Introduction
Extracellular vesicles (EVs) are lipid-bilayered, nanosized vesicles secreted by nearly all cells [1]. Experimental evidence suggests that EVs play a crucial role in intercellular communication, altering the characteristics of recipient cells [2–4]. Notably, EVs can reflect the physiology of their originating cells, rendering them novel markers for various diseases [5–7]. Additionally, studies have been actively conducted on the use of EVs from stem/progenitor cells to treat immunological or degenerative diseases [8,9]. Other research has demonstrated the potential of EVs in mediating the progression of various diseases, including inflammation, apoptosis, fibrosis, and cancer [10–12].
Kidneys play an essential role in the body, including blood filtration, maintenance of fluid/electrolyte and acid/base balance, removal of drug/toxin metabolites, vitamin D synthesis, erythrocyte production, and blood pressure control [13]. The kidneys are composed of multiple cell types, each with distinct roles, including tubular epithelial cells (TECs), glomerular endothelial cells (GECs), peritubular cells, podocytes, mesangial cells, and juxtaglomerular cells [14]. Following filtration, the glomerular filtrate moves along the tubules and undergoes reabsorption, secretion, and dilution/concentration before excretion. During this intra-nephron journey, EVs from TECs play important roles in normal functions, including protein shuttling (e.g., aquaporin 2 [AQP2]), coagulation processes, innate immunity, and regulation of metabolic activities [15]. However, in vivo physiological status changes continuously, making it challenging to fully understand the detailed function of EVs in the glomerular filtrate in vivo [16]. Moreover, EVs secreted from injured renal cells, including podocytes, tubular cells, and interstitial cells, affect other structures within the nephron, leading to kidney disease progression [17,18]. In this review, we explore the functions of EVs in maintaining kidney homeostasis and the progression of kidney diseases. Furthermore, we discuss the current understanding of the function of renal tubular EVs in the pathological progression of kidney diseases. Additional up-to-date information on the key players involved in communication within the kidney is provided in a previous review by Guo et al. [19]. Due to the large amount of study results, we did not include the literatures on the therapeutic role of stem cell-derived EVs for renal diseases [20].
Basic characteristics of extracellular vesicles
EVs are nanosized particles actively synthesized and released by nearly all cells across various kingdoms, including eukaryotes and prokaryotes [21]. In contrast to the old notion that they are merely a means for cells to remove wastes or unwanted contents, recent studies have identified EVs as key players in maintaining homeostasis [22,23]. Moreover, EVs have been increasingly demonstrated to mediate various diseases, including metabolic and cardiovascular diseases, cancer, neurological disorders, and renal diseases [12,24–27]. EVs can reflect the biology of secreting cells and deliver biological messages (e.g., lipids, RNA, and proteins) from these cells to nearby or distant cells, thereby altering the physiology of recipient cells [28–30]. Accordingly, the potential use of EVs for therapeutic purposes, drug delivery, or biomarker development is now under intense investigation [31–33]. Conventionally, EVs are classified as exosomes, microvesicles (MVs), and apoptotic bodies. Exosomes (30–120 nm) originate from multivesicular bodies, which fuse with the plasma membrane, releasing exosomes into the extracellular space [34]. MVs (150–1,000 nm) originate from ectosomes, which protrude and cleave from the plasma membrane [34]. Apoptotic vesicles (100–5,000 nm) are large cytoplasmic fragments produced during apoptotic cell death [34,35]. Detailed mechanisms of their biosynthesis and characteristics are provided in previous reviews [36,37].
Current reports on the role of extracellular vesicle-mediated communication between kidney cells: early proof-of-concept studies
In the kidney, the glomerular compartments mediate blood filtration, whereas the tubular compartments are important for the reabsorption/secretion processes, as well as the concentration of filtrates [38]. EVs released by various renal cells play a crucial role in the communication between different parts of the nephron [11]. The sources of urinary EVs are diverse, including several parts of the nephron, such as glomerular podocytes, proximal/distal tubular cells, Henle’s loop, and the collecting duct. An early report that the proteome contents of EVs isolated from normal human urine are from apical plasma membrane channel and transport proteins, with no expression of basolateral membrane integral membrane proteins. These results indicate that most urinary EVs are derived from the apical plasma membrane, suggesting the potential of the urinary EV for diagnostic uses for tubular diseases [39].
