Mesenchymal stem cell-derived extracellular vesicles for kidney repair: current status and looming challenges

Novel therapies are urgently needed to address the rising incidence and prevalence of acute kidney injury (AKI) and chronic kidney disease (CKD). Mesenchymal stem/stromal cells (MSCs) have shown promising results in experimental AKI and CKD, and have been used in the clinic for more than a decade with an excellent safety profile. The regenerative effects of MSCs do not rely on their differentiation and ability to replace damaged tissues, but are primarily mediated by the paracrine release of factors, including extracellular vesicles (EVs), composed of microvesicles and exosomes. MSC-derived EVs contain genetic and protein material that upon transferring to recipient cells can activate several repair mechanisms to ameliorate renal injury. Recent studies have shown that MSC-derived EV therapy improved renal outcomes in several animal models of AKI and CKD, including ischemia-reperfusion injury, drug/toxin-induced nephropathy, renovascular disease, ureteral obstruction, and subtotal nephrectomy. However, data about the renoprotective effects of EV therapy in patients with renal failure are scarce. This review summarizes current knowledge of MSC-derived EV therapy in experimental AKI and CKD, and discusses the challenges that need to be addressed in order to consider MSC-derived EVs as a realistic clinical tool to treat patients with these conditions.


Background
Kidney disease is a prominent challenge for health care systems. Incidence and mortality rates of both acute kidney injury (AKI) and chronic kidney disease (CKD) have increased in recent decades [1]. It is estimated that during a hospital admission one in five adults and one in three children experience AKI, a sudden episode of kidney failure or kidney damage [2]. CKD, a condition characterized by a gradual loss of kidney function, is estimated to be quite prevalent. In the US alone, its predicted prevalence rate is 13.6%, with more than 670,000 patients in end-stage renal disease (ESRD) [3,4], the final stage of CKD when irreversible loss of renal function mandates dialysis or kidney transplantation. Both AKI and CKD consume considerable healthcare resources and are associated with significant economic costs. AKI is responsible for more than 5% of overall hospital expenses [5], and more than $80 billion of the Medicare budget is spent to care for CKD and ESRD patients, accounting for over 18% of its total expenditure [4,6]. AKI can cause ESRD directly, and increase the risk of developing CKD and worsening of underlying CKD [7]. Importantly, AKI and CKD are risk factors for developing cardiovascular disease and mortality [8]. Therefore, the rising incidence and prevalence of AKI and CKD and their deleterious complications underscore the need to identify more effective therapeutic strategies to attenuate renal injury and prevent its progression to ESRD.
Mesenchymal stem/stromal cells (MSCs) are multipotent cells with robust self-renewal, regenerative, proliferative, and multi-lineage differentiation potential [9]. By definition, MSCs are characterized by the expression of MSC markers and the ability to differentiate into adipocytes, chondrocytes, and osteocytes [10]. Emerging evidence supports the existence of kidney-resident MSCs, which originate from renal pericytes that form an extensive network around the microvasculature [11]. Although the entire spectrum of their function still remains to be elucidated, they play key roles in regulation of renal blood flow, capillary permeability, endothelial survival, and immunologic surveillance [12]. In addition, MSCs with potent proangiogenic and immunomodulatory properties can also be isolated from various extrarenal sources, including adipose tissue, making them ideal candidates for renal regenerative therapy [13,14].
According to ClinicalTrials.gov there are currently 46 ongoing or completed clinical trials using MSC therapy for AKI and CKD, including diabetic nephropathy, focal segmental glomerulosclerosis, systemic lupus erythematous, and kidney transplantation [15][16][17] (Table 1). In an ongoing phase I clinical trial, patients with cisplatininduced AKI and solid organ cancer are followed for 1 month after a single systemic infusion of allogeneic bone marrow-derived MSCs (NCT01275612). Primary and secondary end points include the rate of decline in renal function and urinary injury markers, respectively. Cardiac surgery patients at high risk of postoperative AKI were treated safely with allogeneic MSCs [18,19]. Systemic administration of autologous bone marrowderived MSCs in patients with autosomal dominant polycystic kidney disease did not cause any serious adverse events and decreased serum creatinine levels after 12 months of follow-up [20]. Preliminary results of a randomized clinical trial in patients with diabetic nephropathy also showed stabilized or improved glomerular filtration rate (GFR) after 3 months of treatment with allogenic MSCs [21]. Likewise, intra-arterial infusion of autologous MSCs in patients with renovascular disease (RVD) increased cortical perfusion and renal blood flow (RBF), and reduced renal tissue hypoxia in the poststenotic kidney [22]. Clinical trials are also testing the immunomodulatory and renoprotective properties of MSCs after renal transplantation (NCT02409940). Autologous MSCs were found to be superior to conventional immunosuppressive therapy in preventing acute rejection, decreasing opportunistic infections, and preserving renal function in patients undergoing renal transplant [23]. Taken together, these studies indicate that MSC therapy is safe, feasible, well tolerated, and effectively ameliorates renal pathology in a wide range of diseases.
