

This study was supported by National Institutes of Health grants DK120292, DK122734, and AG062104.

Author Contributions

L.O. Lerman conceptualized the study; W. Huang was responsible for formal analysis; W. Huang was responsible for methodology; L.O. Lerman was responsible for funding acquisition; L.O. Lerman provided supervision; W. Huang wrote the original draft; and W. Huang, A. Lerman, L.O. Lerman, and X.-Y. Zhu reviewed and edited the manuscript.

Supplemental Material

This article contains the following supplemental material online at http://cjasn.asnjournals.org/lookup/suppl/doi:10.2215/CJN.16751221/-/DCSupplemental.

Supplemental Table 1. Promising biomarkers of kidney disease in urinary EVs.

Supplemental Table 2. Circulating EVs as biomarkers in kidney disease.