In 2011, Street et al. [40] showed that the expression of AQP2 protein in exosomes derived from collecting duct cells treated with desmopressin (vasopressin analog) was increased. Moreover, AQP2 from exosomes could be delivered to nearby cells, marking the first in vitro demonstration of cell-to-cell communication between renal cells [40]. Additionally, an in vitro study demonstrated that EVs released from proximal tubule cells have been demonstrated to be taken up by distal tubule and collecting duct cells, reducing reactive oxygen species production in recipient cells [41]. A subsequent in vitro study demonstrated that glyceraldehyde-3-phosphate dehydrogenase–expressing exosomes derived from cultured proximal tubular cells are transferred to the distal tubule and collecting duct cells, leading to a decrease in sodium ion reabsorption [42]. However, it may be important to determine to what extent the horizontal biomolecule transfer is mediated by EVs. For example, non-EV-mediated RNA shuttling in nephron can also occur; in a podocyte-specific uracil phosphoribosyl transferase-expressing mice, the podocyte-specific RNA was detected in the tubular cells upon being injected with 4-thiouracil, which enables the T>C detection after RNA sequencing. It would be interesting to further examine whether the transcripts from podocyte-derived EVs can be incorporated into the TECs, at least in vitro [43].
Functions of extracellular vesicles in renal pathophysiology
Extracellular vesicles from glomerular endothelial and mesangial cells
Prolonged hyperglycemia can induce macro- or microvascular complications in the glomerulus, ultimately leading to chronic kidney disease (CKD). Consequently, several studies have focused on the effects of EVs derived from glomerular cells on other downstream tubular compartments (Table 1). For example, microRNA (miRNA)-200c-3p, transferred via EVs secreted from lipopolysaccharide (LPS)- or high glucose (HG)-treated GECs, have been demonstrated to reduce vascular endothelial growth factor expression in podocytes, leading to podocyte dysfunction [44]. Similarly, exosomal transforming growth factor-beta 1 (TGF-β1) messenger RNA released from GECs in diabetic mice impaired the function of mouse podocytes and induced epithelial-to-mesenchymal transition (EMT) by augmenting alpha-smooth muscle actin (α-SMA) and ferroptosis suppressor protein 1 expression while reducing zonula occludens-1 (ZO-1) and Wilms’ tumor-1 expression [45]. Furthermore, EVs derived from glomerular mesangial cells (GMCs) subjected to HG conditions stimulated the protein expression of p-AKT, p-p65, and TGF-β1, ultimately reducing apoptosis in podocytes [46]. A recent study demonstrated that intercellular shuttling of circular (circ) RNAs from GECs cultured under HG conditions can provoke the EMT of GMCs; exosomes from HG-treated GECs suppressed GMC proliferation and increased α-SMA expression. Mechanistic studies demonstrated that these changes were mediated by circRNF169-2 and circSTRN3-2 located in the exosomes of HG-treated GECs [47]. These results suggest that various glomerular cells release EVs, which induce changes in the expression of recipient cells within the glomerulus.
Effect of extracellular vesicles from podocytes on renal tubular cells
Podocyte injury is a major factor in the onset of diabetic kidney disease (DKD). Therefore, studies have investigated the effects of EVs from podocytes cultured under HG conditions on other renal cells (Table 1). For example, EVs from HG-cultured podocytes have been demonstrated to exhibit higher levels of miR-221 than those cultured under low-glucose conditions. MiR-221 in these EVs downregulated dickkopf-related protein 2 expression in tubular cells, leading to a reduction in ZO-1 and e-cadherin (E-CAD) expression. Additionally, the nuclear translocation of β-catenin increased the expression levels of α-SMA and vimentin (VIM). However, in vivo inhibition of miR-221 using an antagomir reduced proximal tubular injury in streptozotocin (STZ)-induced diabetic mice [48]. Another study demonstrated that HG-treated mouse podocytes induced apoptosis in proximal TECs (PTECs). Functional enrichment analysis revealed that five differentially expressed miRNAs (mmu-miR-1981-3p, mmu-miR-3747, mmu-miR-7224-3p, mmu-miR-6538, and mmu-miR-let-7f-2-3p) from these podocytes were enriched in pathways related to DKD progression [49].