Mounting evidence supports the notion that MSCs exert their reparative effects by releasing extracellular vesicles (EVs), including exosomes with a diameter of 30-120 nm, and micro-vesicles ranging from 100 nm to 1 μm in size [24]. Exosomes arise form endocytic compartments, known as microvesicular bodies, and are released into extracellular space through fusion with plasma membrane [25]. In contrast, microvesicles originate from outward buddings of cell membrane and their release is controlled by calcium influx and cytoskeletal reorganization, among several other factors [25]. We have previously shown that porcine MSCs release EVs ( Fig. 1) that are selectively packed with proteins, mRNAs, and microRNAs [26][27][28]. Furthermore, we recently proposed that genes, proteins, and microRNAs enriched in EVs have the potential to modulate selective cellular pathways in recipient cells [29]. Therefore, MSC-derived EVs may exert trophic and reparative effects, representing an attractive non-cellular approach for treating renal disease. Indeed, recent studies have shown that delivery of MSC-derived EVs is safe and can improve kidney function in several models of AKI and CKD. The purpose of this review is to summarize the current knowledge of MSC-derived EV therapy in experimental AKI and CKD, and discusses the challenges

Ischemia-reperfusion injury
Renal ischemia-reperfusion injury (IRI), a condition caused by initial sudden cessation of blood flow to the kidney followed by restoration of blood flow and reoxygenation, is one of the primary causes of AKI associated with significant morbidity and mortality [30]. Although the pathophysiology of renal IRI remains obscure, both hypoxia at ischemic phase and subsequent generation of reactive oxygen species at reperfusion initiate a cascade of deleterious responses characterized by inflammation and cell death that subsequently leads to AKI [31]. A number of studies have recently tested the efficacy of MSC-derived EVs to blunt experimental IRI-induced AKI ( Table 2). Lindoso et al. [32] tested the biological effect of EVs in an in vitro model of renal IRI induced by ATP depletion of tubular cells, which were subsequently co-incubated with MSC-derived EVs. EVs progressively incorporated into damaged tubular cells, suggesting higher uptake under stressful conditions. EVs decreased cell death and restored proliferation of ATP-depleted tubular cells. This was paralleled with downregulated expression of a specific set of microRNAs involved in apoptosis, hypoxia, and cytoskeletal reorganization, suggesting that EVs can protect tubular cells against metabolic stress by mechanisms involving post-transcriptional regulation. The renoprotective effects of MSC-derived EVs have also been investigated in several in vivo models of renal IRI. In rats subjected to unilateral nephrectomy and renal artery occlusion for 45 min, intravenous MSC-derived EVs immediately after ischemia significantly reduced epithelial tubular cell damage and apoptosis and enhanced their proliferation, improving renal function [33]. Interestingly, the beneficial effect of EVs was mediated in part by the transfer of RNA-based information to recipient cells. Similarly, in rats with renal IRI systemic administration of autologous bone marrow MSC-derived EVs decreased renal injury and improved function, extending the benefits of EVs to ameliorate IRI-induced renal damage and contribute to cellular repair in vivo [34].
EVs harvested from human umbilical cord MSCs have also shown renoprotective benefits in rats with IRI. Intravenous delivery of EVs immediately after the ischemic phase of IRI mitigated renal oxidative damage by decreasing the expression of the pro-oxidant NADPH oxidase-2 [35]. MSC-derived EV-induced attenuation of renal oxidative stress was associated with enhanced renal cell proliferation, decreased apoptosis, and normalized serum creatinine levels 2 weeks after the ischemic insult. Consistent with these findings, intravenous injection of EVs isolated from the conditioned medium of human umbilical cord MSCs after unilateral renal ischemia preserved kidney function and decreased serum levels of the AKI marker neutrophil gelatinase-associated lipocalin [36]. EVs also decreased renal expression of nuclear factor E2-related factor-2, a transcription factor that modulates cellular oxidative stress, which in turn resulted in decreased tubular damage.