The role of podocyte-derived microparticles in the mesenchymal transition of tubular cells has also been demonstrated. For example, it was shown that microparticles from podocytes, as evidenced by podocalyxin-positive nano-size particles by flow cytometry, were increased in both STZ-treated and Akita mice before the onset of albuminuria [50]. Few years later, the same group revealed that microparticles released from a human podocyte cell line increased p38 and Smad3 phosphorylation in human PTECs. Additionally, these EVs increased the expression of extracellular matrix (ECM) proteins such as fibronectin and collagen type IV (COL4) in PTECs, resulting in a profibrotic response in vitro [51]. Puromycin aminonucleosides (PAN) are widely used to induce experimental nephrotic syndrome both in vitro and in vivo. Jeon et al. [52] demonstrated that EVs from PAN-treated human podocytes induced apoptosis and increased the levels of cleaved poly(ADP-ribose) polymerase, phosphorylated extracellular signal-regulated kinase, p-p38, fibronectin, and COL4 in renal TECs. Notably, two upregulated miRNAs (miR-149, -424) have been demonstrated to induce the apoptosis of HK-2 cells [52]. These results indicate that podocyte-driven EVs can trigger fibrotic changes and apoptosis in TECs.
Extracellular vesicles from renal tubular cells
TECs are the most widely used source of EVs, and several studies have explored the interactions between TEC-derived EVs and other renal cells (Table 2). Recent studies have demonstrated that EVs released by injured TECs induce phenotypic changes in other cells and promote disease progression (Fig. 1). For example, EVs derived from TGF-β1–treated renal TECs (NRK-52E) have been demonstrated to stimulate the expression of fibroblast markers α-SMA and fibronectin in renal fibroblasts (NRK-49F) via miR-21 in vitro. Similarly, the expression of fibrotic markers was observed after these EVs were injected into a mouse model of renal fibrosis (unilateral ureteral obstruction, UUO) [53]. Another study demonstrated that EVs derived from TGF-β1–treated human renal epithelial cells (HK-2) decreased apoptotic death in renal interstitial fibroblasts while stimulating fibrotic changes in the renal parenchyma of UUO mice. Specifically, the study revealed that exosomal tumor necrosis factor-alpha (TNF-α)-induced protein 8 (TNFAIP8) enhanced p53 ubiquitination, which inhibited apoptosis and induced the proliferation of renal interstitial fibroblasts [54].
Hyperglycemia can contribute to fibrotic diseases by affecting not only the glomerulus but also renal TECs [55]. Tsai et al. [56] demonstrated that EVs from HK-2 cells cultured in an HG medium induced the transformation of mesangial cells into myofibroblasts. Subsequent analyses revealed that exosomal miR-92a-1-5p was responsible for this change. Moreover, diabetic nephropathy was induced in mice injected with these EVs [56]. Another study suggested that EVs secreted from mouse proximal TECs cultured under HG conditions not only altered cell morphology but also increased the expression of fibroblast markers such as fibronectin, α-SMA, and COL4 in renal fibroblasts (NRK-49F) [57].
Macrophage polarization is also affected by the release of EVs from injured TECs. For example, EVs from TGF-β–treated TECs have been demonstrated to induce the expression of inducible nitric oxide synthase (iNOS), α-SMA, TNF-α, and interleukin (IL)-10 in Raw 264.7 macrophages, leading to their polarization into the M1 type [58]. Another study revealed that the expression of inflammatory cytokines, including TNF-α and IL-6, was increased in Raw 264.7 macrophages co-cultured with EVs secreted from bovine serum albumin-treated mouse TECs. Notably, chemokine (C-C motif) ligand 2 and miR-19b-3p within these EVs were identified as the key contributors to renal inflammation caused by M1-polarized macrophages [59,60].
Prolonged hypoxia induces various renal injuries, with proximal tubular cells being susceptible to hypoxic injury. For example, miR-374b-5p from the exosomes of hypoxic TECs has been demonstrated to promote M1 polarization in Raw 264.7 cells. In a mouse model of renal ischemia-reperfusion, injury was more severe in mice receiving these EVs compared to those receiving EVs from normoxic TECs. Furthermore, miR-374b-5p blockade in mice subjected to ischemia/reperfusion injury improved kidney injury by reducing the expression of proinflammatory cytokines and inhibiting M1-polarization [61]. Another study revealed that forced expression of HIF-1α in TECs led to the shedding of miRNA-23a–enriched exosomes in vivo, which contributed to tubulointerstitial inflammation after ischemic injury. These exosomes promoted the expression of inflammatory markers, such as TNF-α and IL-β1, in Raw 264.7 macrophages, with miR-23a inhibition disrupting these changes. Furthermore, healthy mice injected with these EVs exhibited increased interstitial damage macrophage infiltration [62]. Another study demonstrated that renal fibrosis induced by unilateral ischemia-reperfusion injury worsened upon injection with miR-150-5p–enriched exosomes secreted from hypoxia-cultured TECs (NRK-52E); however, these changes were reversed by miR-150-5p–deficient exosomes. Mechanistically, miR-150-5p regulates suppressor of cytokine signaling 1, which negatively regulates the JAK/STAT pathway, leading to an increase in proinflammatory factors, cell apoptosis, and renal fibrosis [63,64]. These studies showed that TEC-derived EVs have mainly focused on the effects of EVs derived from TECs undergoing hypoxic injury. In vitro studies have shown activation of fibroblasts, upregulation of fibrosis markers, and polarization of macrophages towards a proinflammatory M1 phenotype. Similarly, in vivo, increased inflammation and fibrosis have been observed when TEC-derived EVs are injected into acute kidney injury (AKI), CKD models. In contrast these studies (where hypoxia was given for more than 24 hours), it was reported that short-term ischemic preconditioning (4 hours) of TEC reduced fibrosis followed by renal ischemia/reperfusion injury [65]. The underlying mechanism of this protective mechanism is unknown, but is likely to be due to altered characteristics of TECs by hypoxic conditioning, which had widely used for potentiating the therapeutic effect reported in mesenchymal stem cells (MSCs) [66]. Collectively, these findings suggest that TEC-derived EVs affect the progression of renal diseases.
Extracellular vesicles from other cells
Although located outside the nephron, EVs from blood cells or kidney-resident non-epithelial cells can contribute to kidney diseases [12] (Table 3). Fibroblasts in the kidneys are critical for maintaining organ architecture through ECM homeostasis, water and electrolyte control, and erythropoietin production [67]. These cells express immune receptors, including Toll-like receptors, indicating that they can respond to various injurious stimuli, such as damage-associated molecular patterns [68,69]. Persistent inflammation or fibrotic signals can activate renal fibroblasts into myofibroblasts, a major cell type responsible for excessive ECM production, leading to maladaptive tubular repair and interstitial fibrosis [70]. Zhou et al. [71] demonstrated that the number of MVs from TGF-β1–treated renal fibroblasts was higher than that from untreated cells, with miR-34a being enriched in these MVs. Notably, treatment with these MVs increased tubular cell death, and this apoptotic effect was mediated by miR-34a in vitro. Furthermore, these MVs migrated to the nephron via a disrupted tubular basement membrane [71,72]. These findings indicate that fibrotic signals can activate the fibroblast-myofibroblast transition, which subsequently triggers tubular cell death, tubulointerstitial fibrosis, and decreased renal function.
Endothelial colony-forming cells and myeloid angiogenic cells are proangiogenic hematopoietic cells derived from peripheral blood-derived mononuclear cells [73]. Before this statement report, Cantaluppi et al. [74] demonstrated that MVs from blood-derived undefined pro-angiogenic cells (named ‘endothelial progenitor cells [EPCs]’ in this literature) alleviated chronic renal injury after ischemia-reperfusion injury in mice, as evidenced by improved renal function, reduced apoptosis, and decreased immune cell infiltration. Further analysis revealed that these effects were mediated by miR-126 and miR-296 within the MVs, indicating that the protective role of EPCs is associated with the delivery of RNA contents [74].