Studies in experimental renal IRI have also shown that MSC-derived EVs exert renoprotection by modulating renal angiogenesis. Systemic administration of MSC-derived EVs in rats with renal IRI increased renal capillary density and reduced fibrosis by direct transfer of the proangiogenic factor vascular endothelial growth factor (VEGF) and mRNAs involved in this process [37]. In a similar study, delivery of EVs in rats with IRI increased gene and protein expression of the proangiogenic hepatocyte growth factor, associated with decreased tubular fibrosis [38]. Interestingly, the renoprotective effects of EVs were abolished when EVs were pretreated with RNase, implying that mRNA transfer of proangiogenic AKI acute kidney injury, EV extracellular vesicle, IRI ischemia-reperfusion injury, MSC mesenchymal stem cell, VEGF vascular endothelial growth factor factors mediated EV-induced renal repair. The proangiogenic effects of EVs were not limited to those isolated from umbilical cord MSCs. EVs isolated from kidney resident MSCs have been shown to contain several proangiogenic genes, including VEGF, basic fibroblast growth factor, and insulin-like growth factor (IGF)-1 [39]. Systemic administration of allogeneic kidneyresident MSC-derived EVs into mice with renal IRI was followed by engraftment in ischemic kidneys and improvement in renal function, suggesting that delivery of proangiogenic transcripts may contribute to EV-induced renal repair. Furthermore, administration of MSC-derived EVs has been proved to ameliorate the inflammation that follows IRI. Intravenous delivery of EVs following unilateral renal ischemia in rats decreased the number of kidney macrophages and the expression of the macrophage chemo-attractant factor chemokine C-X-C motif ligand-1 (CXCL1), possibly by transferring into recipient cells microRNAs capable of modulating CXCL1 expression [40]. This treatment boosted tubular proliferation, attenuated fibrosis, and preserved kidney function. Likewise, in rats with IRI induced by bilateral renal artery occlusion and reperfusion, treatment with intravenous MSCs or their EV progeny decreased expression of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1-β [41]. Combined MSC and MSC-derived EV therapy resulted in an additive effect on amelioration of tubular injury, extending their value to preserve the kidney when delivered in conjunction with MSCs.
MSC-derived EVs may also confer protection against IRI that occurs in kidney donation after circulatory death, preserving renal function prior to kidney transplantation. In a recent study, incubation of donated kidneys with EVs in buffering solution after harvest and prior to transplant decreased ischemic damage by altering the expression of genes encoding enzymes known to improve cell energy metabolism and ion transport [42]. However, it remains to be determined whether the renoprotective effect of MSC-derived EVs is confined to a specific cell type or may prolong graft survival after kidney transplantation. Therefore, these studies suggest that the beneficial effect of MSC-derived EVs in renal IRI is attributable to their antioxidant, immunomodulatory, and proangiogenic properties, and their ability to modulate cell metabolism and several cellular pathways.

Drug-induced nephropathy
Drug-induced nephropathy (DIN) is a common etiology of AKI that accounts for as high as 60% of both community-and hospital-acquired episodes [43]. Non-steroidal anti-inflammatory drugs, antibiotics, angiotensin converting enzyme inhibitors, and contrast agents have been associated with renal cell toxicity, and may compromise renal function by promoting tubulo-interstitial nephritis and altering intra-glomerular hemodynamics [44]. Recently, the efficacy of MSC-derived EVs has been tested in models of DIN (Table 3). Co-incubation of cisplatin-damaged tubular cells with MSC-derived EVs increased cell proliferation, partly by transferring IGF-1 and IGF receptor-1 [45]. These observations were supported by in vivo studies in animal models of DIN, in which delivery of MSC-derived EVs prevented tubular cell death and enhanced proliferation. For example, administration of MSC-derived EVs into the renal capsule of rats with cisplatin-induced AKI attenuated renal injury and dysfunction partly by reducing formation of pro-oxidants and suppressing activation of pro-apoptotic pathways [46]. Likewise, in mice after cisplatin-induced [47] and glycerol-induced AKI [48,49] single and multiple intravenous administration of MSC-derived EVs ameliorated tubular injury and improved kidney function. Modulation of apoptosis was implicated in EV-induced renoprotection, which was abolished after degradation of EV mRNA content, suggesting that anti-apoptotic genes shuttled by EVs are the final effectors of their biologic actions.
Modulation of renal inflammation is an important mechanism by which MSC-derived EVs protect the kidney from toxic drug injury. In rats with gentamycininduced AKI, EV delivery preserved renal function by preventing the rise in several pro-inflammatory cytokines, including IL-6 and TNF-α, whereas levels of the anti-inflammatory cytokine IL-10 were restored in EV-treated animals [50]. In line with this observation, in mice with glycerol-induced AKI, EV delivery was associated with downregulation of pro-inflammatory genes [51]. However, these studies did not explore whether renal parenchymal or infiltrating inflammatory cells were direct targets of the immunomodulatory effects of EVs. Interestingly, both studies reported that renoprotective effects of MSC-derived EVs were blunted in mice treated with RNA depleted EVs, suggesting an important role for mRNA and/or microRNA shuttling in mediating EV-induced renal recovery after AKI. In line with this notion, a recent study suggested that the anti-apoptotic and immunomodulatory effects of MSC-derived EVs in DIN-AKI are partly mediated by their ability to transfer genes that activate autophagy [52]. Authors found that administration of MSC-derived EVs in the renal capsule of rats with cisplatin-induced AKI increased renal expression of several autophagy-related genes and improved renal function. Taken together, these results indicate that EVs are capable of modulating several pathways involved in the pathogenesis of DIN, and may serve as a novel therapeutic approach in these patients.

Renovascular disease
Renovascular disease (RVD) is an important cause of secondary hypertension and ESRD in the elderly population [53]. RVD frequently coexists with metabolic syndrome (MetS), a constellation of cardiovascular risk factors that accentuates renal injury and is associated with poor renal outcomes [54]. Recently, our group took advantage of a novel porcine model of coexisting MetS and RVD (MetS + RVD) to test whether intrarenal delivery of autologous MSC-derived EVs would ameliorate structural and functional decline in MetS + RVD kidney [55]. MetS was induced by feeding pigs a high fat/high fructose diet for 16 weeks, whereas RVD was achieved by placing an irritant coil in the main renal artery. We found that a single intrarenal administration of MSC-derived EVs in these pigs attenuated renal inflammation, disclosed by decreased renal vein levels of several pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1-β. Contrarily, renal vein levels of IL-10 increased in EV-treated pigs, associated with a shift from pro-inflammatory to reparative macrophages populating the stenotic kidney, underscoring the immunomodulatory potential of EVs. EVs also improved medullary oxygenation and fibrosis, and restored RBF and GFR, yet animals treated with IL-10 knock-down EVs showed limited renal recovery, implying that this cytokine mediates at least part of their protective effects (Table 4).

Unilateral ureteral obstruction
Although complete ureteral obstruction is not a common cause of human renal disease, the unilateral ureteral obstruction (UUO) model, which promotes renal  CKD chronic kidney disease, EV extracellular vesicle, MSC mesenchymal stem cell, RVD renovascular disease parenchymal inflammation, apoptosis, and fibrosis, offers a unique opportunity to study mechanisms responsible for kidney injury [56]. Lately, studies in mouse models of UUO achieved by unilateral ureteral ligation have tested the efficacy of MSC-derived EVs in preventing renal injury (Table 5). Intravenous administration of MSC-derived EVs mitigated tubular injury and fibrosis and improved renal function 2 weeks after UUO [57]. EVs transferred micro-RNAs capable of modulating fibrosis and epithelial to mesenchymal transition (EMT). In agreement, in vitro experiments in tubular cells treated with the pro-fibrotic transforming growth factor (TGF)-β1 showed that coincubation with kidney-resident MSC-derived EVs reversed EMT and TGF-β1-induced morphological changes. This mechanism was also confirmed by another study on TGF-β1-treated endothelial cells, in which MSCderived EVs ameliorated endothelial to mesenchymal transformation and improved cell proliferation 7 days after UUO [58]. Therefore, these studies underscore important anti-fibrotic and renoprotective properties of MSCderived EVs in experimental UUO.