Macrophages play a crucial role in tissue repair and injury. Tang et al. [75] demonstrated that EVs from IL-10–transfected Raw 264.7 cells, which were subsequently differentiated into M2 macrophages, improved renal tubular injury and inflammation after ischemia-reperfusion injury and CKD transition. This improvement was linked to the suppression of mTOR signaling and the enhancement of mitochondrial function through mitophagy activation in TECs. Furthermore, IL-10+ EVs increased CD206 expression while reducing CD68 levels, indicating their role in driving M2 polarization in the injured kidney [75]. Another study demonstrated that exosomal miR-25-3p secreted by M2 macrophages improved HG-induced podocyte injury, evidenced by increased autophagic activity and higher levels of placental cadherin, ZO-1, and E-CAD, alongside reduced levels of VIM and α-SMA. The same study revealed that exosomal miR-25-5p suppressed the expression of dual specificity phosphatase 1, which promoted podocyte autophagy [76]. Another study demonstrated that EVs derived from macrophages under high hyperglycemic conditions stimulated mouse podocyte apoptosis via miR-25-3p and miR-21a-5p [77]. Similarly, exosomes released from M2 macrophages were enriched with miR-93-5p, targeting the Toll-like receptor 4 pathway, thereby suppressing apoptosis in LPS-induced mouse podocytes [78]. In addition, urinary EVs carrying klotho from healthy individuals demonstrated renoprotective effects, as indicated by improved renal function and reduced expression of neutrophil gelatinase-associated lipocalin (NGAL) and plasminogen activator inhibitor in the kidneys of rhabdomyolysis-induced AKI mice receiving these EVs [79].
The potential of renal extracellular vesicles for clinical application
Since the molecular profile of EVs can represent the traits of the parental cells, EVs hold tremendous potential for biomarker development in various conditions such as AKI, CKD, immunoglobulin A nephropathy, graft function and kidney transplantation, and nephrotic syndrome [80]. Conventional methods for diagnosing renal function include serum creatinine level, urine output, and glomerular filtration rate. However, these parameters can be affected by other factors, and they may not be suitable for early detection of AKI, CKD, and graft rejection after kidney transplantation. Importantly, changes in the biomolecular profile in urinary EVs can occur before creatinine level changes, which makes early diagnosis possible [12]. Moreover, EVs can be easily obtained routinely in noninvasive way, and their contents remain stable even under unfavorable conditions (high/low pH, freeze-thaw cycles, cryopreservation, etc.). Thus, their basic characteristics (numbers and size) and biomolecular contents made EV an ideal material for biomarker development.
For example, Lange et al. [81] has shown that miR-21 is highly expressed in EVs isolated from the urine of CKD patients, correlating with podocyte damage. In type 2 diabetes mellitus, miR-192, an early biomarker for diabetic nephropathy, is significantly upregulated in urinary EVs from patients with microalbuminuria [82]. Another study found that miRNA-181a, a promising biomarker for early diagnosis, is expressed at lower levels in urinary exosomes from CKD patients compared to healthy individuals [83]. In kidney transplantation, detecting or predicting graft rejection is of critical importance to reduce graft loss. It was shown that the proteins in urinary exosomes were superior to those from whole urine in predicting acute rejection. Specifically, eleven proteins were functionally involved in stress response and inflammation, among which three were exclusively detected in urinary exosome [84]. Another study demonstrated that the protein expression of NGAL, one of the markers for delayed graft function (DGF) after KT, was higher in the urinary exosomes of DGF patients compared with non-DGF patients [85]. These ongoing studies into urinary EV biomarkers in kidney disease could greatly enhance the clinical utility of urine tests for disease diagnosis and monitoring.