Subtotal nephrectomy
The renoprotective effects of MSC-derived EVs were also studied in a mouse model of subtotal nephrectomy (STN; Table 6), one of the most widely used experimental models of CKD which is characterized by progressive loss of renal mass and deteriorating renal function [59]. STN was induced by removing one kidney and resecting 5/6 of upper and lower poles of the remaining kidney. Delivery of EVs into the mouse caudal vein 2 days after STN mitigated lymphocyte infiltration and prevented tubular atrophy and fibrosis within 1 week after treatment [60]. Decreased proteinuria, serum creatinine, blood urea nitrogen (BUN), and uric acid levels underscored the potential of MSC-derived EV delivery in preserving the remaining renal function.

Challenges of MSC-derived EV delivery in human CKD
As discussed above, several studies in animal models of AKI and CKD suggest that MSC-derived EVs can effectively preserve renal structure and function. So far, however, only one clinical trial has tested the renoprotective effects of MSC-derived EVs on the progression of CKD [61]. In this phase II/III pilot study, 40 patients with estimated GFR (eGFR) between 15 and 60 ml/min were randomized to receive either placebo or EVs derived from allogenic cord blood MSCs. Patients were treated with two doses of EVs and followed for 12 months. EV therapy improved eGFR, serum creatinine, and BUN levels, as well as urinary albumin/creatinine ratio. Plasma levels of TNF-α decreased, whereas levels of IL-10 increased in EV-treated patients. Renal biopsy findings 3 months after intervention revealed that EV-treated kidneys showed upregulated expression of cell regeneration and differentiation markers. Importantly, participants did not experience any significant adverse events during or after EV therapy throughout the study period. Therefore, this study suggests that MSCderived EV therapy is safe and can ameliorate renal inflammation and improve function in patients with CKD. Nevertheless, future long-term follow-up clinical studies need to confirm the persistence of the beneficial effects of this approach in patients with CKD. Furthermore, significant translational challenges need to be faced before adopting MSC-derived EVs as a useful therapy for AKI and CKD (Table 7). Theoretically, cellfree therapies such as EVs might offer superior advantages over delivery of their parent MSCs in terms of safety. EVs are small particles with no proliferative capacity. Being acellular, EVs should be exempted from adverse effects. Unlike MSCs, EVs can be stored for a long time, allowing their use as "off the shelf" products. Nevertheless, long-term follow-up studies for closely monitoring EVs are needed to determine their safety.
According to recent methodological guidelines [62], several methods could be used to isolate EVs which may impact on EV purity, concentration, morphology, size range, and functional activity [63]. EV handling and storage may also affect their concentration, composition, and function [64]. Therefore, additional studies are needed to test whether renal outcomes vary as a function of EV collection, storage, and CKD chronic kidney disease, EV extracellular vesicle, MSC mesenchymal stem cell, UUO unilateral ureteral obstruction isolation methods, and optimize standard protocols for clinical studies. Few studies have tracked the fate of EVs after systemic in vivo administration, but data from IRI [39] and UUO [58] animal models showed that 24 h after infusion EVs primarily engrafted into the damaged kidney and to a lesser extent in the non-affected kidney [40]. The majority of EVs were taken up by renal tubular epithelial cells (RTECs) and peritubular capillaries [39,58], but some were identified in glomeruli [33]. In our MetS + RVD model, EV retention was higher in post-stenotic kidney than contralateral kidneys, and EVs engrafted tubular cells and macrophages 4 weeks after administration [55]. This suggests enhanced tissue uptake of EVs under stressful conditions, which may be mediated by infiltrated immune cells or altered expression of surface markers on parenchymal cells. EVs were also observed in the heart, and in large quantities in the lungs, liver, and spleen. Development of kidney-targeted EVs can facilitate their systemic delivery and enhance their regenerative benefits.
The duration and long-term term effects of MSCderived EVs are important to consider before moving towards their clinical application. In most experimental studies, follow-up ranged from 1 day to 2 weeks postinjection, and only one study in rats with renal IRI found a lower incidence of CKD 6 months after EV therapy [33]. It is clear that EVs can alter transcription profiles in recipient cells, and modulate tissue metabolism and several cellular pathways. Thus, the long-term implications of these post-transcriptional modifications, especially with continuous or repetitive administration of EVs, need to be elucidated. In this respect, their lack of cellular machinery and inability to proliferate in the recipient tissue might limit the duration of their effects and necessitate repeated administration.
There is also uncertainty regarding the optimal dose regimen of MSC-derived EVs, which might influence their capacity to home and engraft damaged cells, and thereby their efficacy for renal repair. Macrophages may promptly target and remove exogenously administered EVs [65], so multiple doses may be needed to achieve and sustain EV-induced renoprotection. A single study  found that a multiple dose regimen was superior in decreasing mortality and improving renal function [47]. Administration of larger doses of MSCs was not necessarily associated with better outcomes, and even an inverse dose-response relationship may occur following a high MSC dose [66,67]. Administration of both low (1 × 10 5 cells/kg) and high (2.5 × 10 5 cells/kg) dose of autologous MSCs improved renal blood flow and kidney perfusion to the same magnitude in patients with RVD [22]. However, no study has reported the in vivo efficacy of escalating doses of EVs or determined a threshold dose in experimental renal disease. Therefore, a standard regimen of EV delivery needs to be established in order to test their efficacy in randomized clinical trials. Furthermore, the adequate number of EV injections and the interval between them need to be determined in future studies. Cardiovascular risk factors may impair the functionality of MSCs and diminish the regenerative benefits of autologous MSC implantation [68]. However, whether EVs isolated from MSCs are also susceptible remains unknown. We have recently found that MetS interferes with the packaging of cargo of porcine adipose tissue MSC-derived EVs, altering the expression of microRNAs that control genes implicated in the development of MetS and its complications [28]. These observations suggest that cardiovascular risk factors may limit the therapeutic efficacy of autologous MSCs and EVs in subjects with coexisting MetS and renal disease. Further preclinical studies and thoughtfully designed and sufficiently powered clinical trials are urgently needed to clarify these uncertainties and overcome the challenges associated with EV therapy in patients with AKI and CKD.
Lastly, emerging evidence suggests that renal cellderived EVs might also exert tissue protective properties in experimental renal disease. RTECs that line the renal tubules play a crucial role in renal function. Similar to MSCs, RTECs release EVs that serve as intercellular communication messengers and may accelerate renal recovery by eliciting tissue regenerative responses. RTEC-derived EVs similarly contain a rich cargo of mRNAs, microRNAs, and proteins that transmit regenerative signals. TGF-β1-treated RTECs release multiple EVs containing microRNA-21 that enhance PTEN-Akt signaling, which modulates several important biological processes [69]. However, EVs released by injured RTECs also contain TGF-β1 mRNA and microRNAs that activate fibroblasts, and their coincubation with them promoted collagen production [70]. Speculatively, this function might be related to the injury resolution phase. Unfortunately, none of these studies tested the in vivo protective effects of RTEC-derived EVs. More recently, intravenous administration of EVs derived from RTECs in rats with renal IRI improved the renal microvasculature and decreased tubular damage and fibrosis [71]. EVs from hypoxia preconditioned RTECs were more effective compared to those obtained from normoxic cells, possibly due to their inhibitory effects on apoptosis following ATP depletion [72]. Fibroblast-derived EVs failed to ameliorate kidney damage in glycerol-induced AKI [48], suggesting that EV-induced renoprotection depends on their cellular source. Therefore, in vitro modifications of RTECs may enhance the protective properties of their daughter EVs. Future studies are needed to confirm these findings and compare the renoprotective potential of MSC-with non-MSC-derived EVs.

Conclusions
AKI and CKD remain global public health challenges, associated with an increased risk for progression to ESRD and cardiovascular complications. Several characteristics of MSCs tested pre-clinically make them attractive to preserve the kidney suffering from AKI and CKD. There are currently several ongoing or completed clinical trials using MSCs for a wide range of renal diseases and preliminary results suggest that MSCs are safe, well tolerated, and effectively ameliorate renal pathology. MSCs exert their reparative effects by releasing EVs, and recent studies in experimental models of AKI and CKD have shown that MSC-derived EVs offer an effective modern treatment option for these patients. MSCderived EVs contain genetic and protein material that upon transferring to recipient cells can activate several repair mechanisms to ameliorate renal injury (Fig. 2). Furthermore, these particles offer some exciting advantages over MSCs. However, clinical data are limited and several challenges need to be addressed as we move towards clinical translation. To date, the primary uncertainties for MSC-derived EV therapy for renal disease include insufficient scientific data to support their safety, and the need to identify the most appropriate EV cellular source, isolation method, and dose regimen, and to assess the impact of co-morbidities on their cargo and renoprotective effect. Alternatively, RTEC-derived EVs may also contribute to cellular repair in AKI and CKD, but the beneficial effects of this approach in patients with CKD remain unknown. Therefore, further basic and translational studies need to continue exploring the potential therapeutic applications of MSC-derived and renal cell-derived EVs for AKI and CKD.