Controlling EV release or uptake has been proposed as a novel therapeutic approach. For example, the kinin system is essential during vasculitis as well as chronic inflammation [86]. One study observed that a higher number of B1 receptor (kinin receptor)-positive MVs are released from in vitro cultured GECs upon stimulation with the plasma from acute vasculitis. Importantly, blocking the kinin system by a C1 inhibitor reduced the release of B1 receptor-carrying MVs, highlighting its potential in mitigating neutrophil-driven inflammation by MVs [87]. In another study, Liu et al. [88] showed that blocking tubular exosome secretion in UUO mice prevented renal fibrosis, providing strong evidence that early intervention approach by blocking exosome secretion during acute injury could be another stragety for fibrotic diseases. While further research is needed to determine whether modulating EV release and uptake directly impacts the progression of kidney disease, these findings suggest that EVs may serve as promising therapeutic targets. Studies on the development of therapeutics and drug delivery tools utilizing engineered or native EVs are also under extensive research [89].
Despite significant research efforts, several obstacles remain in the clinical application of EVs. Most of all, standardized guidelines for collection, storing, and documenting plasma or urinary EVs should be established. Also, the criteria for selecting biomarker sets should be critically determined based on the large amount of data combined with clinical information. For therapeutic uses, quality controlling of EVs (e.g., purity, concentration, stability, and pharmaceutical activity of active biomolecular cargo, etc.), determination of dosage and administration, efficacy as well as safety study should be critically performed and evaluated according to the regulatory policies. Lastly, developing efficient manufacturing protocols is essential for the consistent and reliable production of high-quality EVs.
Future directions
Although it is becoming evident that EVs play an important role in normal kidney function and the progression of renal diseases, their specific roles in the kidney remain largely unexplored. Understanding how various contexts—such as hypoxia, hyperglycemia, hypertension, nephrotoxins, or nutrient deprivation—affect the physicochemical properties of EVs (e.g., types and sizes) in specific cell types is imperative [36]. Next, the molecular components of renal EVs, such as lipids, proteins, and nucleic acids, and their functions in target cells should be determined. With the advancement of high-throughput multiomic tools, efforts aimed at determining the molecular functions, including cell death, proliferation, inflammation, fibrosis, senescence, and autophagy, associated with the biomolecular fingerprints within EVs are expected to increase. Although not fully covered in this review, the identification of early biomarkers in urinary EVs for renal diseases is expanding. As these EVs reflect the status of kidney malfunction, molecular profiling of urinary EVs can serve as a starting point for investigating how EVs function in various complications as well as developing early biomarkers. It should be noted that a large part of the studies discussed in this review are based on in vitro or animal studies, and further studies are warranted to translate into clinical findings.
Additionally, the potential of stem/progenitor cell-derived EVs for therapeutic purposes has well been documented. Since the first report on the protective role of MVs from human bone marrow-derived mesenchymal stem cells in AKI, it is now becoming evident that the therapeutic effect of MSCs is largely derived from their paracrine actions as well as secreted EVs, not by direct replacement of damaged cells [90,91]. Since these two studies, the renoprotective role of EVs from various cultured cells including MSCs, iPSCs, liver stem cells, macrophages, urinary stem cells, TECs, neutrophils, as well as non-cultured sources (red blood cells, urine) has been reported in various diseases (AKI, CKD, diabetic nephropathy) [9,92]. However, the development of scalable and standardized protocols for generating, storing, functional testing, and delivery methods of EVs are needed [93,94].
Conclusions
EVs are released from all nephronal compartments and mediate cell-to-cell communication within the luminal space. The molecular profile of EVs often reflects the characteristics of their originating cells, rendering them valuable for developing diagnostic markers. In addition, EVs from upstream nephron segments of the nephron can be transported to downstream cells, delivering biological molecules and altering the physiology of recipient cells. This intra-nephron communication is crucial for both normal physiology and disease progression in the kidney. Over the next decade, in-depth studies on the function of the biomolecular profiles of EVs from different nephron segments will enhance the understanding of how initial results can be amplified. Ultimately, identifying specific biomolecules within EVs may provide us insight into how renal diseases are propagated, and this information will contribute to development of early biomarkers for various renal diseases.
PDF Links
PubReader
ePub Link
XML Download
Full text via DOI
Download Citation
Print
